Dendritic cell based personalized immunotherapy based on cancer antigen research.

Human tumor antigens were identified using various immunological and genetic methods, and immune responses to the identified antigens were evaluated in cancer patients. Autologous tumor specific unique antigens derived from genetic alterations in cancer cells were isolated from patients with favorable prognosis after immunotherapy, indicating that they are attractive targets for immunotherapy. Immunogenicity of shared antigens was found to differ among patients due to antigen expression in cancer cells and patients' immunoreactivity. These observations suggest that personalization may be applied for cancer immunotherapy. We therefore developed intratumoral DC administration protocols that are able to induce immune responses to both unique and shared tumor antigens expressed in each individual cancer. By combining cryoablative tumor pretreatment and TLR stimulated DC, the anti-tumor effect of the intratumoral DC administration was significantly augmented in a murine tumor model. This improved protocol enhanced systemic induction of anti-tumor CD8+ CTL, and was able to regress relatively large remote untreated tumors. In clinical trials, systemic immune induction was observed by intratumoral DC administration following cryoablative tumor treatment, although anti-tumor effects are relatively weak, indicating that additional interventions are required for more effective immunotherapy.

Identification of a novel cancer-testis antigen CRT2 frequently expressed in various cancers using representational differential analysis.

PURPOSE: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. EXPERIMENTAL DESIGN: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis-like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. RESULTS: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. A CRT2-derived peptide was identified as an HLA-A24-restricted T-cell epitope that induced tumor-reactive CTLs. CONCLUSION: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24-restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.

A new melanoma antigen fatty acid-binding protein 7, involved in proliferation and invasion, is a potential target for immunotherapy and molecular target therapy.

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.

A novel cancer testis antigen that is frequently expressed in pancreatic, lung, and endometrial cancers.

PURPOSE: To isolate cancer testis antigens that are expressed in pancreatic cancers and may be useful in clinical applications. EXPERIMENTAL DESIGN: To efficiently isolate cancer testis antigens, a testis cDNA library was immunoscreened (SEREX) with serum from a patient with pancreatic ductal adenocarcinoma. The expression of isolated antigens in various cancer cell lines and tissues was evaluated by reverse transcription-PCR and Northern blot analyses. The immunogenicity of the antigen in cancer patients was evaluated by detection of the IgG antibody in sera from patients with various cancers. RESULTS: Of the three clones isolated through screening of a total of 2 x 10(6) cDNA library clones, one clone (KU-CT-1) was found to be expressed in various cancers but only in testis among normal tissues, indicating that it was a novel cancer testis antigen. The KU-CT-1 gene is located on chromosome 10p12 and produces two splice variants, which encode proteins of 397 and 872 amino acids, respectively. KU-CT-1 was expressed in pancreatic cancer tissues (3 of 9, 33%), lung cancer tissues (9 of 24, 38%), and endometrial cancer tissues (7 of 11, 64%). Specific serum IgG antibodies were detected in 3 of 20 pancreatic cancer patients, 2 of 12 endometrial cancer patients, 1 of 18 colon cancer patients, and 1 of 10 prostate cancer patients but not detected in 30 healthy individuals. CONCLUSIONS: KU-CT-1 is a new cancer testis antigen that is expressed in pancreatic, lung, and endometrial cancers and may be useful for diagnosis and immunotherapy for patients with various cancers.

Establishment of pten knockout medaka with transcription activator-like effector nucleases (TALENs) as a model of PTEN deficiency disease.

Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator-like effector nuclease (TALEN) technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.

Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1.

Renal cell carcinoma (RCC) is the most lethal urological malignancy with high risk of recurrence; thus, new prognostic biomarkers are needed. In this study, a new RCC antigen, PTPL1 associated RhoGAP1 (PARG1), was identified by using serological identification of recombinant cDNA expression cloning with sera from RCC patients. PARG1 protein was found to be differentially expressed in RCC cells among patients. High PARG1 expression is significantly correlated with various clinicopathological factors relating to cancer cell proliferation and invasion, including G3 percentage (P = .0046), Ki-67 score (p expression is also correlated with high recurrence of N0M0 patients (P = .0084) and poor prognosis in RCC patients (P = .0345). Multivariate analysis has revealed that high PARG1 expression is an independent factor for recurrence (P = .0149) of N0M0 RCC patients. In in vitro studies, depletion of PARG1by siRNA in human RCC cell lines inhibited their proliferation through inducing G1 cell cycle arrest via upregulation of p53 and subsequent p21Cip1/Waf1, which are mediated by increased RhoA-ROCK activities. Similarly, PARG1 depletion cells inhibited invasion ability via increasing RhoA-ROCK activities in the RCC cell lines. Conversely, overexpression of PARG1 on human embryonic kidney cell line HEK293T promotes its cell proliferation and invasion. These results indicate that PARG1 plays crucial roles in progression of human RCC in increasing cell proliferation and invasion ability via inhibition of the RhoA-ROCK axis, and PARG1 is a poor prognostic marker, particularly for high recurrence of N0M0 RCC patients.

