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Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients 1 , yet harbours mutation-derived T cell neoantigens that are suitable for vaccines 2,3 . Here in a phase I trial of adjuvant autogene cevumeran, an individualized neoantigen vaccine based on uridine mRNA-lipoplex nanoparticles, we synthesized mRNA neoantigen vaccines in real time from surgically resected PDAC tumours. After surgery, we sequentially administered atezolizumab (an anti-PD-L1 immunotherapy), autogene cevumeran (a maximum of 20 neoantigens per patient) and a modified version of a four-drug chemotherapy regimen (mFOLFIRINOX, comprising folinic acid, fluorouracil, irinotecan and oxaliplatin). The end points included vaccine-induced neoantigen-specific T cells by high-threshold assays, 18-month recurrence-free survival and oncologic feasibility. We treated 16 patients with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX. Autogene cevumeran was administered within 3 days of benchmarked times, was tolerable and induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen. Using a new mathematical strategy to track T cell clones (CloneTrack) and functional assays, we found that vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8 + T cells. At 18-month median follow-up, patients with vaccine-expanded T cells (responders) had a longer median recurrence-free survival (not reached) compared with patients without vaccine-expanded T cells (non-responders; 13.4 months, P = 0.003). Differences in the immune fitness of the patients did not confound this correlation, as responders and non-responders mounted equivalent immunity to a concurrent unrelated mRNA vaccine against SARS-CoV-2. Thus, adjuvant atezolizumab, autogene cevumeran and mFOLFIRINOX induces substantial T cell activity that may correlate with delayed PDAC recurrence., (© 2023. The Author(s).)

The majority of JAK2 V617F -negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin ( CALR ), resulting in a common carboxyl-terminal mutant fragment (CALR MUT ), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALR MUT -specific T cells are rare in patients with CALR MUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALR MUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALR MUT neoepitopes with high affinity are underrepresented in patients with CALR MUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALR MUT MPN would not efficiently respond to a CALR MUT fragment cancer vaccine but would when immunized with a modified CALR MUT heteroclitic peptide vaccine approach. We found that heteroclitic CALR MUT peptides specifically designed for the MHC-I alleles of patients with CALR MUT MPN efficiently elicited a CALR MUT cross-reactive CD8 + T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALR MUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8 + T cell response to the CALR MUT fragment upon immunization with a CALR MUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALR MUT MPN.

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., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Irreversible T cell exhaustion limits the efficacy of programmed cell death 1 (PD-1) blockade. We observed that dual CD40-TLR4 stimulation within a single tumor restored PD-1 sensitivity and that this regimen triggered a systemic tumor-specific CD8+ T cell response. This approach effectively treated established tumors in diverse syngeneic cancer models, and the systemic effect was dependent on the injected tumor, indicating that treated tumors were converted into necessary components of this therapy. Strikingly, this approach was associated with the absence of exhausted PD-1hi T cells in treated and distant tumors, while sparing the intervening draining lymph node and spleen. Furthermore, patients with transcription changes like those induced by this therapy experienced improved progression-free survival with anti-PD-1 treatment. Dual CD40-TLR4 activation within a single tumor is thus an approach for overcoming resistance to PD-1 blockade that is unique in its ability to cause the loss of exhausted T cells within tumors while sparing nonmalignant tissues.

The history of immunotherapy is rooted in the treatment of melanoma and therapy with immune checkpoint-blocking agents is now a cornerstone for the treatment of metastatic melanoma. The first effective immunotherapies approved by the US Food and Drug Administration in melanoma included interleukin-2 for metastatic disease and interferon alpha in the adjuvant setting. These were followed by a group of new therapies, including checkpoint-blocking antibodies targeting cytotoxic T lymphocyte-associated protein 4 and programmed cell death protein 1. Therapies intended to 'reeducate' T cells, such as tumor-infiltrating lymphocyte therapy, oncolytic viruses and tumor vaccines, have yielded promising results and are under development. Finally, the integration of the above therapies as well as development of new coinhibitory and costimulatory agents, though in early stages, appear very promising and likely represent the next phase in drug development for the treatment of metastatic melanoma., (© 2015 S. Karger AG, Basel.)

