Mary C. Custer

Global Expression Analysis of Cancer/Testis Genes in Uterine Cancers Reveals a High Incidence of BORIS Expression

Purpose: Cancer/testis (CT) genes predominantly expressed in the testis (germ cells) and generally not in other normal tissues are aberrantly expressed in human cancers. This highly restricted expression provides a unique opportunity to use these CT genes for diagnostics, immunotherapeutic, or other targeted therapies. The purpose of this study was to identify those CT genes with the greatest incidence of expression in uterine cancers. Experimental Design: We queried the expression of known and putative CT gene transcripts (representing 79 gene loci) using whole genome gene expression arrays. Specifically, the global gene expressions of uterine cancers (n = 122) and normal uteri (n = 10) were determined using expression data from the Affymetrix HG-U133A and HG-U133B chips. Additionally, we also examined the brother of the regulator of imprinted sites (BORIS) transcript by reverse transcription-PCR and quantitative PCR because its transcript was not represented on the array. Results: Global microarray analysis detected many CT genes expressed in various uterine cancers; however, no individual CT gene was expressed in more than 25% of all cancers. The expression of the two most commonly expressed CT genes on the arrays, MAGEA9 (24 of 122 cancers and 0 of 10 normal tissues) and Down syndrome critical region 8 (DSCR8)/MMA1 (16 if 122 cancers and 0 of 10 normal tissues), was confirmed by reverse transcription-PCR methods, validating the array screening approach. In contrast to the relatively low incidence of expression of the other CT genes, BORIS expression was detected in 73 of 95 (77%) endometrial cancers and 24 of 31 (77%) uterine mixed mesodermal tumors. Conclusions: These data provide the first extensive survey of multiple CT genes in uterine cancers. Importantly, we detected a high frequency of BORIS expression in uterine cancers, suggesting its potential as an immunologic or diagnostic target for these cancers. Given the high incidence of BORIS expression and its possible regulatory role, an examination of BORIS function in the etiology of these cancers is warranted.

Potential Use of T Cell Receptor Genes to Modify Hematopoietic Stem Cells for the Gene Therapy of Cancer

The purpose of this review is to illustrate some of the technical and biological hurdles that need to be addressed when developing new gene therapy based clinical trials. Gene transfer approaches can be used to “mark” cells to monitor their persistence in vivo in patients, to protect cells from toxic chemotherapeutic agents, correct a genetic defect within the target cell, or to confer a novel function on the target cell. Selection of the most suitable vector for gene transfer depends upon a number of factors such as the target cell itself and whether gene expression needs to be sustained or transient. The TCR gene transfer approach described here represents one innovative strategy being pursued as a potential therapy for metastatic melanoma. Tumor reactive T cells can be isolated from the tumor infiltrating lymphocytes (TIL) of melanoma patients. A retroviral vector has been constructed containing the T cell receptor (TCR) α and β chain genes from a MART-1(27-35)-specific T cell clone (TIL 5). Jurkat cells transduced with this virus specifically release cytokine in response to MART-1(27-35) peptide pulsed T2 cells, showing that the virus can mediate expression of a functional TCR. HLA-A2 transgenic mice are being used to examine whether transduced bone marrow progenitor cells will differentiatein vivo into mature CD8+ T cells expressing the MART-1(27-35)-specific TCR. Expression of the human TCR α and β chain genes has been detected by RT-PCR in the peripheral blood of HLA-A2 transgenic mice reconstituted with transduced mouse bone marrow. Expression of the TIL 5 TCR genes in the peripheral blood of these mice was maintained for greater than 40 weeks after bone marrow reconstitution. TIL 5 TCR gene expression was also maintained following transfer of bone marrow from mice previously reconstituted with transduced bone marrow to secondary mouse recipients, suggesting that a pluripotent progenitor or lymphocyte progenitor cell has been transduced.

