MAGE-12 and MAGE-6 are frequently expressed in malignant melanoma.

MAGE proteins have been identified as potential specific targets for cancer vaccination. Although MAGE-6 and MAGE-12 were originally identified in malignant melanoma there are no studies reporting the frequency of expression of these antigens in this malignancy. These are of relevance particularly for MAGE-6 as recent studies have identified CTL activity against several epitopes. We have studied MAGE-1, -2, -3, -4, -6 and -12 gene expression using reverse transcription-polymerase chain reaction in 47 melanoma samples and 11 melanoma cell lines established from these tumours. The tumour samples expressed MAGE-12 (74%) and MAGE-6 (64%) mRNA at much higher frequencies than the other MAGE genes. MAGE-12 and MAGE-6 were expressed at the highest frequencies, relative to the other MAGE antigens, in early stage lesions. The frequency of expression of all the MAGE genes was found to be higher in samples from metastatic deposits compared to those from locoregional disease. The cell lines all expressed the same or more MAGE antigens than the tumours from which they were derived. In only one cell line was expression of a MAGE antigen lost. Certain recurring patterns of MAGE expression were observed in the tumour samples. MAGE-6 and/or -12 expression were detected in all of those 26 tumour samples that were positive for one or more of MAGE-1, -2, -3 and -4. Twenty of these 26 samples expressed both antigens. These findings suggest that protocols targeting MAGE-12 and -6 would permit many more patients to be included into clinical cancer vaccination trials.

The immune response of mice and cynomolgus monkeys to macaque mucin 1-mannan.

Mice immunised with human epithelial mucin MUC1 coupled to oxidised mannan produce MUC1 specific MHC Class 1 restricted CD8(+) cytotoxic T cells and are completely protected from the development of MUC1(+) tumours; such therapy may be applicable to humans. In this light we describe pre-clinical studies in cynomolgus monkeys (Macaca fascicularis), to test the efficacy of mannan-MUC1 in higher primates. Monkey MUC1 genomic clones were isolated from a macaque library, peptides and fusion protein synthesised and mice and monkeys immunised with macaque MUC1-mannan. In mice CTL responses were induced (as has been found with human MUC1 mannan conjugates), but in contrast monkeys produced a humoral response, with no T cell proliferative, cytotoxic responses or CTLp found. In spite of the presence of anti-MUC1 auto-antibodies, there was no toxicity or induction of autoimmunity.

Induction of humoral and cellular responses in cynomolgus monkeys immunised with mannan-human MUC1 conjugates.

Mice immunised with oxidised mannan conjugated to the human mucin 1 (MUC1), produce MHC Class 1 restricted CD8+ cytotoxic T-cells which eradicate MUC1 + tumours, indicating potential for the immunotherapy of MUC1 + cancers in humans. We now describe preclinical studies performed in cynomolgus monkeys immunised with human or murine MUC1 conjugated to oxidised mannan, where immune responses and toxicity were examined. High titred antibodies specific for MUC1 were produced, MUC1 specific CD4+ and CD8+ T-cell proliferative responses and specific cytotoxic precursor cells (CTLp) were found, but not MUC1 specific cytotoxic T-cells (CTL). There was no toxicity and monkeys can be immunised against human MUC1 with mannan-MUC1 conjugates, but a humoral response (Th2 type) predominates. The results contrast with those obtained in mice when a CTL response (Th1 type) predominates.

Natural human anti-Gal alpha(1,3)Gal antibodies react with human mucin peptides.

We have recently demonstrated that both antibodies to Gal alpha(1,3)Gal, and the Gal alpha(1,3)Gal binding lectin (IB4), bind a synthetic peptide (DAHWESWL), there being a similar recognition of carbohydrate and peptide structures. We now report that the anti-Gal alpha(1,3)Gal antibodies and IB4 lectin also react with peptides encoded by mucin genes (MUC 1, 3, 4)-sequences known to be rich in serine, threonine and proline. This activity was demonstrated (1) by the ability of mucin derived peptides to block the reaction of anti-Gal alpha(1,3)Gal antibodies and IB4 lectin with a Gal alpha(1,3)Gal+ pig endothelial cell line; the reactions were specific and did not occur with a random peptide containing the same sequences or with other mucin peptides; (2) by the fact that anti-mucin1 antibodies could react with the Gal alpha(1,3)Gal expressed after transfection of COS cells (Gal alpha(1,3)Gal-,Muc1-) with cDNA encoding the pig alpha, 3galactosyltransferase; and (3) that the IB4 lectin and anti-Gal alpha(1,3)Gal antibodies could react with mucin 1 found on the surface of human breast cancer cells. Thus natural occurring anti-Gal alpha(1,3)Gal antibodies found in all human serum can react with self (Muc1) peptides expressed in large amounts on the surface of tumour cells but not on normal cells. The findings are of interest and serve to explain the previously reported findings that human cells can, at times, express Gal alpha(1,3)Gal; such expression is an artefact, the reaction is due to the phenomenon described herein, i.e. that anti-Gal alpha(1,3)Gal antibodies react with mucin peptides.