Frequent immune responses to a cancer/testis antigen, CAGE, in patients with microsatellite instability-positive endometrial cancer.

PURPOSE: Identification of cancer/testis antigens useful for diagnosis or immunotherapy of cancers was attempted by cDNA expression cloning with patients' sera (SEREX). EXPERIMENTAL DESIGN: cDNA expression libraries made from testis or endometrial cancer cell lines were screened using sera from patients with endometrial cancer or melanoma patients immunized with dendritic cells pulsed with autologous tum or lysates. Tissue-specific expression by RT-PCR and immunogenicity by Western blotting of the bacterial recombinant antigen with sera from cancer patients were evaluated. RESULTS: A cancer/testis antigen, CAGE, was isolated by two independently performed SEREX. CAGE was expressed in various cancer cell lines including endometrial cancer, colon cancer, and melanoma in 7 of 10 endometrial cancer tissues and in 1 of 3 atypical endometrial hyperplasia, but not in normal tissues including the endometrium and testis. The protein expression on cancer cells was confirmed by Western blot analysis with the recombinant CAGE protein, anti-CAGE IgG antibody was detected in sera from 5 of 45 endometrial cancer, 2 of 24 melanoma, and 2 of 33 colon cancer patients, but not in sera from healthy individuals. By ELISA analysis, anti-CAGE antibody was detected in 12 of 45 endometrial cancer, 2 of 20 melanoma, and 4 of 33 colon cancer patients. Intriguingly, anti-CAGE antibody was highly positive in 7 of the 13 (53.8%) microsatellite instability (MSI)-H patients with endometrial cancer, but negative in 20 non-MSI-H patients (P = 0.001). CONCLUSION: CAGE may be useful for immunotherapy and diagnosis of various cancers particularly MSI-positive endometrial cancer.

Establishment of HRAS(G12V) transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs.

Most targeted anticancer drugs have been identified by screening at the molecular or cellular level in vitro. However, many compounds selected by such costly and time-consuming screening do not prove effective against tumors in vivo. The development of anticancer drugs would thus be facilitated by the availability of an in vivo screening system based on a multicellular organism. We have now established a transgenic line of the freshwater fish medaka in which melanophores (melanocytes) proliferate in a manner dependent on heat shock-induced signaling by a human RAS oncoprotein. The human HRAS(G12V) oncogene was expressed under the control of a melanophore-specific gene promoter in order to allow visualization of tumor growth in live fish maintained in a water tank. The expression of HRAS(G12V) was induced as a result of Cre-mediated recombination by exposure of the fish to a temperature of 37°C for 30 min, given that the Cre gene was placed under the control of a medaka heat shock promoter. One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. The transgenic medaka established here thus represents a promising in vivo system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes.

Reversible whole-organism cell cycle arrest in a living vertebrate.

In vivo cell cycle analysis in higher eukaryotes has been limited by the challenge of preserving the integrity of the living organism while visualizing dividing cells. Here, we propose a new model, which uses the unique combination of features of the Japanese medaka in order to visualize and manipulate the cell cycle progression in a live vertebrate. Our stable transgenic histone H2B-GFP medaka line allows fluorescence-based monitoring of the chromosomes. The system has a high specificity, with a strong GFP signal labeling the chromatin architecture. The subcellular resolution ensures detection of both normal and abnormal divisions in live recordings. This translates into the possibility to quantify temporal and spatial aspects of the cell cycle, such as length or nuclear size, as well as to expose drug toxicity at the earliest stage. We also show that acclimation to cold, a prominent feature of the eurytherm medaka, is a valuable natural way of inducing a reversible cell cycle arrest in the entire living organism. Our results suggest that this manipulation can be performed from the early stages of development, has no toxicity and does not alter the cell cycle profile of the embryo.

Dendritic cells transduced with autoantigen FCRLA induce cytotoxic lymphocytes and vaccinate against murine B-cell lymphoma.