Retroviral transduction of interferon-γ cDNA into a nonimmunogenic murinefibrosarcoma: generation of T cells in draining lymph nodes capable of treating established parental metastatic tumor

Gene modification of tumor cells with the cDNA for interferon γ (IFNγ) has been shown to increase the immunogenicity of some tumor cells. In order to explore further the possible therapeutic relevance of these previous findings, two clones of the nonimmunogenic MCA-102 fibrosarcoma of C57BL/6 origin were retrovirally transduced with the cDNA encoding murine IFNγ: 102.4JK (4JK), a clone with relatively high major histocompatibility complex (MHC) class I expression, and 102.24JK (24JK), a clone with low expression of surface MHC class I molecules. Retroviral transduction of tumor cells with the cDNA encoding for IFNγ resulted in a substantial up-regulation of MHC class I surface expression in the 24JK clone but little change of class I in the 4JK clone. In an attempt to generate antitumor lymphocytes, these gene-modified cells were inoculated into mouse footpads and draining lymph nodes (DLN) were removed, dispersed, and cultured in vitro for 10 days with irradiated tumor cells and interleukin-2. DLN from mice bearing either unmodified tumor or tumor transduced with cDNA encoding neomycin resistance (NeoR) or IFNγ, were used to treat recipients harboring 3-day pulmonary metastases induced by the parental, unmodified tumor. Treatment with DLN cells obtained following the injection of 24JK tumor cells modified with the gene for IFNγ significantly reduced the number of pulmonary metastases in four separate experiments, compared to groups treated by DLN cells generated from inoculation of either the unmodified, parental 24JK clone or the same clone transduced with theNeoR gene only. In contrast, DLN cells induced either by IFNγ-transduced 4JK (high expression of MHC class I) or an unmodified 4JK tumor (moderate expression of MHC class I) had significant but equal therapeutic efficacy. Although the in vitro growth rate of tumor cell lines was unaffected by the insertion of the mouse IFNγ cDNA, their in vivo (s.c.) growth rates were significantly slower than those of the nontransduced tumors. Thus, after retroviral transduction of the murine IFNγ cDNA into a nonimmunogenic tumor with a very low level of surface expression of MHC class I, modified tumor cells could elicit therapeutic T cells from DLN capable of successfully treating established pulmonary metastases upon adoptive transfer. This strategy significantly confirms previous observations on the potential therapeutic effects of gene modification of tumor cells with IFNγ and extends the realm of therapeutic possibilities to include the use of DLN cells for the development of T-cell based immunotherapies against nonimmunogenic human tumors.

The persistence of human peripheral lymphocytes, tumor infiltrating lymphocytes, and colon adenocarcinomas in immunodeficient mice

The reconstitution of severely immunodeficient mice with human peripheral blood mononuclear cells (PBMCs) may represent a unique model system to evaluate human antitumor responses. To evaluate this possibility, we studied human PBMC reconstitution, human tumor infiltrating lymphocyte (TIL) persistence, and human colon adenocarcinoma propagation in beige/nude/xid (BNX) and in severe combined immunodeficient (SCID) mice. To evaluate human PBMC reconstitution, 75 mice received 1 x 10(7)-1 x 10(9) human PBMCs i.p. or i.v. and were studied at intervals ranging from 1 to 8 weeks by fluorescence-activated cell sorting (FACS) analysis and by measurement of circulating human immunoglobulin levels. By FACS analyses, only one of 75 mice had evidence of human PBMC persistence at 2 weeks in the spleen. Moreover, liver and peritoneum showed evidence of human cells in only 13 of 56 and 16 of 55 mice, respectively. In these mice, human cells comprised 1-77% of total cells recovered. Human immunoglobulin levels in mouse serum ranged from 0 to 34,000 micrograms/ml and correlated only weakly with evidence of human PBMC reconstitution in peripheral organs, but were generally higher in SCID mice than in BNX mice. Human TIL persistence was evaluated in BNX and SCID mice that were given 3 x 10(7) TILs i.v. (in divided doses) or 1 x 10(8) i.p. TILs along with interleukin-2 administration. At 1, 2, 7, and 14 days following TIL delivery, evidence of human TIL persistence in liver, lung, peritoneum, and spleen was evaluated by FACS analysis. Fresh organ suspensions did not contain human TILs. In mice given cyclophosphamide followed by human TILs i.p., the TILs were demonstrated at 7 days in the SCID peritoneum (leu 4 = 4%) and at 2 days in the SCID spleen (leu 4 = 2%). In BNX mice, 12 of 14 fresh human colon adenocarcinomas were propagated successfully at subcutaneous sites with latency periods ranging from 1 to 13 weeks. Enzymatic disaggregation of tumors greater than 1 cm following one passage yielded 6.5-47 x 10(6) cells with viabilities ranging from 13 to 85%. We conclude that limitations and variability exist in the use of BNX and SCID mice for human PBMC reconstitution, TIL persistence, and propagation of human colon adenocarcinomas.