Biochemical studies of pig xenoantigens detected by naturally occurring human antibodies and the galactose alpha(1-3)galactose reactive lectin.

The xenotransplantation of pig organs to humans is now receiving serious consideration because of the shortage of human donors for organ transplants. However, such xenografts would be hyperacutely rejected due to naturally occurring antibodies, present in all human sera, that react with pig antigens on the surface of endothelial cells, leading to complement fixation and the rapid onset of intravascular coagulation. A major target of these human natural antibodies is the terminal nonreducing disaccharide Gal alpha (1,3)Gal, and we now report on the array of molecules that are galactosylated by the alpha 1,3-galactosyltransferase. Pig lymphocytes and endothelial cells (both of which bear Gal alpha(1,3)Gal epitopes) were surface iodinated and the 125I-labeled molecules were precipitated with either human antibodies or the lectin from Griffonia simplicifolia (IB4, which binds to Gal alpha(1,3)Gal epitopes). The precipitated molecules were analyzed by gel electrophoresis and autoradiography. Five major groups of molecules were identified by one-dimensional SDS/PAGE (alpha 220 kDa, beta 160-180 kDa, gamma 120 kDa, delta 64 kDa, epsilon 40 kDa); the beta molecule was different in the 2 cell types (beta 1 of lymphocytes and beta 2 of endothelial cells). Two-dimensional SDS/PAGE analysis revealed that each of these groups of molecules resolved into further species of different charge (presumably due to different glycosylation) and also different molecular mass to give at least 20 different Gal alpha(1,3)Gal+ surface molecules. None of these molecules appeared to be present as disulfide-associated dimers. It is clear that there are many galactosylated molecules on the cell surface; indeed, using longer exposures of the autoradiographs, at least 40 different Gal alpha (1,3)Gal+ molecules could be identified. Several of these molecules are likely to have been identified by others, e.g., the 115-kDa, 125-kDa, and 135-kDa triad identified by Platt. Strategies to overcome hyperacute rejection could include modification or deletion of the alpha 1,3-galactosyltransferase gene, which would simultaneously delete all the Gal alpha(1,3)Gal epitopes on these molecules.

Gal alpha(1,3)Gal is the major xenoepitope expressed on pig endothelial cells recognized by naturally occurring cytotoxic human antibodies.

Hyperacute rejection, mediated by natural antibody, is the major barrier to xenotransplantation. The studies reported herein were aimed at evaluating antibody-mediated cytotoxicity and the role of the Gal alpha(1,3)Gal epitope, which we had previously demonstrated was the major epitope of pig cells detected by naturally occurring human antibodies. Also, we had shown that this epitope could be induced in non-expressing cells by the transfection of a cDNA clone encoding alpha(1,3)galactosyl transferase, the enzyme that produces this epitope. The importance of the Gal alpha(1,3)Gal epitope was supported by (1) sugar inhibition studies; (2) complete absorption of cytotoxic antibodies by melibiose-sepharose columns; and (3) the ability of normal human serum to lyse COS cells after transfection with a cDNA clone encoding alpha(1,3)galactosyl transferase. These findings strongly suggest that the majority of cytotoxic human antibodies that would recognize a xenogeneic graft are directed to the Gal alpha(1,3)Gal epitope.

Distribution of the major xenoantigen (gal (alpha 1-3)gal) for pig to human xenografts.

We have previously demonstrated that the major epitope in pig tissues detected by naturally occurring human IgM antibodies is galactose (alpha 1-3)galactose. Subsequent biochemical studies demonstrated this epitope to be present on molecules (Mr40-220kDa) on both endothelial cells and lymphocytes. The objective of the present study was to define the distribution of gal(alpha 1-3)gal in different pig tissues, concentrating on those of relevance for the potential transplantation of pig organs or tissues to humans. Adult pig tissues were obtained fresh, fixed, and stained by the immunoperoxidase technique using biotinylated Griffonia simplicifolia lectin (IB4) which binds only to gal(alpha 1-3)gal, and examined histologically. Endothelial cells in all small vessels (capillaries, arterioles and venules) had a unifrom and dense expression of gal(alpha 1-3)gal; in larger vessels, like the aorta, they were less reactive. The highest concentrations were found in the liver parenchyma which stained uniformly, and in the kidney, where the highest amounts were found in the brush border of the proximal convoluted tubules. There was no staining of collecting ducts or glomeruli (except for endothelium) and moderate staining of the distal convoluted tubules. Heart muscle was nonreactive, although the high density of capillaries indicated a reasonable content of gal(alpha 1-3)gal. In contrast to these tissues was the distribution in the pancreas, which, apart from vessels and the lining of ducts, was nonreactive, i.e. islet cells were essentially lacking in gal(alpha 1-3)gal. Other tissues such as the lung contained moderate amounts of material lining the alveoli and bronchioles.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes.

