Agonist Epitope Research Articles

A Fentanyl Hapten-Displaying Lipid Nanoparticle Vaccine that Non-Covalently Encapsulates a TLR7/8 Agonist and T-Helper Epitope Induces Protective Anti-Fentanyl Immunity.

Opioid use disorder - particularly involving fentanyl - has precipitated a public health crisis characterized by a significant increase in addiction and overdose-related deaths. Fentanyl-specific immunotherapy, which aims at inducing fentanyl-specific antibodies capable of binding fentanyl molecules in the bloodstream, preventing their entry in the central nervous system, is therefore gaining momentum. Conventional opioid designs rely on the covalent conjugation of fentanyl analogues to immunogenic carrier proteins that hold the inherent capacity of mounting immunodominant responses. Here, we present an alternative fentanyl vaccine design that utilizes a non-covalent assembly of lipid nanoparticles (LNPs) to deliver fentanyl haptens in conjunction with a CD4+ T-helper peptide epitope and an imidazoquinoline TLR7/8 agonist. Our results demonstrate that a single intramuscular administration of the LNP-based nanovaccine elicits fentanyl-specific antibodies, significantly mitigating the effects of opioid overdose in preclinical mouse models. Furthermore, we analyzed the immunobiological behavior of the vaccine in vivo in mouse models, providing evidence that covalent attachment of a fentanyl hapten to a carrier proteins or peptide epitope is not necessary for inducing an effective immune response. However, co-delivery - specifically, the physical assembly of all immune cues into an LNP - remains essential for inducing hapten-specific immunity.

Characterization of recombinant gorilla adenovirus HPV therapeutic vaccine PRGN-2009.

There are approximately 44,000 cases of human papillomavirus–associated (HPV-associated) cancer each year in the United States, most commonly caused by HPV types 16 and 18. Prophylactic vaccines successfully prevent healthy people from acquiring HPV infections via HPV-specific antibodies. In order to treat established HPV-associated malignancies, however, new therapies are necessary. Multiple recombinant gorilla adenovirus HPV vaccine constructs were evaluated in NSG-β2m–/– peripheral blood mononuclear cell–humanized mice bearing SiHa, a human HPV16+ cervical tumor, and/or in the syngeneic HPV16+ TC-1 model. PRGN-2009 is a therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T cell enhancer agonist epitopes. PRGN-2009 treatment reduced tumor volume and increased CD8+ and CD4+ T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6–specific T cells, and increased multifunctional CD8+ and CD4+ T cells in the tumor microenvironment. These studies provide the first evaluation to our knowledge of a therapeutic gorilla adenovirus HPV vaccine, PRGN-2009, showing promising preclinical antitumor efficacy and induction of HPV-specific T cells, along with the rationale for its evaluation in clinical trials.

Phase I study of a multitargeted recombinant Ad5 PSA/MUC-1/brachyury-based immunotherapy vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC)

BackgroundAntitumor vaccines targeting tumor-associated antigens (TAAs) can generate antitumor immune response. A novel vaccine platform using adenovirus 5 (Ad5) vectors [E1–, E2b–] targeting three TAAs—prostate-specific antigen (PSA), brachyury, and MUC-1—has...

Anti-tumor efficacy and immune responses induced by a novel therapeutic HPV-specific gorilla adenovirus off-the-shelf immunotherapeutic (PRGN-2009)

Abstract There are about 44,000 cases of Human Papilloma Virus (HPV) associated cancer each year in the US, most commonly caused by HPV 16 and 18. Prophylactic vaccines have been successful at preventing healthy patients from acquiring HPV infections via the induction of HPV specific antibodies. To treat established HPV-associated malignancies, however, the continued development of new therapies is necessary. One promising new modality is HPV immunotherapeutics, which target the HPV 16 and 18 oncoproteins E6 and E7 with the goal to activate antigen specific cytotoxic T cells. Here, we evaluated PRGN-2009, a novel gorilla adenovirus GAd HPV off-the-shelf immunotherapeutic containing multiple CTL agonist epitopes of E6 and E7 to enhance the immune responses. Three weekly administrations with PRGN-2009 in the TC-1 mouse model led to decreases in tumor volume and weight, and significant increases in HPV16 E6-specific splenocytes evaluated by IFNg ELISPOT. PRGN-2009 also increased the tumor-infiltrating CD8 and CD4 T cells, as well as multi-functional (IFNg+, GzmB+) CD8 T cells in the tumor microenvironment (TME). Additionally, PRGN-2009 was tested in SiHa, a human HPV16+ cervical tumor, in the NSG β2m−/− PBMC humanized mouse model. Weekly PRGN-2009 administrations led to a reduction in tumor and a trending increase in CD8 and CD4 T cells in the tumor; IHC confirmed the increase of CD8 T cells into the TME. These studies provide the first evaluation of the GAd HPV off-the-shelf immunotherapeutic PRGN-2009 and its therapeutic impact against HPV-associated cancers.

