Matthew J. Scanlan

Cancer/testis antigens: an expanding family of targets for cancer immunotherapy

Summary: Cancer/testis (CT) antigens are a category of tumor antigens with normal expression restricted to male germ cells in the testis but not in adult somatic tissues. In some cases, CT antigens are also expressed in ovary and in trophoblast. In malignancy, this gene regulation is disrupted, resulting in CT antigen expression in a proportion of tumors of various types. Since their initial identification by T‐cell epitope cloning, the list of CT antigens has been greatly expanded through serological expression cloning (SEREX) and differential mRNA expression analysis, and approximately 20 CT antigens or antigen families have been identified to date. Characteristics commonly shared by CT antigens, aside from the highly tissue‐restricted expression profile, include existence as multigene families, frequent mapping to chromosome X, heterogeneous protein expression in cancer, likely correlation with tumor progression, induction of expression by hypomethylation and/or histone acetylation, and immunogenicity in cancer patients. Spontaneous humoral and cell‐mediated immune responses have been demonstrated against several CT antigens, including NY‐ESO‐1, MAGE‐A, and SSX antigens. Since CT antigens are immunogenic and highly restricted to tumors, their discovery has led directly to the development of antigen‐specific cancer vaccines, and clinical trials with MAGE‐A and NY‐ESO‐1 are in progress.

Characterization of human colon cancer antigens recognized by autologous antibodies

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) has proven to be a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. In the present study, 48 distinct antigens (NY‐CO‐1–NY‐CO‐48) reactive with autologous IgG were identified by SEREX analysis in 4 patients with colon cancer. Sequencing analysis showed that 17 of the cDNA clones were previously uncharacterized molecules and 31 represented known gene products. The individual cDNA clones were analyzed in the following manner: a search for mutations or other structural changes; an analysis of mRNA expression in a panel of normal tissues; and a frequency analysis of the antibody response to the expressed product in the sera of colon cancer patients and normal individuals. The initial analysis showed NY‐CO‐13 to be a mutated version of the p53 tumor suppressor gene. Three of the 48 antigens showed a differential pattern of mRNA expression, with NY‐CO‐27 (galectin‐4) expressed primarily in gastrointestinal tract, and NY‐CO‐37 and ‐38 showing a pattern of tissue‐specific isoforms. With regard to immunogenicity, 20 of the 48 antigens were detected by allogeneic sera; 14 of these were reactive with sera from both normal donors and cancer patients, and 6 other clones (NY‐CO‐8, ‐9, ‐13, ‐16, ‐20 and ‐38) reacted exclusively with sera from colon cancer patients (ranging from 14% to 27%). Our results on colon cancer illustrate both the complexity and the potential of the SEREX approach for analysis of the humoral immune response against human cancer. Int. J. Cancer76:652–658, 1998.© 1998 Wiley‐Liss, Inc.

A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene

Usher syndrome type 1 describes the association of profound, congenital sensorineural deafness, vestibular hypofunction and childhood onset retinitis pigmentosa. It is an autosomal recessive condition and is subdivided on the basis of linkage analysis into types 1A through 1E (refs 2–6). Usher type 1C maps to the region containing the genes ABCC8 and KCNJ11 (encoding components of ATP-sensitive K + (KATP) channels), which may be mutated in patients with hyperinsulinism. We identified three individuals from two consanguineous families with severe hyperinsulinism, profound congenital sensorineural deafness, enteropathy and renal tubular dysfunction. The molecular basis of the disorder is a homozygous 122-kb deletion of 11p14–15, which includes part of ABCC8 and overlaps with the locus for Usher syndrome type 1C and DFNB18 (ref. 11). The centromeric boundary of this deletion includes part of a gene shown to be mutated in families with type 1C Usher syndrome, and is hence assigned the name USH1C. The pattern of expression of the USH1C protein is consistent with the clinical features exhibited by individuals with the contiguous gene deletion and with isolated Usher type 1C.