The development of successful cancer vaccines is contingent on the ability to induce effective and persistent anti-tumor immunity against self-antigens that do not typically elicit immune responses. In this study, we examine the effects of a non-myeloablative dose of total body irradiation on the ability of tumor-naïve mice to respond to DNA vaccines against melanoma. We demonstrate that irradiation followed by lymphocyte infusion results in a dramatic increase in responsiveness to tumor vaccination, with augmentation of T cell responses to tumor antigens and tumor eradication. In irradiated mice, infused CD8(+) T cells expand in an environment that is relatively depleted in regulatory T cells, and this correlates with improved CD8(+) T cell functionality. We also observe an increase in the frequency of dendritic cells displaying an activated phenotype within lymphoid organs in the first 24 hours after irradiation. Intriguingly, both the relative decrease in regulatory T cells and increase in activated dendritic cells correspond with a brief window of augmented responsiveness to immunization. After this 24 hour window, the numbers of dendritic cells decline, as does the ability of mice to respond to immunizations. When immunizations are initiated within the period of augmented dendritic cell activation, mice develop anti-tumor responses that show increased durability as well as magnitude, and this approach leads to improved survival in experiments with mice bearing established tumors as well as in a spontaneous melanoma model. We conclude that irradiation can produce potent immune adjuvant effects independent of its ability to induce tumor ablation, and that the timing of immunization and lymphocyte infusion in the irradiated host are crucial for generating optimal anti-tumor immunity. Clinical strategies using these approaches must therefore optimize such parameters, as the correct timing of infusion and vaccination may mean the difference between an ineffective treatment and successful tumor eradication.

Modulation of the immune system by targeting coinhibitory and costimulatory receptors has become a promising new approach of immunotherapy for cancer. The recent approval of the CTLA-4-blocking antibody ipilimumab for the treatment of melanoma was a watershed event, opening up a new era in the field of immunotherapy. Ipilimumab was the first treatment to ever show enhanced overall survival (OS) for patients with stage IV melanoma. However, measuring response rates using standard Response Evaluation Criteria in Solid Tumors (RECIST) or modified World Health Organization criteria or progression-free survival does not accurately capture the potential for clinical benefit for ipilimumab-treated patients. As immunotherapy approaches are translated into more tumor types, it is important to study biomarkers, which may be more predictive of OS to identify the patients most likely to have clinical benefit. Ipilimumab is the first-in-class of a series of immunomodulating antibodies that are in clinical development. Anti-PD1 (nivolumab and MK-3475), anti-PD-L1 (BMS-936 559, RG7446, and MEDI4736), anti-CD137 (urelumab), anti-OX40, anti-GITR, and anti-CD40 monoclonal antibodies are just some of the agents that are being actively investigated in clinical trials, each having the potential for combination with the ipilimumab to enhance its effectiveness. Development of rational combinations of immunomodulatory antibodies with small-molecule pathway inhibitor therapies such as vemurafenib makes the discovery of predictive biomarkers even more important. Identifying reliable biomarkers is a necessary step in personalizing the treatment of each patient's cancer through a baseline assessment of tumor gene expression and/or immune profile to optimize therapy for the best chance of therapeutic success., (©2013 AACR.)

Objective: To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs., Animals: 111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved., Procedures: 58 dogs received an initial series of 4 injections of huTyr vaccine (102 μg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death., Results: Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising., Conclusions and Clinical Relevance: Results support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM., Impact for Human Medicine: Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

Despite significant scientific knowledge in the field of cancer immunology, therapeutic strategies using cancer vaccines to generate anti-tumor immunity have historically resulted in only modest clinical benefit. Disappointing results from prior cancer vaccine trials are likely due to multifactorial causes. Perhaps the most important is the role of inherent tumor-induced immune suppression and enhanced immunologic tolerance. Current research directed toward understanding the mechanisms of immunologic tolerance has led to the development of promising therapeutic immune regulatory antibodies that inhibit immunologic checkpoints and subsequently enhance immunologic anti-tumor activity. This review discusses the prior challenges associated with cancer vaccines and describes how, by breaking immune inhibition and facilitating immune stimulation, immune regulatory antibodies show great promise in the treatment of a variety of tumors.

Background: Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response., Methods: To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100(209-217) and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100(209-217) (ITDQVPFSV), tyrosinase(369-377) (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape., Results: Following vaccination, patients generated weak to no CD4(+) or CD8(+) T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7(lo)CD45RA(lo)) tetramer(+)CD8(+) T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules., Conclusion: Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially "boosting" the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.