Suboptimal stimulation by weak agonist epitope variants does not drive dysfunction of HIV-1-specific cytotoxic T lymphocyte clones.

To assess whether weakly recognized epitope variants induce anergy in HIV-1-specific CD8 T lymphocyte (CTL) clones as a mechanism of dysfunction. HIV-1-specific CTL clones were exposed to suboptimally recognized epitope variants, and screened for anergy and other T-cell dysfunction markers, and subsequent capability to kill target cells bearing index epitope. In addition to the optimally recognized index epitope, two suboptimally recognized epitope variants were selected based on titration curves for killing of peptide-labeled target cells by three HIV-1-specific CTL clones targeting the epitopes SLYNTVATL (Gag 77-85, A02-restricted), RPAEPVPLQL (Rev 66-75, B07-restricted), and KRWIIMGLNK (Gag 263-272, B27-restricted). Consequences of suboptimal stimulation were assessed by cytokine secretion, gene expression, and capacity to kill index epitope-labeled target cells upon rechallenge. Suboptimal recognition of epitope variants reduced cytokine production by CTL similarly to reduction in killing of target cells. Gene expression profiles after suboptimal stimulation demonstrated no patterns consistent with T-cell dysfunction due to anergy, exhaustion, or apoptosis. Preexposure of CTL to epitope variants had no discernable impact on their subsequent capacity to kill index epitope-bearing target cells. Our data explore the hypothesis that poorly recognized epitope variants not only facilitate HIV-1 evasion of CTL recognition, but also induce CTL dysfunction through suboptimal signaling causing anergy. However, the results do not suggest that suboptimal signaling induces anergy (or exhaustion or apoptosis), indicating that the major role of CTL epitope variation is likely viral escape.

Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen–A2 allele (HLA-A2)–Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer–cell platforms.

The generation and analysis of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic

We have reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]), in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. CEA, MUC1, and brachyury are tumor-associated antigens (TAA) expressed on a wide range of human tumors. Ad5 [E1-, E2b-]–CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. The Ad5 [E1-, E2b-]–CEA vector encodes the entire CEA sequence modified to express an enhancer T-cell epitope. We report here the development of novel Ad5 [E1-, E2b-]–brachyury and Ad5 [E1-, E2b-]–MUC-1 vaccine constructs. The Ad5 [E1-, E2b-]–brachyury vector was constructed to encode the entire brachyury gene devoid of 25 amino acids involved in DNA binding, and modified to express an enhancer T cell epitope. The Ad5 [E1-, E2b-]–MUC-1 vector was constructed to encode the entire MUC-1 transgene with eight agonist epitopes, including five in the C-terminus. Our results show that these constructs (CEA, MUC1 and brachyury) are capable of activation, as well as generation of antigen specific T cells in vitro, and of inducing antigen-specific T cells in vaccinated mice. We have also demonstrated that the use of a combination of the three vaccines (designated Tri-Ad5) displays little, if any, antigenic competition in in vitro studies of human dendritic cells for antigen-specific T cell activation and generation, or in murine vaccination studies. The studies reported here support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.

