Kenneth O. Lloyd

Ovarian cancer antigen CA125 is encoded by the MUC16 mucin gene

Serum assays based on the CA125 antigen are widely used in the monitoring of patients with ovarian cancer; however very little is known about the molecular nature of the CA125 antigen. We recently cloned a partial cDNA (designated MUC16) that codes for a new mucin that is a strong candidate for being the CA125 antigen. This assignment has now been confirmed by transfecting a partial MUC16 cDNA into 2 CA125‐negative cell lines and demonstrating the synthesis of CA125 by 3 different assays. Of the 3 antibodies (OC125, M11 and VK‐8) tested on the transfected cells, only the first 2 were strongly positive, indicating the differential expression of the CA125 epitopes in these cells. The cloning and expression of CA125 antigen opens the way to an understanding of its function in normal and malignant cells.

Immunization of ovarian cancer patients with a synthetic Lewisy-protein conjugate vaccine : A phase 1 trial

As the initial step in developing carbohydrate‐based vaccines for the treatment of ovarian cancer patients in an adjuvant setting, 25 patients were immunized with a Lewisy pentasaccharide (Ley)‐keyhole limpet hemocyanin (KLH)‐conjugate vaccine together with the immunological adjuvant QS‐21. Four different doses of the vaccine, containing 3, 10, 30, and 60 μg of carbohydrate were administered s.c. at 0, 1, 2, 3, 7, and 19 weeks to groups of 6 patients. Sera taken from the patients at regular intervals were assayed by ELISA for reactivity with naturally occurring forms of Ley (Ley‐ceramide and Ley mucin) and by flow cytometry and a complement‐dependent cytoxicity assay for reactivity with Ley‐expressing tumor cells. The majority of the patients (16/24) produced anti‐Ley antibodies as assessed by ELISA, and a proportion of these had strong anti‐tumor cell reactivity as assessed by flow cytometry and complement‐dependent cytotoxicity. One serum, analyzed in detail, was shown to react with glycolipids but not with glycoproteins or mucins expressed by ovarian cancer cell line OVCAR‐3. The vaccine was well tolerated and no gastrointestinal, hematologic, renal, or hepatic toxicity related to the vaccine was observed. On the basis of this study, Ley‐KLH should be a suitable component for a polyvalent vaccine under consideration for the therapy of epithelial cancers. Int. J. Cancer 87:79–85, 2000.

A FULLY SYNTHETIC GLOBO H CARBOHYDRATE VACCINE INDUCES A FOCUSED HUMORAL RESPONSE IN PROSTATE CANCER PATIENTS : A PROOF OF PRINCIPLE

Human trials on the globo H carbohydrate vaccine (see picture, KLH=the carrier protein keyhole limpet hemocyanin) show that it produces strong IgM, and in some cases IgG, responses in patients with progressive and recurrent prostate cancer. Furthermore, these antibodies not only recognize synthetic antigens, but also globo H-positive tumors in biopsy extracts and tumor tissues.

A novel and efficient method for synthetic carbohydrate conjugate vaccine preparation: synthesis of sialyl Tn-KLH conjugate using a 4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide (MMCCH) linker arm

STn (NeuAcα2→6GalNAcα-O-Ser/Thr) is a carbohydrate epitope overexpressed in various human carcinomas. Clinical trials are underway using synthetic STn or STn trimeric glycopeptides [STn, cluster; STn(c) conjugated with keyhole limpet hemocyanin (KLH) as active specific immunotherapy for these cancers. These vaccines have been prepared by conjugating a crotyl ethyl amide derivative of STn or STn(c) to KLH by direct reductive amination after ozonolysis. In the case of STn(c) the conjugation efficiency and the resulting epitope ratios were low. This may be due to steric hinderance of the short spacer arm. To overcome these difficulties, without resynthesis, the STn(c) glycopeptide was modified by attachment of an MMCCH (4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazide) spacer arm to the aldehyde derivative, and then conjugated with thiolated KLH. This method gave a higher epitope ratio and yield than the direct method. The STn(c)-MMCCH-KLH conjugate induced high titer antibodies in mice against STn(c). This method may be generally applicable for large synthetic oligosaccharides.