HER2/neu is an oncogene amplified and over-expressed in 20-30% of breast adenocarcinomas. Treatment with the humanized monoclonal antibody trastuzumab has shown efficacy in combination with cytotoxic agents, although resistance occurs over time. Novel approaches are needed to further increase antibody efficacy. In this study, we provide evidence in a mouse breast cancer therapeutic tumor model that the combination of active immunization with a modified HER2/neu DNA vaccine and passive infusion of an anti-HER2/neu monoclonal antibody leads to significant regression of established tumors. Our data indicate that combination therapy with a HER2/neu DNA vaccine and trastuzumab may have clinical activity in breast cancer patients., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: We compared the efficacy of human Langerhans cells (LC) as tumor immunogens in vivo with monocyte-derived dendritic cells (moDC) and investigated how interleukin 15 (IL15) supports optimal DC-stimulated antitumor immunity., Experimental Design: American Joint Committee on Cancer stage III/IV melanoma patients participated in this first clinical trial comparing melanoma peptide-pulsed LC with moDC vaccines (NCT00700167, www.ClinicalTrials.gov). Correlative studies evaluated mechanisms mediating IL15 support of DC-stimulated antitumor immunity., Results: Both DC vaccines were safe and immunogenic for melanoma antigens. LC-based vaccines stimulated significantly greater tyrosinase-HLA-A*0201 tetramer reactivity than the moDC-based vaccines. The two DC subtypes were otherwise statistically comparable, in contrast to extensive prior data in vitro showing LC superiority. LCs synthesize much more IL15 than moDCs and stimulate significantly more antigen-specific lymphocytes with a cytolytic IFN-γ profile even without exogenous IL15. When supplemented by low-dose IL15, instead of IL2, moDCs stimulate 5 to 6 logs more tumor antigen-specific effector memory T cells (T(EMRA)) over 3 to 4 weeks in vitro. IL2 and IL15 can be synergistic in moDC stimulation of cytolytic T cells. IL15 promotes T-cell expression of the antiapoptotic bcl-2 and inhibits candidate regulatory T-cell (Treg) expansion after DC stimulation, countering two effects of IL2 that do not foster tumor immunity., Conclusions: MoDC-based vaccines will require exogenous IL15 to achieve clinical efficacy. Alternatively, LCs can couple the endogenous production of IL15 with potent T-cell stimulatory activity. Optimization of full-length tumor antigen expression for processing into multiple immunogenic peptides for presentation by both class I and II MHC therefore merits emphasis to support more effective antitumor immunity stimulated by LCs.

Since the development of the first vaccines, modern medicine has been consistently aiming to improve the efficacy of immune responses. Traditionally, adjuvants have been used as non-specific immune modulators to enhance recognition and activation against a desired antigen. By providing 'danger' signals to the immune system, adjuvants activate innate immunity, which enhances the development of protective and therapeutic adaptive immune responses. The newest class of immune modulators bypasses the innate response and targets cells of the adaptive response directly. Targeted immunomodulatory therapy is focused primarily on the activation of costimulatory receptors (eg, 4-1BB, OX40 and GITR [glucocorticoid-induced TNF receptor-related gene]) or the blockade of co-inhibitory receptors (eg, CTLA-4, PD-1 and PD-L1) on T-cells during activation and/or effector responses. With promising clinical results obtained to date, immunomodulatory therapy is becoming an integral part of immunotherapeutic approaches. The modulation of GITR is listed as one of the top 25 most promising research areas by the NCI, and has demonstrated potential in both antitumor and vaccine settings. This review discusses the role of GITR as a potential target for immunomodulatory therapy, as well as the research involved in understanding the mechanisms of anti-GITR therapy and current progress in translation into the clinic.

Background: An improvement in overall survival among patients with metastatic melanoma has been an elusive goal. In this phase 3 study, ipilimumab--which blocks cytotoxic T-lymphocyte-associated antigen 4 to potentiate an antitumor T-cell response--administered with or without a glycoprotein 100 (gp100) peptide vaccine was compared with gp100 alone in patients with previously treated metastatic melanoma., Methods: A total of 676 HLA-A*0201-positive patients with unresectable stage III or IV melanoma, whose disease had progressed while they were receiving therapy for metastatic disease, were randomly assigned, in a 3:1:1 ratio, to receive ipilimumab plus gp100 (403 patients), ipilimumab alone (137), or gp100 alone (136). Ipilimumab, at a dose of 3 mg per kilogram of body weight, was administered with or without gp100 every 3 weeks for up to four treatments (induction). Eligible patients could receive reinduction therapy. The primary end point was overall survival., Results: The median overall survival was 10.0 months among patients receiving ipilimumab plus gp100, as compared with 6.4 months among patients receiving gp100 alone (hazard ratio for death, 0.68; P<0.001). The median overall survival with ipilimumab alone was 10.1 months (hazard ratio for death in the comparison with gp100 alone, 0.66; P=0.003). No difference in overall survival was detected between the ipilimumab groups (hazard ratio with ipilimumab plus gp100, 1.04; P=0.76). Grade 3 or 4 immune-related adverse events occurred in 10 to 15% of patients treated with ipilimumab and in 3% treated with gp100 alone. There were 14 deaths related to the study drugs (2.1%), and 7 were associated with immune-related adverse events., Conclusions: Ipilimumab, with or without a gp100 peptide vaccine, as compared with gp100 alone, improved overall survival in patients with previously treated metastatic melanoma. Adverse events can be severe, long-lasting, or both, but most are reversible with appropriate treatment. (Funded by Medarex and Bristol-Myers Squibb; ClinicalTrials.gov number, NCT00094653.)