Abstract 2561: Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses

Abstract The tumor-associated antigen MUC1 is overexpressed on various human hematological and epithelial malignancies. The MUC1 molecule, which has an N-terminal (MUC1-N) and a C-terminal (MUC1-C, which has been shown to act as an oncogene), is an attractive target for cancer immunotherapy. We have identified and reported 10 agonist epitopes (7 in the C-terminus, 2 in the N-terminus VNTR region, and 1 in the non-VNTR region) that enhance production of CD8 cytotoxic T lymphocytes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 MHC class I alleles, thus encompassing the majority of the North American population. Compared to native epitopes, these agonist epitopes more efficiently generate MUC1-specific T cells, induce production of IFN-gamma by MUC1-specific T cells, and lyse human tumor cells expressing MUC1 native epitopes in an MHC-restricted manner. We have previously shown that heat-killed recombinant Saccharomyces cerevisiae yeast genetically modified to express carcinoembryonic antigen can efficiently activate human dendritic cells (DCs) and stimulate CEA-specific CD8+ T cells. Here, we investigated the ability of a Saccharomyces cerevisiae vector containing the MUC1 transgene (yeast-MUC1) and encoding MUC1 with 8 agonist epitopes to activate human DCs, stimulate MUC1-N- and MUC1-C-specific T cells, and generate MUC1-specific T cells. We show here for the first time that human DCs treated with yeast-MUC1 vectors can activate MUC1-N and MUC1-C agonist-specific T-cell lines and can act as antigen-presenting cells to generate MUC1-N- and MUC1-C-specific T cells specific to each agonist epitopes, and that these T cells are capable of lysing MUC1-expressing tumor cells. Together, these findings provide a rationale for further clinical evaluation of yeast-MUC1 constructs encoding MUC1 agonist epitopes in cancer vaccine immunotherapy. Citation Format: Kwong Y. Tsang, Benjamin Boyerinas, Caroline Jochems, Tim Rodell, Thomas King, Jeffey Schlom. Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2561. doi:10.1158/1538-7445.AM2014-2561

Identification and characterization of a cytotoxic T-lymphocyte agonist epitope of brachyury, a transcription factor involved in epithelial to mesenchymal transition and metastasis.

The transcription factor brachyury is a major driver of epithelial to mesenchymal transition in human carcinoma cells. It is overexpressed in several human tumor types versus normal adult tissues, except for testes and thyroid. Overexpression is associated with drug resistance and poor prognosis. Previous studies identified a brachyury HLA-A2 cytotoxic T-lymphocyte epitope. The studies reported here describe an enhancer epitope of brachyury. Compared to the native epitope, the agonist epitope: (a) has enhanced binding to MHC class I, (b) increased the IFN-γ production from brachyury-specific T cells, (c) generated brachyury-specific T cells with greater levels of perforin and increased proliferation, (d) generated T cells more proficient at lysing human carcinoma cells endogenously expressing the native epitope, and(e) achieved greater brachyury-specific T-cell responses in vivo in HLA-A2 transgenic mice. These studies also report the generation of a heat-killed recombinant Saccharomyces cerevisiae (yeast) vector expressing the full-length brachyury gene encoding the agonist epitope. Compared to yeast-brachyury (native) devoid of the agonist epitope, the yeast-brachyury (agonist) enhanced the activation of brachyury-specific T cells, which efficiently lysed human carcinoma cells. In addition to providing the rationale for the recombinant yeast-brachyury (agonist) as a potential vaccine in cancer therapy, these studies also provide the rationale for the use of the agonist in (a) dendritic cell (DC) vaccines, (b) adjuvant or liposomal vaccines, (c) recombinant viral and/or bacterial vaccines, (d) protein/polypeptide vaccines, (e) activation of T cells ex vivo in adoptive therapy protocols, and (f) generation of genetically engineered targeted T cells.

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein

The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8⁺ cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.

Generation of human T cells directed against an agonist epitope of Brachyury, a transcription factor involved in human tumor cell epithelial to mesenchymal transition (EMT)

Meeting abstracts The T-box family transcription factor Brachyury is overexpressed in a variety of human carcinomas, including lung, breast, colon, ovarian and prostate. Brachyury has been shown to promote epithelial to mesenchymal transition (EMT) in tumor cells, a critical step in the path to

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein

Meeting abstracts The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated VNTR region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are

Abstract 1260: Generation of human T cells directed against an agonist epitope of a transcription factor involved in epithelial to mesenchymal transition (EMT).