SSX: A multigene family with several members transcribed in normal testis and human cancer

Analysis of t(X;18) translocation in synovial sarcoma had previously led to the definition of the SSX2 gene, the fusion partner on chromosome X. Subsequent screening of testicular cDNA libraries identified 2 highly homologous genes, SSX1 and SSX3. Among these 3 genes, SSX2 has been found to be identical to HOM‐MEL‐40, which codes for an immunogenic tumor antigen expressed in various human cancers. SSX2 thus belongs to the family of cancer/testis (CT) antigens, i.e., immunogenic protein antigens with characteristic mRNA expression in normal testis and in cancer. To define additional CT antigens, we have immuno‐screened a testicular cDNA expression library with an allogeneic serum from a melanoma patient, and both SSX2 and SSX3 were isolated. Further studies using testicular cDNA and SSX probes defined 2 new members of this gene family, SSX4 and SSX5, while a shorter cDNA variant of SSX4 was also identified. All 5 members of the SSX family shared strong sequence homology, with nucleotide homology ranging from 88 to 95% and amino acid homology ranging from 77 to 91%. Genomic cloning of a prototype SSX gene (SSX2) showed that its coding region is encoded by 6 exons, and the shortened form of SSX4 cDNA represents an alternatively spliced product lacking the 5th exon. Analysis of SSX mRNA expression by gene‐specific RT‐PCR confirmed that all 5 SSX genes are expressed in testis. In addition, analysis of a panel of 12 melanoma cell lines showed strong mRNA expression of either SSX1 (3/12), SSX2 (3/12), SSX4 (1/12), or SSX5 (1/12), indicating variable activation of the genes in malignant cells. Int. J. Cancer 72:965–971, 1997.

Antigens recognized by autologous antibody in patients with renal-cell carcinoma

The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty‐five distinct antigens (NY‐REN‐1 to NY‐REN‐65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN‐9, ‐10, ‐19, and ‐26 have a known association with human cancer. REN‐9 (LUCA‐15) and REN‐10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN‐19 is equivalent to LKB1/STK11, a gene that is defective in Peutz‐Jeghers syndrome and cancer. REN‐26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN‐3 displays a pattern of tissue‐specific isoforms, and REN‐21 and REN‐43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty‐three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5–25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer. Int. J. Cancer 83:456–464, 1999.

A monoclonal antibody recognizing human cancers with amplification/overexpression of the human epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) has attracted considerable attention as a target for cancer therapy. Wild-type (wt)EGFR is amplified/overexpressed in a number of tumor types, and several mutant forms of the coding gene have been found, with ΔEGFR, a deletion mutation lacking exons 2–7 of the external domain, being the most common and particularly associated with glioblastoma. We generated monoclonal antibodies (mAbs) against NR6ΔEGFR (mouse fibroblast line NR6 transfected with ΔEGFR). mAb 806 with selective reactivity for NR6ΔEGFR in mixed hemadsorption assays, fluorescence-activated cell sorting, Western blot, and immunohistochemistry was analyzed in detail and compared with mAbs 528 (anti-wtEGFR) and DH8.3 (anti-ΔEGFR). In xenograft tumors and molecularly pretyped glioblastomas, the reactivity pattern was as follows: 528 reactive with amplified and nonamplified wtEGFR; DH8.3 reactive with ΔEGFR; and 806 reactive with amplified/overexpressed wtEGFR (with or without ΔEGFR). In normal tissues, 528 but not DH8.3 or 806 was widely reactive with many organs, e.g., liver expressing high EGFR levels. In glioblastoma and non-CNS tumor panels, 806 was reactive with a high proportion of glioblastomas and a substantial number of epithelial cancers of lung and of head and neck. DH8.3 reactivity was restricted to ΔEGFR-positive glioblastoma. Thus, 806 represents a category of mAbs that recognizes tumors with EGFR amplification/overexpression but not normal tissues or tumors with normal EGFR levels. Our study also indicates that ΔEGFR is restricted to glioblastoma, in contrast to other reports that this mutation is found in tumors outside the brain.

Frequent and specific immunity to the embryonal stem cell–associated antigen SOX2 in patients with monoclonal gammopathy

Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138− compartment in MGUS. SOX2 expression is also detected in a proportion of CD138+ cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.

Identification of cancer/testis genes by database mining and mRNA expression analysis.