Purpose: Prior studies show that i.m. injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) induces CD8(+) T-cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a pilot clinical trial comparing i.m. injection with particle-mediated epidermal delivery (PMED)., Experimental Design: Human leukocyte antigen (HLA)-A*0201(+) disease-free melanoma patients were randomized to the PMED or i.m. arm, receiving eight vaccinations over 4 months. Patients received 4 microg or 2,000 microg per injection, respectively, of mouse gp100 DNA. Peripheral blood mononuclear cells were collected, cultured with gp100 peptides, and analyzed by tetramer and intracellular cytokine staining for responses to HLA-A*0201-restricted gp100 epitopes [gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA)]., Results: Twenty-seven patients with stage IIB-IV melanoma were analyzable for immune response. The only common toxicity was grade 1 injection site reaction in nine patients with no intergroup difference, and one dose-limiting toxicity of acute hypersensitivity occurred in a PMED patient with undiagnosed gold allergy. Four of 27 patients produced gp100 tetramer(+)CD8(+) T cells, all carrying the CCR7(lo)CD45RA(lo) effector-memory phenotype. Five of 27 patients generated IFN-gamma(+)CD8(+) T cells, one who was also tetramer-positive. Overall, vaccination induced a response in 30% of patients, which was not significantly associated with study arm or clinical outcome. However, the PMED group showed a trend toward increased IFN-gamma(+)CD8(+) T-cell generation (P = 0.07)., Conclusion: A comparable efficacy and safety profile was shown between the i.m. and PMED arms, despite a significantly decreased dose of DNA used for PMED injection., ((c) 2010 AACR.)

A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 microg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-gamma release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-gamma-secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia.

A differentiation antigen commonly expressed on melanoma cells, gp100 is the target of infiltrating T cells. We conducted a phase I randomized cross-over trial of melanoma patients with either xenogeneic (mouse) or human gp100 plasmid DNA injected intramuscularly at three dosages (100, 500 or 1,500 microg) every three weeks for three doses. After the first three injections, patients were then immunized three times with gp100 from the other species. Peripheral blood samples were analyzed at various time points following 10-day culture with gp100 peptides using multi-parametric flow cytometry. A total of 19 patients were enrolled, with 18 assessable for immune function and survival. 14 (74%) were male, with a median age of 56 years (range, 20-82). All patients had no evidence of disease; 10 (53%) had stage III disease, 3 each (16%) had stage IIB and IV disease, 2 (11%) had choroidal and 1 (5%) had anal mucosal involvement. With a median follow-up of 30 months, median progression-free survival (PFS) is 44 months. Median survival is not reached. There was no grade 3/4 toxicity; the most common grade 1/2 toxicity was an injection site reaction in 12 patients (63%, all grade 1). Five patients developed CD8+ cells binding gp100(280-288) HLA-A2-restricted tetramer. One patient had an increase in CD8+ IFN-gamma+ cells. This xenogeneic immunization strategy was safe and associated with minimal toxicity. There was also evidence of immune response.

Keratinocyte growth factor (KGF), which is given exogenously to allogeneic bone marrow transplantation (allo-BMT) recipients, supports thymic epithelial cells and increases thymic output of naive T cells. Here, we demonstrate that this improved T-cell reconstitution leads to enhanced responses to DNA plasmid tumor vaccination. Tumor-bearing mice treated with KGF and DNA vaccination have improved long-term survival and decreased tumor burden after allo-BMT. When assayed before vaccination, KGF-treated allo-BMT recipients have increased numbers of peripheral T cells, including CD8(+) T cells with vaccine-recognition potential. In response to vaccination, KGF-treated allo-BMT recipients, compared with control subjects, generate increased numbers of tumor-specific CD8(+) cells, as well as increased numbers of CD8(+) cells producing interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). We also found unanticipated benefits to antitumor immunity with the administration of KGF. KGF-treated allo-BMT recipients have an improved ratio of T effector cells to regulatory T cells, a larger fraction of effector cells that display a central memory phenotype, and effector cells that are derived from a broader T-cell-receptor repertoire. In conclusion, our data suggest that KGF can function as a potent vaccine adjuvant after allo-BMT through its effects on posttransplantation T-cell reconstitution.