A major problem with pig-to-human-tissue xenograft studies is that humans have natural antibodies to pig cells; these antibodies would cause hyperacute rejection if pig tissues were xenografted to humans. Here we show that most of human IgM antibodies present in the serum of healthy donors and reactive with pig cells react with galactose in an (alpha 1-3) linkage with galactose--i.e., Gal(alpha 1-3)Gal. Absorption studies demonstrated that the antibodies detected the same or similar epitopes on the surface of pig erythrocytes, blood and splenic lymphocytes, and aortic endothelial cells (EC). The antibodies were sensitive to 2-mercaptoethanol (2ME) treatment, did not bind to protein A or G, and were present in the high molecular weight fraction of serum; they are clearly IgM antibodies. Further, the antibodies did not react with human ABO blood group substances and are not related to human blood group A or B, which carry a terminal galactose. The reaction of human serum with pig erythrocytes was specifically inhibited by mono- and disaccharides: D-galactose, melibiose, stachyose, methyl-alpha-D-galactopyranoside, and D-galactosamine but not by D-glucose or methyl-beta-D-galactopyranoside; demonstrating that the reaction is with galactose in an alpha and not a beta linkage. A cDNA clone encoding the murine alpha-1,3-galactosyltransferase (which transfers a terminal galactose residue with an (alpha 1-3) linkage to a subterminal galactose) was isolated by polymerase chain reaction (PCR), cloned, and transfected into COS cells, which are of Old World monkey origin and, like humans, do not express Gal(alpha 1-3)Gal. After transfection, COS cells became strongly reactive with human serum and with IB4 lectin [which reacts only with Gal(alpha 1-3)Gal]; this reactivity could be removed by absorption with pig erythrocytes. As most of the antibody reacting with pig cells can be removed by absorption with either melibiose or Gal(alpha 1-3)Gal+ COS cells, most of these react with Gal(alpha 1-3)Gal. These findings provide the basis for genetic manipulation of the pig alpha-1,3-galactosyltransferase for future transplantation studies.

CD48 is a low affinity ligand for human CD2.

COS cells transiently transfected with human CD48 were found to bind human PBL, whereas mock or CD7-transfected COS cells failed to bind human lymphocytes. Binding of PBL to CD48 transfectants was almost totally inhibited by either CD48 mAb pretreatment of COS cells or CD2 mAb pretreatment of PBL, implying an interaction between CD2 and CD48. This conclusion was confirmed by the demonstration that a highly fluorescent, multimeric form of rCD2 bound to CD48 transfected COS cells in a CD48-dependent manner. Additional mAb blocking studies revealed that CD48 interacts with the T11(1) region of CD2, the same region of CD2 that binds LFA-3 (CD58). Thus, CD48 and CD58 represent alternative and possibly competing ligands for CD2, although based on blocking studies with soluble CD2, CD48 interacts with CD2 with approximately a 100-fold lower affinity that CD58.

Isolation and characterization of cDNA clones for mouse Ly-9.

We describe the production and characterization of a mouse mAb, S-450-33.2, recognizing the Ly-9.2 specificity. This mAb was used to purify Ly-9 molecules from lymphoid cell lines, and the amino-terminal amino acids were determined. The mAb was also used in a eukaryotic expression system, to isolate cDNA clones encoding Ly-9. Analysis of RNA showed that Ly-9 expression is lymphocyte specific, as determined by the presence of a single hybridizing 2.4-kb species found only in lymphoid cells. Genomic DNA analysis indicated that Ly-9 is encoded by a single-copy gene of 10 to 15 kb. The predicted polypeptide belongs to the Ig superfamily of cell surface molecules with four extracellular Ig-like domains, i.e., a non-disulfide-bonded V domain, a truncated C2 domain with two disulfide bonds, a second non-disulfide-bonded V domain, and a truncated C2 domain with two disulfide bonds (V-C2-V-C2). The sequence data also support the view that Ly-9 belongs to the subgroup of the Ig superfamily that includes Bcm-1, CD2, and LFA-3.

The isolation of cDNA clones for CD48.

HuLy-m3 is an Mr 47,000 pan-leukocyte antigen detected by the monoclonal antibody (mAb) 5-4.8. This report describes the isolation and analysis of a cDNA clone encoding HuLy-m3. Serological analysis demonstrated that antibodies of the CD48 cluster also reacted with transfected cells expressing HuLy-m3. The DNA sequence of the clone suggests linkage to the cell membrane through a glycosyl phosphatidylinositol tail and this was verified experimentally. Sequence similarity with the human B-cell activation antigen Blast-1 was noted.