Abstract Purpose: The T-box family transcription factor Brachyury is overexpressed in a variety of human carcinomas, including lung, breast, colon, ovarian and prostate. Brachyury has been shown to promote epithelial to mesenchymal transition (EMT) in tumor cells, a critical step in the path to metastasis. An HLA-A2 epitope of Brachyury has been shown to expand human T cells that are capable of lysing Brachyury-expressing tumor cells in an HLA-dependent manner. This study sought to define an agonist of this epitope in order to increase T cell activation and tumor lysis. Experimental Design: A novel agonist epitope of Brachyury was generated by residue substitution of the native epitope. Characterization of this epitope as an agonist included; comparison of HLA binding affinity and stability, interferon γ production by epitope-specific T cell lines, as well as Brachyury-and HLA-specific lysis of tumor cells. The presence of Brachyury-specific T cells that would recognize the agonist peptide, within the circulating PBMC of cancer patients, was determined by ELISPOT. Results: The agonist epitope was shown to bind HLA-A2 with higher affinity and stability than the native. T cell lines generated from both the native and agonist epitopes produced higher levels of interferon γ in response to stimulation with the agonist epitope. The agonist-specific T cell line was able to lyse a variety of Brachyury-expressing tumor cells more efficiently than the T cell line generated with the native epitope, and specificity of lysis was confirmed by cold-target inhibition and HLA-A2 blocking. Tetramer staining revealed Brachyury agonist-specific T cells in the PBMC of a prostate cancer patient after in vitro stimulation with the agonist peptide. PBMC from colon and ovarian cancer patients reacted to the agonist peptide in an interferon γ ELISPOT assay. Conclusions: An agonist epitope for Brachyury has been identified, which increased T cell activation and function as compared to the native epitope. T cells that react to the agonist peptide were detected in patients with carcinomas known to express high levels of Brachyury. This study supports the use of Brachyury as a cancer vaccine strategy, including the Brachyury agonist epitope. Citation Format: Jo A. Tucker, Diane J. Poole, Claudia Palena, Caroline Jochems, Jeffrey Schlom, Kwong Y. Tsang. Generation of human T cells directed against an agonist epitope of a transcription factor involved in epithelial to mesenchymal transition (EMT). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1260. doi:10.1158/1538-7445.AM2013-1260

Abstract 5524: Generation of cellular immune responses to MUC1-C to target the portion of MUC1 that is most biologically relevant to the transformation process

Abstract MUC1 is overexpressed in diverse human carcinomas and hematological cancers, and therefore serves as a potential target for immunotherapy. The MUC1 molecule has two distinctive subunits, formed by autocleavage with a SEA domain. The N-terminal (MUC1-N) is the large extracellular domain, and the C-terminal (MUC1-C) has three subunits: a small extracellular domain that is covalently bound to MUC1-N, a single transmembrane domain and a cytoplasmic tail. It has been reported by Kufe et al that the MUC1-C subunit transduces signals that confer growth and survival responses. The MUC1-C cytoplasmic tail located downstream of the SEA domain is sufficient for the induction of anchorage independent growth and tumorigenicity. MUC1-C overexpression is also highly associated with poor prognosis in patients with lung cancer and breast cancer. We and others have previously reported on HLA-A2 binding agonist epitopes to MUC1-N. The objective of this investigation is to identify and characterize HLA-A3 binding cytotoxic T-lymphocyte (CTL) epitopes and agonist epitopes of the MUC1-C subunit. The intention is to add these peptides to vaccines against MUC1 positive carcinomas, as well as using them for monitoring patients’ immune responses in clinical MUC1 vaccine trials. The generation of cellular immune responses to MUC1-C would thus target the portion of MUC1 that is most biologically relevant to the transformation process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2011-5524

Stimulation of Naive CD8+ T Cells by a Variant Viral Epitope Induces Activation and Enhanced Apoptosis