Vaccines are often inefficient in old people and old mice. Few studies have focused on testing vaccines in old populations. Here we used DNA tumor antigen vaccines against melanoma and showed that old mice were not protected. Vaccines incorporating fusions of the tumor antigen with microbial adjuvant proteins OmpA (E. Coli) or Vp22 (Herpes simplex virus-1) dramatically improved protection of old mice. The mechanisms by which these adjuvant proteins act are distinct. TLR2 was not required for either OmpA or Vp22. Antigen processing and presentation were not boosted by these fusion constructs. However, fusion constructs with Vp22 gave a strong CD4 response to B16 melanoma and the OmpA response is MHC-II dependent. Both adjuvant fusion constructs stimulated CD4 and CD8 responses otherwise diminished in old mice.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances immune responses by inducing proliferation, maturation, and migration of dendritic cells (DCs) as well as expansion and differentiation of B and T lymphocytes. The potency of DNA vaccines can be enhanced by the addition of DNA encoding cytokines, acting as molecular adjuvants. We conducted a phase I/II trial of human GM-CSF DNA in conjunction with a multipeptide vaccine (gp100 and tyrosinase) in stage III/IV melanoma patients. Nineteen human leukocyte antigen (HLA)-A*0201+ patients were treated. Three dose levels were studied: 100, 400, and 800 microg DNA/injection, administered subcutaneously every month with 500 microg of each peptide. In the dose-ranging study, three patients were treated at each dose level. The remaining patients were then treated at the highest dose. Most toxicities were grade 1 injection-site reactions. Eight patients (42%) developed CD8+ T-cell responses, defined by a > or =3 SD increase in baseline reactivity to tyrosinase or gp100 peptide in tetramer or intracellular cytokine staining (ICS) assays. There was no relationship between dose and T-cell response. Responding T cells had an effector memory cell phenotype. Polyfunctional T cells were also demonstrated. At a median of 31 months follow-up, median survival has not been reached. Human GM-CSF DNA was found to be a safe adjuvant.

Passive immunization with monoclonal antibody TA99 targeting melanoma differentiation antigen tyrosinase-related protein-1 (Tyrp1; gp75) and active immunization with plasmid DNA encoding altered Tyrp1 both mediate tumor immunity in the B16 murine melanoma model. We report here that TA99 enhances Tyrp1 DNA vaccination in the treatment of B16 lung metastases, an effect mediated by immunologic mechanisms as Tyrp1 has no known role in regulating tumor growth. TA99 is shown to increase induction of anti-Tyrp1 CD8+T-cell responses to DNA vaccination against Tyrp1 as assessed by IFN-gamma ELISPOT assays. Immunohistochemistry studies reveal that TA99 localizes rapidly and specifically to B16 lung nodules. Augmentation of T-cell responses is dependent on the presence of tumor as well as on activating Fc receptors. Furthermore, TA99 enhances DNA vaccination against a distinct melanoma antigen, gp100(pmel17/silver locus), improving antitumor efficacy, augmenting systemic CD8+ T-cell responses to gp100, and increasing CD8+ T-cell infiltration at the tumor site. Epitope spreading was observed, with CD8+ T-cell responses generated to Tyrp1 peptide in mice receiving gp100 DNA vaccination in the presence of TA99. Finally, we show that TA99 improves therapeutic efficacy of DNA vaccination combined with adoptive T-cell transfer in treatment of established subcutaneous B16 melanoma. In conclusion, TA99 enhances DNA vaccination against both the target antigen Tyrp1 and a distinct melanoma antigen gp100 in an Fc receptor-dependent mechanism, consistent with enhanced cross-presentation of tumor-derived antigen. Monoclonal antibodies should be tested as vaccine adjuvants in the treatment of cancer.

