Hilary A. Vaughan

Antibody and T cell responses of patients with adenocarcinoma immunized with mannan-MUC1 fusion protein.

Mucin 1 (MUC1) is a large complex glycoprotein that is highly expressed in breast cancer, and as such could be a target for immunotherapy. In mice, human MUC1 is highly immunogenic, particularly when conjugated to mannan, where a high frequency of CD8(+) MHC-restricted cytotoxic T lymphocytes is induced, accompanied by tumor protection. On this basis, a clinical trial was performed in which 25 patients with advanced metastatic carcinoma of breast, colon, stomach, or rectum received mannan-MUC1 in increasing doses. After 4 to 8 injections, large amounts of IgG1 anti-MUC1 antibodies were produced in 13 out of 25 patients (with antibody titers by ELISA of 1/320-1/20,480). Most of the antibodies reacted to the epitopes STAPPAHG and PAPGSTAP. In addition, T cell proliferation was found in 4 out of 15 patients, and CTL responses were seen in 2 out of 10 patients. Mannan-MUC1 can immunize patients, particularly for antibody formation, and to a lesser extent, cellular responses. It remains to be seen whether such responses have antitumor activity.

Mannan-MUC1–Pulsed Dendritic Cell Immunotherapy: A Phase I Trial in Patients with Adenocarcinoma

Purpose: Tumor antigen-loaded dendritic cells show promise for cancer immunotherapy. This phase I study evaluated immunization with autologous dendritic cells pulsed with mannan-MUC1 fusion protein (MFP) to treat patients with advanced malignancy. Experimental Design: Eligible patients had adenocarcinoma expressing MUC1, were of performance status 0 to 1, with no autoimmune disease. Patients underwent leukapheresis to generate dendritic cells by culture ex vivo with granulocyte macrophage colony-stimulating factor and interleukin 4 for 5 days. Dendritic cells were then pulsed overnight with MFP and harvested for reinjection. Patients underwent three cycles of leukapheresis and reinjection at monthly intervals. Patients with clinical benefit were able to continue with dendritic cell-MFP immunotherapy. Results: Ten patients with a range of tumor types were enrolled, with median age of 60 years (range, 33-70 years); eight patients were of performance status 0 and two of performance status 1. Dendritic cell-MFP therapy led to strong T-cell IFNγ Elispot responses to the vaccine and delayed-type hypersensitivity responses at injection sites in nine patients who completed treatments. Immune responses were sustained at 1 year in monitored patients. Antibody responses were seen in three patients only and were of low titer. Side effects were grade 1 only. Two patients with clearly progressive disease (ovarian and renal carcinoma) at entry were stable after initial therapy and went on to further leukapheresis and dendritic cell-MFP immunotherapy. These two patients have now each completed over 3 years of treatment. Conclusions: Immunization produced T-cell responses in all patients with evidence of tumor stabilization in 2 of the 10 advanced cancer patients treated. These data support further clinical evaluation of this dendritic cell-MFP immunotherapy.

Humans lack iGb3 due to the absence of functional iGb3-synthase: implications for NKT cell development and transplantation.

The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galα(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galα(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.

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.

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

We have recently demonstrated that both antibodies to Galα(1,3)Gal, and the Galα(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-Galα(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-Galα(1,3)Gal antibodies and IB4 lectin with a Galα(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 Galα(1,3)Gal expressed after transfection of COS cells (Galα(1,3)Gal-, Muc1-) with cDNA encoding the pig α,3galactosyltransferase; and (3) that the IB4 lectin and anti-Galα(1,3)Gal antibodies could react with mucin 1 found on the surface of human breast cancer cells. Thus natural occurring anti-Galα(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 Galα(1,3)Gal; such expression is an artefact, the reaction is due to the phenomenon described herein, i.e. that anti-Galα(1,3)Gal antibodies react with mucin peptides. Abbreviations: HPLC, high performance liquid phase chromatography; HRP, horse radish peroxidase; mAb, monoclonal antibody; NHS, normal human serum; PBS, phosphate buffered saline; VNTR, variable number of tandem repeats

Biochemical studies of pig xenoantigens detected by naturally occurring human antibodies and the galactoseα(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.

Recognition of an octapeptide sequence by multiple Galα(1,3)Gal-binding proteins

Abstract: Strategies are now being designed to overcome the hyperacute rejection of vascularized pig xenografts by human natural anti‐Galα(1,3)Gal antibodies. We now demonstrate that a synthetic octapeptide, Gal pep 1 (DAHWESWL), isolated from a peptide epitope library using the α‐galactosyl specific lectin IB4, “mimics” the carbohydrate epitope Galα(1,3)Gal. In vitro studies demonstrated that the binding of the IB4 lectin or human natural antibodies to pig cells could be specifically blocked by Gal pep 1, in several different serological assays: a) the hemagglutnation of pig erythrocytes; b) cytofluorographic analysis of pig lymphocytes and endothelial cells; c) cytotoxicity of human serum against pig endothelial cells; d) an ELISA assay. The relative affinity of the peptide for the IB4 lectin was similar to that of α‐galactosyl sugars, although it was bound at a lower affinity by human antibodies. The implications of these observations to xenotransplantation are that peptides could theoretically be used to block hyperacute rejection.

Description of a mouse monoclonal anti-HLA-B27 antibody HLA-ABC-m3

The production and characterization of a new anti-HLA-B27 monoclonal antibody HLA-ABC-m3 is described. This cytotoxic IgG2a antibody binds protein A and is able to precipitate cell surface molecules of 43,000 and 12,000 daltons corresponding to the HLA heavy chain and beta 2-microglobulin. Population testing revealed that the HLA-ABC-m3 antibody reacted with the peripheral blood lymphocytes of 47/47 individuals conventionally typed as HLA-B27+ and with 5/105 HLA-B27- individuals. These five extra reactions were with individuals expressing the cross-reactive HLA-B7 alloantigen, although the affinity of the monoclonal antibody for B27 heterozygous individuals (approx 10(9) M-1) was tenfold greater than with B7 individuals (approx 10(8) M-1). In addition, HLA-ABC-m3 reactivity segregated with HLA-B27 in two families. This monoclonal antibody should be of value in the investigation of the role of HLA-B27 in disease.

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.

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.