Classically, naive T cells recognize a specific peptide-MHC complex resulting in their activation and differentiation. However, it is known that T cells also have the ability to interact productively with variant ligands, indicating a flexibility in TCR Ag recognition. These altered peptide ligands have been shown to trigger responses ranging from complete activation to full inhibition of T cell responses, and thus may play an important role in initiating or sustaining T cell-mediated immunity. We have found that influenza virus-specific CD8(+) TCR transgenic T cells differentially respond to a native (agonist) and variant viral epitope, differing in two amino acids that are thought to alter TCR recognition. In response to stimulation with the agonist epitope, these cells activate, proliferate, and differentiate into effector CTLs. Conversely, stimulation with the variant epitope results in activation, proliferation, and development of effector activity followed by rapid and extensive apoptotic cell death. Stimulation of the T cells with the altered ligand results in an inability to sustain the expression of the prosurvival molecules, Bcl-2 and Bcl-xL. These data suggest that the response to the agonist and variant epitopes may reflect TCR avidity-dependent differential signaling through the TCR, resulting either in activation-dependent T cell proliferative expansion and survival or in the accelerated death of acutely activated differentiating T cells. This process of CD8(+) T cell activation, proliferation, and differentiation followed by rapid cell death may represent a novel mechanism of altered peptide ligand-induced apoptosis programmed by initial Ag receptor engagement.

Identification of cytotoxic T‐lymphocyte epitope(s) and its agonist epitope(s) of a novel target for vaccine therapy (PAGE4)

PAGE4 is an X chromosome-linked cancer testis antigen and is a potential new tumor-associated antigen that is overexpressed in prostate and uterine cancers. The purpose of this study was to identify a human CTL epitope and a corresponding agonist epitope of PAGE4 to determine if PAGE4 is a potential target for vaccine-mediated immunotherapy against PAGE4-expressing tumors. A class I PAGE4 epitope was identified with a high level of binding to HLA-A2. PAGE4 peptide-pulsed dendritic cells were then used to generate human PAGE4-specific T-cell lines. Further studies demonstrated the generation of an enhancer agonist epitope. Compared with the native peptide, the agonist (i) bound to HLA-A2 molecules at lower peptide concentrations, (ii) demonstrated a higher stability of the peptide HLA-A2 complex, (iii) induced higher levels of production of IFN-gamma, Granzyme B, TNF-alpha, IL-2 and lymphotactin by PAGE4-specific T-cell lines and (iv) T-cell lines generated against the agonist peptide were more efficient to lyse HLA-A2 human tumor cells expressing native PAGE4. The studies reported here are the first to describe a PAGE4 CTL epitope and its agonist epitope, and thus identify PAGE4 as a potentially useful target for vaccine-mediated therapy of prostate cancer.

An altered peptide ligand for naïve cytotoxic T lymphocyte epitope of TRP-2(180-188) enhanced immunogenicity.

Tyrosinase-related protein-2 (TRP-2) is a non-mutated melanocyte differentiation antigen. The TRP-2-recognizing CD8(+) T cells can evoke immune responses to melanoma in both humans and mice. Developing epitopes with amino acid replacements in their sequences might improve the low immunogenicity against this 'self' tumor antigen. We designed altered peptide ligands (APLs) of TRP-2((180-188)) (SVYDFFVWL) with preferred primary and auxiliary HLA-A*0201 molecule anchor residue replacement. These APLs were screened for MHC-affinity by affinity prediction plots and molecular dynamics simulation, and analyzed in vitro for stability and binding-affinity to molecular HLA-A*0201. We also investigated the CTLs activities induced by TRP-2 wild-type epitope and the APLs both in vitro in human PBMCs and HLA-A2.1/K(b) transgenic mice. The results indicate that TRP-2 2M analog simultaneously had stronger binding-affinity and a lower dissociation rate to HLA-A*0201, than wild-type peptide. In addition, the analog 2M was superior to other APLs and wild-type epitope in terms of immunological efficacy ex vivo as measured by the ELISPOT assays of IFN-gamma and granzyme B. These results demonstrate that TRP-2 2M is an agonist epitope that can induce anti-tumor immunity superior to its wild-type epitope, and has potential application in peptide-mediated immunotherapy.

A pilot study of a PANVAC-V and PANVAC-F in patients (pts) with metastatic carcinoma