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

Purpose: HER2/neu is frequently overexpressed in breast cancer. In a mouse model, vaccination with HER2/neu DNA elicits antibodies that confer partial protection against tumor challenge., Experimental Design: To enhance antitumor immunity, we fused cDNA encoding Flt-3 ligand (FL) to the rat HER2/neu extracellular domain (neu), generating a chimeric FLneu molecule. FLneu and neu DNA vaccines were compared for immunogenicity and their ability to protect mice from tumor challenge., Results: The neu vaccine generated a HER2/neu-specific antibody response. In contrast, vaccination with FLneu induced CD8+ T cells specific for HER2/neu but a negligible anti-HER2/neu antibody response. The switch from an antibody-mediated to T cell-mediated response was due to different intracellular localization of neu and FLneu. Although the neu protein was secreted, the FLneu protein was retained inside the cell, co-localizing with the endoplasmic reticulum, facilitating processing and presentation to T cells. The neu and FLneu vaccines individually conferred only weak tumor immunity. However, efficient tumor rejection was seen when neu and FLneu were combined, inducing both strong anti-HER2/neu-specific antibody and T cell responses. Adoptive transfer of both immune CD8+ T cells and immune sera from immunized mice was required to confer tumor immunity in naïve hosts., Conclusions: These results show that active induction of both humoral and cellular immunity to HER2/neu is required for efficient tumor protection, and that neither response alone is sufficient.

One strategy to generate T-cell responses to tumors is to alter subdominant epitopes through substitution of amino acids that are optimal anchors for specific MHC molecules, termed heteroclitic epitopes. This approach is manually error-prone and time-consuming. In here, we describe a computer-based algorithm (EpitOptimizer) for the streamlined design of heteroclitic epitopes. Analysis of two cancer-related proteins showed that EpitOptimizer-generated peptides have enhanced MHC-I binding compared with their wild-type counterparts; and were able to induce stronger CD8+ T-cell responses against their native epitope. These data demonstrate that this approach can serve as the basis of epitope-engineering against cancer and intracellular pathogens.

Antitumor immune responses can be elicited in preclinical mouse melanoma models using plasmid DNA vaccines encoding xenogeneic melanosomal differentiation antigens. We previously reported on a phase I clinical trial of human tyrosinase (huTyr) DNA vaccination of 9 dogs with advanced malignant melanoma (World Health Organization stages II-IV), in which we demonstrated the safety of the treatment and the prolongation of the expected survival time (ST) of subjects as compared to historical, stage-matched controls. As a secondary goal of the same study, we report here on the induction of tyrosinase-specific antibody responses in three of the nine dogs vaccinated with huTyr DNA. The antibodies in two of the three responders cross-react with syngeneic canine tyrosinase, demonstrating the ability of this vaccine to overcome host immune tolerance and/or ignorance to or of "self" antigens. Most interestingly, the onset of antibody induction in these three dogs coincides with observed clinical responses and may suggest a means to account for their long-term tumor control and survival.

Purpose: Immunization of mice with xenogeneic DNA encoding human tyrosinase-related proteins 1 and 2 breaks tolerance to these self-antigens and leads to tumor rejection. Viral vectors used alone or in heterologous DNA prime/viral boost combinations have shown improved responses to certain infectious diseases. The purpose of this study was to compare viral and plasmid DNA in combination vaccination strategies in the context of a tumor antigen., Experimental Design: Using tyrosinase as a prototypical differentiation antigen, we determined the optimal regimen for immunization with plasmid DNA. Then, using propagation-incompetent alphavirus vectors (virus-like replicon particles, VRP) encoding tyrosinase, we tested different combinations of priming with DNA or VRP followed by boosting with VRP. We subsequently followed antibody production, T-cell response, and tumor rejection., Results: T-cell responses to newly identified mouse tyrosinase epitopes were generated in mice immunized with plasmid DNA encoding human (xenogeneic) tyrosinase. In contrast, when VRP encoding either mouse or human tyrosinase were used as single agents, antibody and T-cell responses and a significant delay in tumor growth in vivo were observed. Similarly, a heterologous vaccine regimen using DNA prime and VRP boost showed a markedly stronger response than DNA vaccination alone., Conclusions: Alphavirus replicon particle vectors encoding the melanoma antigen tyrosinase (self or xenogeneic) induce immune responses and tumor protection when administered either alone or in the heterologous DNA prime/VRP boost approaches that are superior to the use of plasmid DNA alone.

Xenogeneic DNA vaccination can elicit tumor immunity through T cell and antibody-dependent effector mechanisms. Blockade of CTLA-4 engagement with B7 expressed on APCs has been shown to enhance T cell-dependent immunity. We investigated whether CTLA-4 blockade could increase T-cell responses and tumor immunity elicited by DNA vaccines. CTLA-4 blockade enhanced B16 tumor rejection in mice immunized against the melanoma differentiation antigens tyrosinase-related protein 2 and gp100, and this effect was stronger when anti-CTLA-4 was administered with booster vaccinations. CTLA-4 blockade also increased the T-cell responses to prostate-specific membrane antigen (PSMA) when given with the second or third vaccination. Based on these pre-clinical studies, we suggest that anti-CTLA-4 should be tested with xenogeneic DNA vaccines against cancer and that special attention should be given to sequence and schedule of administration.