Mechanisms of immunologic antitumor therapy: lessons from the laboratory and clinical applications

The use of interleukin 2-based immunotherapies for cancer has been associated with significant responses in tumor models in both mouse and humans. Further definition of the elements responsible for response is now possible. It appears that the response is associated with T-cell infiltration of the tumor, and transfer of tumor-infiltrating lymphocytes expanded in tissue culture with interleukin 2 is associated with significant antitumor effects. Further expansion of cultured human melanoma tumor-infiltrating lymphocytes with suppression of lymphokine-activated killer activity as well as the modulation of monocyte activity by interleukin 4 suggests that this cytokine may be clinically useful alone or in combination with interleukin 2. Other means of enhancing the activity of interleukin 2-based immunotherapy are suggested by the finding that tumor cell susceptibility to lysis by natural killer cells is depressed following treatment with interferon gamma and tumor necrosis factor, but susceptibility to lysis by tumor-infiltrating lymphocytes is markedly enhanced. Further development of these therapies will require innovative interpretation and application of findings related to the processing and presentation of human tumor antigens and the nature of tumor antigens and careful analysis of the T-cell receptor in antitumor effectors.

Normal viability of Kai1/Cd82 deficient mice

The KAI1/CD82 tetraspanin is a widely expressed cell surface molecule thought to organize diverse cellular signaling processes. KAI1/CD82 suppresses metastasis but not tumorigenicity, establishing it as one of a class of metastasis suppressor genes. In order to further assess its functions, we have characterized the phenotypic properties of Kai1/Cd82 deleted mice, including viability, fertility, lymphocyte composition, blood chemistry and tissue histopathology, and of their wild‐type and heterozygote littermates. Interestingly, Kai1/Cd82−/− showed no obvious genotype associated defects in any of these processes and displayed no genotype associated histopathologic abnormalities after 12 or 18 months of life. Expression profiles of non‐immortal, wild‐type and Kai1/Cd82−/− mouse embryo fibroblast (MEFs) indicated distinct sex‐specific and genotype‐specific profiles. These data identify 191 and 1,271 differentially expressed transcripts (by twofold at P < 0.01) based on Kai1/CD82 genotype status in female and male MEFs, respectively. Differentially expressed genes in male MEFs were surprisingly enriched for cell division related processes, suggesting that Kai1/Cd82 may functionally affect these processes. This suggests that Kai/Cd82 has an unappreciated role in the early establishment of proliferation and division when challenged with a new environment that might play a role in adaptability to new metastatic sites.

Identification of a T-cell receptor from a therapeutic murine T-cell clone.

Tumor-infiltrating lymphocytes (TIL) have been successfully used for the treatment of metastatic malignancies in clinical trials and in experimental animal models. Tumor-specific reactivity by TIL is mediated via receptors expressed on the surface of T cells (TcRs), which recognize tumor-associated antigens (TAA) presented in the context of MHC molecules on the surface of tumor cells. The current study was performed to identify the TcR alpha and beta chains from a tumor-specific therapeutic TIL clone that can be used to develop a preclinical animal model for genetically modifying lymphocytes and hematopoietic progenitors with TcR genes. TIL 205 was generated from a subcutaneous implant of MCA-205 fibrosarcoma and at 21 days was cloned by limiting dilution. TIL clone 8, obtained from a culture seeded at one cell/well, mediated specific lysis and specific secretion of gamma-interferon to MCA-205 and WP6, a subclone of MCA 205. No reactivity was observed against other syngeneic sarcoma lines. Anchor polymerase chain reaction analysis determined that antigen recognition by clone 8 was mediated by a TcR consisting of V alpha 3/J alpha 27 and V beta 8.2/D beta 2.1/D beta 2.4. Immunofluorescent staining with V beta subfamily specific monoclonal antibodies revealed that > 95% of the T cells in TIL clone 8 expressed V beta 8.2, confirming that TIL clone 8 was indeed a clone. In contrast, approximately 30% of the T cells in the parental TIL 205 expressed V beta 8.2. The transfer of as few as 500,000 TIL clone 8 cells in conjunction with the systemic administration of recombinant human interleukin-2 mediated regression of established 3-day WP6 lung metastases. Thus, clone 8 recognizes a biologically relevant tumor rejection antigen, making the V alpha 3/J alpha 27-V beta 8.2/D beta 2.1/J beta 2.4 TcR isolated from this clone useful as a probe for cloning the tumor-rejection antigen in the WP6 tumor as well as modeling, in mice, the TcR-based gene therapies being developed for humans.