We previously reported early evidence of the human Fc receptor-like A (hFCRLA), an antigen (Ag) that was specifically expressed in melanocytes, melanoma cells, and some B-cell states, and was recognized by IgG antibodies from melanoma patients. Recently, it has been demonstrated that hFCRLA is expressed in most human B-cell lymphoma tissues. In this report, we investigated the potential of FCRLA as a tumor-associated Ag of B-cell lymphoma for immunotherapy. We confirmed that murine FCRLA (mFCRLA) was expressed and distributed in murine tissues similar to hFCRLA. Recombinant mFCRLA fusion protein was constructed with a polyarginine (R9)-protein-transduction domain (PTD) (rR9-HA-mFCRL), and was transduced into bone marrow-derived dendritic cells (DC) ex vivo. Mice immunized with rR9-HA-mFCRL-treated DC primed cytotoxic T-lymphocyte (CTL) that killed the B-cell lymphoma cell line (A20), which express mFCRLA abundantly. In a tumor challenging study, A20 tumor growth inoculated in skin was significantly suppressed in mice vaccinated with rR9-HA-mFCRL-treated DC, compared with control mice. These results indicated that FCRLA is a potential target Ag in immunotherapy for B-cell lymphoma. In addition, our experimental system using R9-PTD-containing full-length proteins might be a useful method to analyze the immunogenicity of novel candidates of tumor-associated Ags in vivo.

Immunological detection of altered signaling molecules involved in melanoma development.

To understand immune responses to human cancer and develop more effective immunotherapy, human tumor antigens has been isolated using various immunological methods with tumor reactive T cells or antibodies obtained from patients with melanoma. During the process of tumor antigen isolation, various molecules with genetic alterations or over-expression in tumor cells, which may be involved in proliferation, differentiation, or survival of various cancer cells, were identified. In melanoma, abnormal molecules with mutations including beta -catenin, CDK4, and BRAF, and molecules with increased expression including Survivin, were immunologically detected. Therefore, immunological isolation of human tumor antigens contributes to the identification of important molecules including altered signaling molecules involved in melanoma formation.

Systematic identification of human melanoma antigens using serial analysis of gene expression (SAGE).

To identify new melanoma antigens using systematic gene expression analysis combined with rapid screening of patient sera for immunogenicity, a serial analysis of gene expression (SAGE) method was applied to profile transcripts in a highly pigmented melanoma cell line SKmel23. 25,997 SAGE tags consisting of 10,382 unique transcripts were sequenced. This melanoma SAGE library was compared with a testis SAGE library and the colon SAGE database, and to the cDNA database obtained by random sequencing of a melanocyte cDNA library. Among the 15 tags finally selected with criteria of preferential expression on melanoma and melanocytes at relatively high frequency, two tags were further analyzed for their structure and immunogenicity. One was identified as PAX3, and its isoform, PAX3d, was found to be dominantly expressed in melanoma and melanocytes. The other was derived from a novel gene and its full-length cDNA clone was isolated. Preferential expression of these genes in melanoma and melanocytes was confirmed by RT-PCR and Northern blot analysis. The recombinant bacterial PAX3d protein was recognized by serum IgG from some patients with melanoma and Vogt-Koyanagi-Harada (VKH) disease but not from healthy individuals, indicating that PAX3d is a new melanocyte-specific antigen immunogenic in patients with melanoma or VKH disease. The authors report two melanocyte/melanoma-specific molecules, which may be useful for development of diagnosis or treatment of these pigment disorders. In addition, a system using SAGE and immunoscreening with patients' sera is shown to be an efficient method for the systematic identification of tumor antigens.

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients.

We applied a strategy that utilized a combination of systematic gene expression analysis with various tissues and immunological detection with sera from melanoma patients to identify melanoma antigens expressed preferentially in melanoma and melanocytes. We selected 101 genes by comparing cDNA profiles obtained by GeneChip analysis of a highly pigmented melanoma cell line, SKmel23, primary cultured melanocytes, HUVECs cultured endothelial cells, keratinocytes, liver and stomach. After the additional selection with criterion of high registered frequency of each cDNA in melanocyte-related cDNA libraries in the NCBI database, 15 genes including 12 known melanocyte specific genes were identified. One of the remaining 3 genes, FCRL/FREB, encoding a member of the Fc receptor family that was previously reported to express in germinal center B cells, was found to express preferentially in melanocytes and melanoma tissues by RT-PCR and Northern blot analysis. The FCRL/FREB protein was detected in the cytoplasm of melanoma cells by staining with the murine polyclonal antibody and by transfection with GFP-fused FCRL/FREB cDNA. The bacterial recombinant protein was recognized by serum IgG antibody obtained from some patients with melanoma. These results suggest that FCRL/FREB may function in melanocytes and melanoma and may be useful for development of diagnostic methods for various pigment disorders and immunotherapy of melanoma.