Purpose: Melanomas, sarcomas, and neuroblastomas abundantly express the ganglioside GD2 on the cell surface where it is susceptible to immune attack by antibodies. Overexpression of GD2 on these tumors is striking, as is the frequency of clinical responses after treatment of neuroblastoma with monoclonal antibodies against GD2. In addition, preclinical models have demonstrated the ability of a GD2-keyhole limpet hemocyanin (KLH) conjugate vaccine to induce antibodies that eliminate micrometastases. However, vaccination of patients with GD2-KLH has previously failed to induce a consistent relevant antibody response. We test here whether the use of GD2 lactone-KLH can overcome the low immunogenicity of GD2-KLH., Experimental Design: Eighteen patients with melanoma were vaccinated s.c. in the adjuvant setting on weeks 0, 1, 2, 3, 10, and 24. Groups of 6 patients were entered at three dose levels (3, 10, or 30 micro g) of GD2 lactone (GD2L) in vaccines containing GD2L-KLH plus the immunological adjuvant QS-21. Blood was drawn at regular intervals to assess the antibody response., Results: The vaccine was well tolerated. The majority of patients in all three dose levels produced anti-GD2 antibodies detectable by ELISA assay. Specificity for GD2 was also confirmed by immune thin-layer chromatography. Although there was no statistical difference in terms of titers between the three groups, patients at the 30- micro g dose level had higher titers and longer lasting antibody responses overall by ELISA (median IgM/IgG peak titer 1:640/1:80) and generated the strongest cell surface reactivity by fluorescence-activated cell sorting (median IgM peak percentage positive cells/mean fluorescence intensity for pre- and postvaccination sera is 10%/63 and 70%/135). Patients vaccinated with the 30- micro g GD2 dose also had the most potent complement dependent cytotoxicity using human complement, with 5 of 6 patients showing strong cell surface reactivity by fluorescence-activated cell sorting and >30% cytotoxicity by chromium release with a serum dilution of 1/100., Conclusions: GD2L-KLH conjugate vaccine plus adjuvant QS-21 induces antibodies against GD2 that bind to the cell surface and induce complement-dependent cytotoxicity in the majority of patients with melanoma.

Successful active immunization against cancer requires induction of immunity against self or mutated self Ags. However, immunization against self Ags is difficult. Xenogeneic immunization with orthologous Ags induces cancer immunity. The present study evaluated the basis for immunity induced by active immunization against a melanoma differentiation Ag, gp100. Tumor rejection of melanoma was assessed after immunization with human gp100 (hgp100) DNA compared with mouse gp100 (mgp100). C57BL/6 mice immunized with xenogeneic full-length hgp100 DNA were protected against syngeneic melanoma challenge. In contrast, mice immunized with hgp100 DNA and given i.p. tolerizing doses of the hgp100 D(b)-restricted peptide, hgp100(25-33), were incapable of rejecting tumors. Furthermore, mice immunized with DNA constructs of hgp100 in which the hgp100(25-27) epitope was substituted with the weaker D(b)-binding epitope from mgp100 (mgp100(25-27)) or a mutated epitope unable to bind D(b) did not reject B16 melanoma. Mice immunized with a minigene construct of hgp100(25-33) rejected B16 melanoma, whereas mice immunized with the mgp100(25-33) minigene did not develop protective tumor immunity. In this model of xenogeneic DNA immunization, the presence of an hgp100 heteroclitic epitope with a higher affinity for MHC created by three amino acid (25 to 27) substitutions at predicted minor anchor residues was necessary and sufficient to induce protective tumor immunity in H-2(b) mice with melanoma.

Purpose: Canine malignant melanoma (CMM) is a spontaneous, aggressive, and metastatic neoplasm. Preclinical mouse studies have shown that xenogeneic DNA vaccination with genes encoding tyrosinase family members can induce antibody and cytotoxic T-cell responses, resulting in tumor rejection. These studies provided the rationale for a trial of xenogeneic DNA vaccination in CMM using the human tyrosinase gene., Experimental Design: Three cohorts of three dogs each with advanced (WHO stage II, III, or IV) CMM received four biweekly i.m. injections (dose levels 100, 500, or 1500 micro g, respectively/vaccination) of human tyrosinase plasmid DNA i.m. via the Biojector2000 delivery device., Results: Mild local reactions at injection sites were the only toxicities observed, with no signs of autoimmunity. One dog with stage IV disease had a complete clinical response in multiple lung metastases for 329 days. Two dogs with stage IV disease had long-term survivals (421 and 588+ days) in the face of significant bulky metastatic disease, and two other dogs with locally controlled stage II/III disease had long-term survivals (501 and 496 days) with no evidence of melanoma on necropsy. Four other dogs were euthanized because of progression of the primary tumor. The Kaplan-Meier median survival time for all nine dogs was 389 days., Conclusions: The results of this trial demonstrate that xenogeneic DNA vaccination of dogs with advanced malignant melanoma is a safe and potentially therapeutic modality. On the basis of these results, additional evaluation of this novel therapeutic is warranted in locally controlled CMM and advanced human melanoma.