2512 Background: The vast majority of carcinomas express either MUC-1 or CEA. Preclinical and clinical studies have demonstrated that induction of T-cell responses directed against CEA and MUC-1 can lead to anti-tumor activity without toxicity. Two novel vaccines have been developed: (a) PANVAC-V (V) a recombinant vaccinia virus containing CEA and MUC-1 with modified agonist epitopes and 3 costimulatory molecule transgenes, and (b) PANVAC-F (F) a similar recombinant fowlpox virus. Methods: This pilot study enrolled 25 pts with metastatic carcinoma who had progressive disease after chemotherapy. Pts received V priming followed by F boosts every 2 weeks × 3 then monthly with restaging scans every 2 months while on study. All vaccines were given with sargramostim at the vaccine site. The primary endpoint was safety. Class I and class II immune responses to CEA were analyzed. Class I responses were assayed by ELISPOT, intracellular cytokine, and tetramer binding; and class II responses were assayed by ELISA using CEA protein and class II peptides. Results: Treatment was well tolerated with 135 cycles given. Only one grade 3 toxicity (syncope during a flu-like illness) was possibly related to vaccine. Apart from injection site reactions, only 9 cycles (7%) were associated with transient grade 2 toxicity (generally flu-like syndromes). Class I and class II immunologic responses were demonstrated. A pt with metastatic breast cancer and 5 large liver metastasis had a 24% decrease in unidimensional measurement of disease for 6 months. A pt with metastatic gastric cancer had stable disease for 5 months. Furthermore, a pt with metastatic clear cell ovarian cancer s/p chemotherapy had complete disappearance of large volume, symptomatic ascites accompanied by marked improvement in the appearance of mesenteric stranding and a CA-125 reduction from 281 U/mL to continued sustained normal values (< 20 U/mL). Conclusions: This vaccine can be given safely and can generate therapy specific immunologic responses. In addition 1 pt with metastatic ovarian cancer had a dramatic clinical response. Based on this, we have initiated a pilot study specifically for ovarian cancer pts to gain more information on which to base a large clinical endpoint trial. No significant financial relationships to disclose.

Class B GPCRs: A Hidden Agonist Within?: Fig. 1.

Class B G protein-coupled receptors (GPCRs) regulate a wide range of endocrine and neuroendocrine functions and are endogenously stimulated by moderately large peptide hormones. Current evidence suggests that the carboxyl termini of cognate peptides bind to the amino terminus of their G protein-coupled receptors (GPCRs) and that the peptides9 amino terminal segments then dock to the heptahelical receptor portion to induce signaling. In this issue of <i>Molecular Pharmacology</i>, Dong et al. (p. 206) propose an alternative model of ligand-induced class B GPCR activation. Based primarily on studies with the secretin receptor, a prototype class B family member, they provide evidence that the endogenous peptide hormone does not function as an activator per se. Instead, this hormone (secretin) exposes a hidden, built-in agonist epitope that is present within the amino terminus of its target GPCR. Isolated oligopeptide fragments containing this epitope act as full agonists on the secretin receptor despite their lack of amino acid homology with the secretin hormone. These nonconventional agonists can be minimized to tripeptide molecules and still maintain biological activity. The study to be discussed introduces a novel paradigm of class B GPCR function, and may facilitate the elusive goal of finding small molecule agonist drugs for this therapeutically attractive group of receptors.

Identification of Novel Human CTL Epitopes and Their Agonist Epitopes of Mesothelin

Mesothelin is overexpressed in many pancreatic and ovarian cancers, mesotheliomas, and other tumor types. Clinical trials are ongoing using immunotoxins to target mesothelin, and patients immunized with allogeneic pancreatic tumor cell lines have shown immune responses to previously defined mesothelin epitopes. The purpose of this study was to define novel mesothelin CTL epitopes and, more importantly, agonist epitopes that would more efficiently activate human T cells to more efficiently lyse human tumors. Two novel mesothelin HLA-A2 epitopes were defined. T-cell lines generated from one of these epitopes were shown to lyse pancreatic and ovarian tumor cells. Several agonist epitopes were defined and were shown to (a) have higher affinity and avidity for HLA-A2, (b) activate mesothelin-specific T cells from normal individuals or cancer patients to a greater degree than the native epitope in terms of induction of higher levels of IFN-gamma and the chemokine lymphotactin, and (c) lyse several mesothelin-expressing tumor types in a MHC-restricted manner more effectively than T cells generated using the native peptide. External beam radiation of tumor cells at nontoxic levels was shown to enhance the expression of mesothelin and other accessory molecules, resulting in a modest but statistically significant increase in tumor cell lysis by mesothelin-specific T cells. The identification of novel CTL agonist epitopes supports and extends observations that mesothelin is a potential target for immunotherapy of pancreatic and ovarian cancers, as well as mesotheliomas.