How the immune system recognizes endogenously arising tumors and elicits adaptive immune responses against nonmutated tumor-associated Ags is poorly understood. In search of intrinsic factors contributing to the immunogenicity of the tumor-associated Ag NY-ESO-1, we found that the NY-ESO-1 protein binds to the surface of immature dendritic cells (DC), macrophages, and monocytes, but not to that of B cells or T cells. Using immunoprecipitation coupled with tandem mass spectrometry, we isolated DC surface calreticulin as the receptor for NY-ESO-1. Calreticulin Abs blocked NY-ESO-1 binding on immature DC and its cross-presentation to CD8+ T cells in vitro. Calreticulin/NY-ESO-1 interactions provide a direct link between NY-ESO-1, the innate immune system, and, potentially, the adaptive immune response against NY-ESO-1.

Monitoring the spontaneous antibody (Ab) response against a panel of relevant tumor-associated antigens (TAA) in cancer patients may provide useful information regarding the clinical status of cancer. However, current Ab detection approaches require the purification of recombinant proteins, which is often difficult to achieve. In order to bypass the purification of recombinant proteins, we identified a dominant B-cell epitope from a shared tumor antigen NY-ESO- In the last 10 years, molecular identification of TAA has clearly demonstrated that the human immune system can react with endogenously arising cancer cells. 1 . Both the cellular and humoral arms of the human immune system recognize autologous TAA derived from cancer cells. 2,3 Of particular interest to the serological analysis of human cancers is the identification of TAA recognized by antibodies (Ab) present in the sera of cancer patients. 4 SEREX, or the serological analysis of recombinant cDNA expression libraries of human cancers, has been the primary approach to identify TAA based on their recognition by Ab. Up to now, there are at least 1,800 TAA identified based on recognition by Ab present in patients' sera. 5 . These TAA include targets from many cancer types, such as melanoma, renal cancer, Hodgkin's disease, esophageal cancer, lung cancer, colon cancer, gastric cancer, breast cancer, prostate cancer and so on. 3 The discovery of these TAA provides molecular details of the humoral immune response to autologous tumors. More importantly, the discovery of these molecules awakens the old hope of finding serological markers for cancer detection, diagnosis and prognosis. However, most of the SEREX-defined antigens do not or only react with few allogeneic sera. To investigate Ab responses against a large panel of TAA that covers a wide spectrum of patients with a particular cancer requires the purification of individual TAA protein, which is expensive and difficult to achieve. To circumvent the requirement of purifying individual protein, we propose identifying dominant B-cell epitopes from TAA for detection of Ab against TAA. We choose NY-ESO-1 as a prototype for identifying B-cell epitopes for the following reasons: 1) NY-ESO-1 is a cancer/testis antigen, which is expressed in a number of human cancers including those of the esophageal, 6 prostate, head and neck, lung, ovarian, breast, neuroblastoma, 7 gastric, as well as melanoma, hepatocellular carcinoma (HCC), T cell lymphoma and sarcoma. 8 Approximately 20 -30% of the above-mentioned cancers express the protein, and moreover, nearly 40 -50% of the patients with NY-ESO-1 expressing tumors develop antibodies against the protein. 9 2) Ab titers against NY-ESO-1 are driven by antigen expression of the endogenously arising tumor, and several studies have suggested that Ab titers against NY-ESO-1 correlated with advanced stages of the antigen-positive tumor in melanoma, transitional cell carcinoma and prostate cancer. 10 -12 3) Owing to its high immunogenicity and broad expression in a variety of ca...

In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T celldependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-toSer) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern.It has been speculated that the initiation of spontaneous adaptive immune responses against tumor-associated antigens (TAA) 3 may involve receptors of the innate immune system (1, 2). Necrotic cancer cells release endogenous factors or a damage-associated molecular pattern such as heat shock proteins (3), high mobility group box-1 protein (HMGB-1) (4), and uric acid (5). These damage-associated molecular pattern factors alert the innate immune system through CD91, receptor for advanced glycation end products and possibly TLR2/TLR4, as well as the IL-1 receptor (6), respectively. TAA themselves are generally perceived as by-standers of the above "danger signals," which serve as adjuvants in the initiation of anti-tumor immune responses. According to this paradigm, spontaneous immune responses may favor neo-peptides resulting from an estimated 11,000 genetic alterations within a cancer cell (7). However, human TAA identified to date are commonly nonmutated self-proteins (8). We thus hypothesize that intrinsic factors from some of the known TAA may also play a role in the initiation of anti-tumor immune responses in vivo.In search of intrinsic factors derived from the host and the tumor that contribute to anti-tumor immune responses, we focused on NY-ESO-1, a non-mutated cancer/testis antigen with distinctively strong immunogenicity (9). Spontaneous Ab and T cell-based immune response against NY-ESO-1 were readily detectable in a wide spectrum of cancer patients with NY-ESO-1-expressing tumors, including older patients with late stage cancers whose immune systems were known to be less responsive (9). Furthermore, spontaneous NY-ESO-1 antigen loss in melanoma suggested that anti-NY-ESO-1 responses could affect the natural history of cance...

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