DNA vaccination against tissue-restricted antigens is a strategy for cancer therapy. Immune tolerance and ignorance of self antigens has been a hurdle for this approach. We have shown that immunization with xenogeneic DNA orthologues elicits tumor immunity. One model that we have developed entails immunization of mice against tyrosinase-related protein-2 (Tyrp2) using cDNA encoding homologous human Tyrp2. A subset of mice immunized with human Tyrp2 developed antibody responses to Tyrp1. Unexpectedly, this was not simply due to cross-reactivity, as mice with anti-Tyrp1 antibodies were not usually the same animals with anti-Tyrp2 antibodies. Although autoimmune vitiligo was frequently observed in mice that had been immunized with Tyrp2, its occurrence was not correlated with the development of antibodies to Tyrp1. This implies that the appearance of anti-Tyrp1 antibodies was not simply a consequence of the destruction of melanocytes by T-cells recognizing Tyrp2. This represents an example of intermolecular determinant recognition, but is not simply due to epitope spreading since antibodies against the antigen targeted by DNA vaccination are not typically detected.

The identification of antigens on tumor cells has led to significant contributions to the field of immunotherapy. One of the most active areas under investigation in cancer immunotherapy is the development of vaccines against melanoma antigens. Induction of immunity against tumor antigens can follow multiple routes using different mechanisms. Crucial to the development of active immunization and other immunotherapies is the discovery and understanding of the molecular identity of antigens and the mechanisms involved in tumor immunity, as well as escape from immunity. In this review, we will discuss strategies to induce active immunity against melanoma.

Purpose:We conducted a phase I vaccine trial to determine safety, toxicity, and immunogenicity of autologous Langerhans-type dendritic cells (LCs), electroporated with murine tyrosinase-related peptide-2 (mTRP2) mRNA in patients with resected AJCC stage IIB, IIC, III, or IV (MIa) melanoma.Experimental Design: Nine patients received a priming immunization plus four boosters at three week intervals. Vaccines comprised 10 × 106mRNA-electroporated LCs, based on absolute number of CD83+CD86brightHLA-DRbrightCD14negLCs by flow cytometry. Initial vaccines used freshly generated LCs, whereas booster vaccines used viably thawed cells from the cryopreserved initial product. Post-vaccination assessments included evaluation of delayed-type hypersensitivity (DTH) reactions after booster vaccines and immune response assays at one and three months after the final vaccine.Results:All patients developed mild DTH reactions at injection sites after booster vaccines, but there were no toxicities exceeding grade 1 (CTCAE, v4.0). At one and three months post-vaccination, antigen-specific CD4 and CD8 T cells increased secretion of proinflammatory cytokines (IFN-γ, IL-2, and TNF-α), above pre-vaccine levels, and also upregulated the cytotoxicity marker CD107a. Next-generation deep sequencing of the TCR-V-β CDR3 documented fold-increases in clonality of 2.11 (range 0.85–3.22) for CD4 and 2.94 (range 0.98–9.57) for CD8 T cells at one month post-vaccines. Subset analyses showed overall lower fold-increases in clonality in three patients who relapsed (CD4: 1.83, CD8: 1.54) versus non-relapsed patients (CD4: 2.31, CD8: 3.99).Conclusions:TRP2 mRNA-electroporated LC vaccines are safe and immunogenic. Responses are antigen-specific in terms of cytokine secretion, cytolytic degranulation, and increased TCR clonality, which correlates with clinical outcomes. [ABSTRACT FROM PUBLISHER]

The article discusses research on identifying antigens for cancer vaccines. It references a study by R. Vile et al published in "Nature Biotechnology." The researchers showed that melanoma in mice can be treated by vaccination with a human melanoma cDNA library delivered by a vesicular stomatitis virus (VSV) vector. Findings of the study also opened several additional avenues for future research on areas such as the pattern of antigen expression in individual tumors.