We have explored the isoelectronic replacement of the C═C double bond found at the core of many nonsteroidal estrogen ligands with a simple Schiff base (C═N). Di- and triaryl-substituted imine derivatives were conveniently prepared by the condensation of benzophenones with various anilines without the need for phenolic hydroxy protection. Most of these imines demonstrated high affinity for the estrogen receptors, which, in some cases exceeded that of estradiol. In cell-based assays, these imines profiled as ERα agonists but as ERβ antagonists, showing preferential reliance on the N-terminal activation function (AF1), which is more active in ERα. X-ray analysis revealed that the triaryl-imines distort the ligand-binding pocket in a new way: by controlling the separation of helices 3 and 11, which appears to alter the C-terminal AF2 surface that binds transcriptional coactivators. This work suggests that C═N for C═C substitution might be more widely considered as a general strategy for preparing drug analogues.

2525 Background: Effective vaccines that mediate clinical responses in cancer patients may require generation of broadly specific cytotoxic T lymphocytes (CTLs) directed against multiple epitopes and tumor-associated antigens (TAAs). The EP-2101 vaccine was designed to induce CTLs against epitopes in carcinoembryonic antigen (CEA), p53, HER-2/neu, and MAGE 2/3, four TAAs frequently over-expressed in NSCLC and colon cancer. Using a rational epitope identification process, 2 epitopes from each TAA were selected for vaccine development. All 8 epitopes displayed high HLA-A2 supertype binding affinity and immunogenicity in a primary in vitro induction assay. Six of the epitopes were analogs containing a single substitution which either enhanced MHC binding or stimulated heteroclitic T cell activation, and 2 epitopes were native sequences. The 8 epitopes together with CAP1–6D, a previously described heteroclitic CEA analog, and the PADRE helper T cell epitope were emulsified in Montanide ISA51 adjuvant and tested for safety and immunogenicity in HLA-A2+ NSCLC (stage IIb/IIIa) and colon (stage III) cancer patients with no detectable disease following standard treatment. Methods: Patients received 6 EP-2101 treatments at 3 wk intervals, at a dose of 5 mg total peptide (0.5 mg/epitope). CTL responses in the peripheral blood of patients were measured using a validated interferon-gamma (IFN-γ) ELISPOT assay. Results: Based on 7 patients who have completed treatment (4 colon, 3 lung), EP-2101 appears to be safe and well-tolerated. To date, IFN-γ-secreting CTL responses were observed in all three patients tested, with 1, 4, and 6 epitope responses being detected respectively. Vaccine responses against 1 native epitope and 5 analog epitopes in EP-2101 have ranged from 23–260 net spots/50,000 cells with <8 net spots detected in pre-vaccination samples. Conclusions: A cancer vaccine delivering 9 TAA epitopes can simultaneously induce CTL responses against multiple epitopes and TAAs in patients. This vaccine consisting of native and analog epitopes may provide effective immunotherapy in patients with diverse types of cancer expressing common TAAs. No significant financial relationships to disclose.

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Dendritic Cells and Vaccines

Recognition by CD8+ T lymphocytes (CTL) of epitopes that are derived from conserved gene products, such as Gag and Pol, is well documented and conceptually supports the development of epitope-based vaccines for use against diverse HIV-1 subtypes. However, many CTL epitopes from highly conserved regions within the HIV-1 genome are highly variable, when assessed by comparison of amino acid sequences. The TCR is somewhat promiscuous with respect to peptide binding, and, as such, CTL can often recognize related epitopes. In these studies, we evaluated CTL recognition of five sets of variant HIV-1 epitopes restricted to HLA-A*0201 and HLA-A*1101 using HLA transgenic mice. We found that numerous different amino acid substitutions can be introduced into epitopes without abrogating their recognition by CTL. Based on our findings, we constructed an algorithm to predict those CTL epitopes capable of inducing responses in the HLA transgenic mice to the greatest numbers of variant epitopes. Similarity of CTL specificity for variant epitopes was demonstrated for humans using PBMC from HIV-1-infected individuals and CTL lines produced in vitro using PBMC from HIV-1-uninfected donors. We believe the ability to predict CTL epitope immunogenicity and recognition patterns of variant epitopes can be useful for designing vaccines against multiple subtypes and circulating recombinant forms of HIV-1.

omega-Iodoalkyl(methyl)malonate esters have potential utility in the bio-conjugate chemistry of steroids. A representative set of these esters with a variety of protecting groups and alkyl groups has been prepared. The compounds offer a range of elution values on silica, as well as several convenient deprotection options.

The demonstration that highly active anti-retroviral therapy (HAART) can control human immunodeficiency virus type 1 (HIV-1) viral replication and the associated destruction of the immune system provides an opportunity to implement therapeutic vaccine strategies. The Epimmune approach is based on separate vaccine immunogens designed to induce, or augment, helper T-lymphocyte (HTL) or cytotoxic T-lymphocyte (CTL) responsiveness when administered in conjunction with HAART. The vaccines are composed of carefully selected, minimal HTL or CTL epitopes. Vaccines composed of multiple epitopes can be produced and delivered using different formats, including deoxyribonucleic acid (DNA) plasmid-based vaccines and recombinant proteins.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-gamma ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.