Cancer/testis (CT) antigens are immunogenic proteins expressed predominantly in gametogenic tissue and cancer; they are considered promising target molecules for cancer vaccines. The identification of new CT genes is essential to the development of polyvalent cancer vaccines designed to overcome tumor heterogeneity and antigen loss. In the current study, a search for new CT genes was conducted by mining the Unigene database for gene clusters that contain expressed sequence tags derived solely from both normal testis and tumor‐derived cDNA libraries. This search identified 1,325 different cancer/testis‐associated Unigene clusters. The mRNA expression pattern of 73 cancer/testis‐associated Unigene clusters was assessed by reverse transcriptase polymerase chain reaction. Three gene products, CT15/Hs.177959, CT16/Hs.245431 and CT17/Hs.178062, were detected only in testis and in tumor tissue. CT15 is equivalent to ADAM2/fertilin‐β. CT16, an uncharacterized gene product, has homology (30–50%) to members of the GAGE gene family and is 89% identical to CT16.2/Hs.293317, indicating that CT16 and CT16.2 are members of a new GAGE gene family. The uncharacterized gene product, CT17, has homology (30%) to phospholipase A1. RT‐PCR analysis showed that CT15 is expressed exclusively in renal cancer, whereas CT16 and CT17 are expressed in a range of human cancers. Real‐time RT‐PCR analysis of newly defined CT genes and the prototype CT antigens, MAGE‐3 and NY‐ESO‐1, revealed low levels (less than 3% of the level detected in testis) of CT15, CT16 and NY‐ESO‐1 in a limited range of normal, non‐gametogenic tissues. This study demonstrates the merits of database mining with respect to the identification of tissue‐restricted gene products expressed in cancer.

Expression of cancer-testis antigens in lung cancer: definition of bromodomain testis-specific gene (BRDT) as a new CT gene, CT9

In an effort to define new cancer-testis (CT) genes, we investigated whether BRDT, a testis-restricted member of the RING3 family of transcriptional regulators, is also expressed in cancer. Standard RT-PCR expression analysis detected BRDT transcripts in 12 of 47 cases of non-small cell lung cancer and single cases of both squamous cell carcinoma of the head and neck (1/12) and esophagus (1/12) but not in melanoma or in cancers of the colon, breast, kidney and bladder. Typing of 33 non-small cell lung cancers for coexpression of a panel of CT antigens revealed a high incidence (60%) of MAGE-3 mRNA expression, followed by MAGE-1 (36%), CT7/MAGE-C1 (30%), CT10 (30%), SSX4 (23%), BRDT (21%), NY-ESO-1 (21%) and HOM-MEL-40/SSX2 (15%). The coexpression pattern of these antigens provides a foundation for developing a polyvalent lung cancer vaccine.

CT10: a new cancer-testis (CT) antigen homologous to CT7 and the MAGE family, identified by representational-difference analysis.

Assays relying on humoral or T‐cell‐based recognition of tumor antigens to identify potential targets for immunotherapy have led to the discovery of a significant number of immunogenic gene products, including cancer‐testis (CT) antigens predominantly expressed in cancer cells and male germ cells. The search for cancer‐specific antigens has been extended via the technique of representational‐difference analysis and SK‐MEL‐37, a melanoma cell line expressing a broad range of CT antigens. Using this approach, we have isolated CT antigen genes, genes over‐expressed in cancer, e.g., PRAME and KOC, and genes encoding neuro‐ectodermal markers. The identified CT antigen genes include the previously defined MAGE‐A6, MAGE‐A4a, MAGE‐A10, CT7/MAGE‐C1, as well as a novel gene designated CT10, which shows strong homology to CT7/MAGE‐C1 both at cDNA and at genomic levels. Chromosome mapping localized CT10 to Xq27, in close proximity to CT7/MAGE‐C1 and MAGE‐A genes. CT10 mRNA is expressed in testis and in 20 to 30% of various human cancers. A serological survey identified 2 melanoma patients with anti‐CT10 antibody, demonstrating the immunogenicity of CT10 in humans. Int. J. Cancer 85:726–732, 2000.