T CELL-RECEPTOR V GENE USE BY CD4+ MELANOMA-REACTIVE CLONAL AND OLIGOCLONAL T-CELL LINES

Tumor-reactive CD4+ T cells can be isolated and expanded from the peripheral blood and tumor lesions of patients with melanoma. In contrast to CD8+ T cells, little is known about the antigens recognized by these CD4+ T cells. As a consequence, little is known about the diversity of the T-cell receptor (TcR) use by melanoma-reactive CD4+ T cells. To address these questions, a panel of clonal or highly oligoclonal CD4+ T-cell lines was established from a patient with metastatic melanoma. A CD4+ tumor-infiltrating lymphocyte (TIL) line was established that was highly oligoclonal and recognized only autologous melanoma cells but not allogeneic melanomas, suggesting the expression of a mutated or uniquely expressed antigen by this melanoma. The antigen recognized by the CD4+ TILs could be presented by intact melanoma cells or by autologous Epstein-Barr virus (EBV) B cells pulsed with melanoma cell lysates. A panel of CD4+ clonal and highly oligoclonal T-cell lines was isolated from peripheral blood mononuclear cells (PBMC) from this patient; these were also reactive with autologous melanoma cells or tumor extracts pulsed on autologous EBV B cells. Despite their reactivity with the autologous melanoma, we found no evidence of restricted TcR V gene use, because all six T-cell lines recognized antigen via different TcR α/β rearrangements. Furthermore, there were no conserved amino acids in the CDR3 regions of these TcRs, indicating that multiple TcR clono-types could mediate recognition of a single unique major histocompatibility (MHC) complex class II restricted melanoma antigen or that multiple MHC class II restricted melanoma antigens are expressed by the melanoma.

Use of soluble recombinant TNF receptor to improve detection of TNF secretion in cultures of tumor infiltrating lymphocytes

The accurate quantitation of picogram amounts of TNF is possible by ELISA and is useful in many areas of biomedical research, including studies of TNF release in vitro by stimulated lymphocytes and macrophages, and of serum levels in patients with cancer and sepsis. However, we show in this report that the detection of recombinant TNF standards by ELISA falls over time with incubation at 37 degrees C, and is further decreased when incubated with tumor infiltrating lymphocytes (TIL), making accurate quantitation difficult. We demonstrate that the soluble dimeric form of the TNF receptor can prevent this decrease, both in the presence and absence of TIL. In contrast, the soluble monomeric TNF receptor was much less effective in preventing this decrease. In addition, the dimeric but not the monomeric TNF receptor was found to inhibit bioactivity of TNF as measured by L929 cytotoxicity. The dimeric TNF receptor does not interfere with the detection of recombinant TNF standards by ELISA, and entirely stabilizes TNF levels incubated over 48 h at 37 degrees C in the presence and absence of TIL. This protection is specific, and the TNF receptor does not stabilize interferon-gamma. The dimeric form of the soluble TNF receptor has proven useful in detecting TNF released by TIL transduced with the TNF cDNA that are currently being used in studies of the gene therapy of cancer with TIL. The dimeric TNF receptor may also prove useful in the accurate quantitation of TNF released by stimulated lymphocytes and macrophages in vitro, and in the quantitation of serum TNF levels in patients.