Immune responses to DNA mismatch repair enzymes hMSH2 and hPMS1 in patients with pancreatic cancer, dermatomyositis and polymyositis.

To identify tumor antigens useful for diagnosis and immunotherapy of patients with pancreatic ductal adenocarcinoma, we applied a SEREX approach with a cDNA library made from 5 pancreatic cancer cell lines and sera obtained from 8 patients with pancreatic cancer, and isolated total 32 genes, including 14 previously characterized genes and 18 genes with unknown functions. Among these isolated antigens, serum IgG antibodies for 2 isolated DNA mismatch repair enzymes, Homo sapiens mutS homolog 2 (hMSH2) and Homo sapiens postmeiotic segregation increased 1 (hPMS1), were detected in patients with pancreatic ductal adenocarcinoma and dermatomyositis (DM), and polymyositis (PM), but not in sera from healthy individuals. Immunohistochemical study demonstrated that hMSH2 and hPMS1 were over-expressed in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. These results suggested that hMSH2 and hPMS1 may be useful as CD4+ helper T cell antigens for immunotherapy of pancreatic cancer patients and that serum IgG antibodies may be useful for diagnosis of patients with pancreatic ductal adenocarcinoma and DM/PM.

Identification of human tumor antigens and its implications for diagnosis and treatment of cancer.

Human tumor antigens recognized by T cells have been identified by means of various molecular biological and immunological methods, including cDNA expression cloning with patients' T cells and antibodies, cDNA subtraction using RDA and PCR differential display, systematic gene analysis such as DNA sequencing, CGH, DNA chip/microarray and SAGE, in vitro T cell induction and immunization of HLA transgenic mice. The identification of human tumor antigens has led to a better understanding of the nature of tumor antigens, anti-tumor immune responses in patients before and after immunotherapy, and tumor escape mechanisms. The information obtained from these researches has enabled us to develop and improve immunotherapy by attempting to overcome the identified problems, including intrinsically low immunogenicity of tumor antigens and several escape mechanisms, such as regulatory T cell induction. The existence of immunogenic unique antigens derived from genetic alterations in tumor cells, and the varied immunogenicity of shared tumor antigens among patients due to differing expression in tumor cells and immunoreactivity of patients, indicates that individualized immunotherapy should ideally be performed. The identified antigens will also be useful for development of diagnostic methods and molecular targeting therapy for cancer.

Identification of bladder cancer antigens recognized by IgG antibodies of a patient with metastatic bladder cancer.

To identify tumor antigens useful for the diagnosis and treatment of patients with bladder cancer, a lambda phage cDNA library constructed from a high-grade bladder cancer cell line was screened with autologous serum from a patient with metastatic bladder cancer. Forty-eight distinct antigens were isolated. By evaluating the immunogenicity and the tissue-specific expression, KU-BL-1 and KU-BL-2 were identified as immunogenic antigens with restricted tissue expression. KU-BL-1 was found to be a putative human lipoic acid synthetase with a metal-binding site, CXXXCXXC, that was expressed in bladder cancer cell lines and most bladder cancer tissues, as well as normal bladder mucosa and testis tissues. Immunoglobulin (Ig)G antibody to KU-BL-1 was detected in 2 of 28 patients with bladder cancer, but not in 30 healthy individuals. KU-BL-2 was found to be a putative human kelch-like protein that was homologous to Drosophila kelch, with a BTB/POZ domain and kelch repeats. KU-BL-2 was expressed in bladder cancer cell lines, most bladder cancer tissues, testis and heart, but not in normal bladder mucosa. IgG antibody to KU-BL-2 was detected in 8 of 28 patients with bladder cancer, but not in 16 healthy individuals. Tumor reactive T cells were induced from peripheral blood mononuclear cells (PBMC) by stimulation with one of the HLA-A24 binding KU-BL-2 peptides. Therefore, KU-BL-1 and KU-BL-2, which showed preferential expression in bladder cancer with restricted expression in normal tissues, as well as immunogenicity in multiple patients with bladder cancer, may be useful for the development of diagnostic and therapeutic methods for patients with bladder cancer.