The chemistry and immunochemistry of blood group A, B, H, and Lewis antigens: Past, present and future

This article traces reseach on the chemistry and immunochemistry of blood group A, B, H, and Lewis antigens from early work on the identification of soluble sources of these antigens, through the elucidation of the structures of the carbohydrate epitopes responsible for these specificities, to recent work on exploring their possible use as cancer vaccines. The various approaches used in the isolation of oligosaccharides from mucins for use in structural studies are discussed, as are recent efforts in the chemical systhesis of blood group-active oligosaccharides.

Characterization of a mouse monoclonal IgG3 antibody to the tumor-associated globo H structure produced by immunization with a synthetic glycoconjugate.

Globo H (Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glc) is a carbohydrate structure that shows enhanced expression in many human carcinomas. From mice immunized with a globo H-KLH (keyhole limpet hemocyanin) synthetic conjugate an IgG3 monoclonal antibody (mAb VK-9) was derived that recognizes the globo H structure. Serological analysis showed that the minimal structure recognized by this mAb was the tetrasaccharide sequence Fucα1→2Galβ1→3GalNAcβ1→3Gal. An isomeric structure with an internal αGalNAc linkage was also recognized but less efficiently. mAb VK-9 did not react with many related structures, such as galactosylgloboside, globoside, H type 1, H type 2 blood group structures or fucosyl-gangliotetraosyl ceramide, but did react weakly with globo A ceramide. Not only did mAb VK-9 react with carbohydrate-protein conjugates but it could also recognize globo H-ceramide and human tumor cells expressing globo H. These results suggest that globo H-KLH could be explored as a vaccine in the treatment of carcinoma patients.

CA125 and UQCRFS1 FISH studies of ovarian carcinoma.

OBJECTIVE With the gene CA125 having recently been cloned, we chose to investigate the gene copy number of various ovarian cancer samples by FISH. As a control we chose BACs close to the chromosome 19 centromere. One of these BACs carries the gene UQCRFS1. METHODS We developed FISH probes for CA125 and the UQCRFS1 region. We studied 22 touch preparations and 14 paraffin-embedded samples of ovarian carcinomas with known CA125 serum levels, two ovarian cancer cell lines, and one ascites sample from an ovarian cancer patient. The average copy number per cell of both probes was calculated. Metaphase analyses were done on cell lines and ascites cells to localize the signals. RESULTS The CA125 gene mapped to 19p13.2. Three of 22 (13.6%) touch preparations and 1 of 14 (7.1%) paraffin samples had amplified levels of CA125. The cell lines and ascites sample did not have amplified CA125. Unexpectedly, 3 of 22 (13.6%) touch preparations, 1 of 14 (7.1%) paraffin samples, one cell line, and the ascites sample had amplification of the UQCRFS1 region. The amplification of the UQCRFS1 region occurred in the form of homogeneously staining regions (HSRs). Only one sample had coamplification of CA125 and UQCRFS1. CONCLUSIONS CA125 was only sometimes modestly amplified in ovarian carcinoma, even when the serum CA125 level was highly elevated. Unexpectedly, the UQCRFS1 region was also sometimes amplified as HSRs. The UQCRFS1 protein is also known as complex III of the mitochondrial respiratory chain. This product may have an important role in malignant cells.

Melanoma tumor antigen and autologous antibody

The present invention concerns novel immunoprecipitating autologous antibodies which recognize the Class 1 gp90 antigen on melanoma cells. These antibodies, optionally tagged with a chromophoric or radioactive label and immobilized on an inert support, may be used to recognize and isolate the gp90 antigen from melanoma cell extracts. Monoclonal antibodies to melanoma may be screened with the gp90 antigen for those which recognize epitopes other than the FD antigenic system. The cell line containing the gp90 antigen which has been cultured in vitro is a source of gp90 antigen for generation of monoclonal antibodies which will be useful in analyzing the gp90 antigen for those epitopes which may be of diagnostic value in immunoassay of melanoma.