Use of the mannan receptor to selectively target vaccine antigens for processing and antigen presentation through the MHC class I and class II pathways.

Extensive studies have shown that synthetic and recombinant vaccines developed against hemoparasites have not been as effective as whole parasites or crude membrane fractions in eliciting protective immunity. A possible reason is that synthetic vaccines are not being presented in a form that induces the appropriate immune response. We have developed a bovine model system to evaluate the ability of adjuvant compounds to induce an immune response to peptide antigens dominated by a cytokine profile with a Type 1 (cell-mediated) or Type 2 (humoral) bias. In the initial testing of this system, we found that mRNA expression of certain cytokines (interleukin [IL]-1beta, IL-6, IL-12, IL-15, GM-CSF, iNOS, and tumor necrosis factor [TNF]-alpha) is enhanced when monocyte-derived macrophages are stimulated with peptide antigen conjugated with mannan under oxidizing conditions compared to peptide conjugated with reduced mannan. The data suggest this model will be useful in identifying adjuvant systems that selectively modulate the cytokine profile of antigen presenting cells at the time of antigen presentation and the consequent downstream maturation of naive T cells to effector cells with Type 1 or Type 2 cytokine bias.

Getting into the groove: unusual features of peptide binding to MHC class I molecules and implications in vaccine design.

The major histocompatibility complex presents antigenic peptides on the surface of antigen presenting cells to T cell receptors. Recognition of peptide-MHC by T cells initiates a cascade of signals in T cells which maintains a T cell dependent immune response. An understanding of the how peptides bind to MHC class I molecules is an important prerequisite in the design of vaccines. Herein, we will discuss, with special emphasis on MUC1, unusual features of MUC1 peptide binding to MHC class I, obtained from vaccine studies including a MUC1 peptide mimic and the crystal structures of low and high affinity peptides lacking canonical anchor motifs in complex with H-2Kb.

Anti-mucin monoclonal antibodies.

Mucins are of major interest in cell biology, not only are they highly over-expressed in many adenocarcinomas (up to 40-fold increase), but also have important physiological function, and probably more to be determined (1-3). There is much information available on mucins - doubtless because of their unusual structure being heavily glycosylated, but also containing a repeat region rich in the amino acids serine, threonine and proline. This repeat region confers high immunogenicity of the mucins, and as a result, many antibodies (Abs) have been made to mucins of different species (4). Furthermore, the production of Abs led to the cloning of the cDNAs and armed with these reagents (antibodies, cDNA and genomic structures), advances in the knowledge of the structure and function of mucins has been rapid, together with the development of transgenic and gene knockout animals for biological studies (1-9). Here we describe monoclonal antibodies (Mabs) made to the different mucins, including Mucins 1-4, concentrating on human Mucin 1 (MUC1), to variants of MUC1, to regions outside the VNTR of MUC1, mouse Mucin1 (muc1), unusual features and cross reactions of anti-MUC1 Mabs and Abs made by patients in clinical trials. We will especially describe the Mabs produced in our laboratory.

Tolerance in baboon kidney transplantation with total lymphoid irradiation (TLI) and anti-CD3/CD4-idarubicin conjugates.

BACKGROUND: We previously reported the induction of transplantation tolerance by a modified wide field method of pretransplant total lymphoid irradiation (TLI), cumulative dose 800 cGy, given as 80 or 100 cGy fractions twice/week, in approximately one-third of chacma baboons receiving liver or kidney allografts (1-4) and in vervet monkeys receiving baboon kidney xenografts (5). In this study, the effects of the administration of brief courses of anti-CD3 or CD4-Idarubicin conjugates on the frequency and predictability of tolerance induction by TLI were examined. METHODS: TLI was administered pretransplant in doses of 800, 600, or 400 cGy. The conjugates were administered either after transplantation in doses of 0.25 mg/kg body weight, 3 times/week for 2 weeks, or as a single dose of 1.0 mg/kg body weight 24 hr before transplantation. RESULTS: Operational tolerance, defined as normal graft function >1 year after transplantation, was obtained in one-half of six baboons receiving the single dose of 1 mg/kg of Idarubicin conjugate pretransplant after 800 cGy of TLI and also in one of four baboons treated with 400 cGy of TLI and a single dose of anti-CD3 conjugate before transplantation. By contrast, administration of the conjugated antibodies 3 times/week for 2 weeks after transplantation prevented tolerance induction in all animals, providing further evidence for the involvement of active mechanisms, capable of inhibition by immunosuppressive agents, in tolerance induction with TLI, and of relevance to our reported clinical experience with TLI (6). CONCLUSIONS: These promising findings invite further studies with a larger number of animals and additional brief regimens of irradiation and antibody dosages and specificities.

Characteristics of immunoglobulin gene usage of the xenoantibody binding to gal-alpha(1,3)gal target antigens in the gal knockout mouse.

BACKGROUND: Natural antibodies that react with galactose-alpha(1,3)galactose [galalpha(1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibodies to environmental microbes that express the galalpha(1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-galalpha(1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to this carbohydrate epitope. METHODS: Six hybridomas that produce monoclonal antibodies (mAbs) to galalpha(1,3)gal were generated in Gal o/o mice. The mAbs were tested to characterize the binding activity with flow cytometry using pig aortic endothelial cells and ELISA with galalpha(1,3)gal carbohydrates. The VH and VK genes of these hybridomas were cloned, sequenced, and analyzed. RESULTS: The mAbs showed distinct patterns of antibody binding to galalpha(1,3)gal antigens. The VH genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configuration. Four of six clones used closely related progeny of the same VH germline gene (VH441). Comparison of the mouse gene VH441 to the human gene IGHV3-11, a gene that encodes antibody activity to galalpha(1,3)gal in humans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (complimentary determining regions 1 and 2) of their immunoglobulin VH genes. CONCLUSIONS: These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to galalpha(1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of antibody/antigen interactions associated with the humoral response to pig xenografts in humans.

Mannan mucin-1 peptide immunization: influence of cyclophosphamide and the route of injection.

The mucin MUC1 is greatly increased in breast cancer and is a potential target for immunotherapy. In mice, MUCI conjugated to oxidized mannan (MUC1-mannan fusion protein [M-FP]) targets the mannose receptor and induces a high frequency of cytotoxic T lymphocytes and anti-tumor responses. On this basis, three phase I trials were performed in patients with adenocarcinoma to evaluate the toxicity and the immunologic responses to mannan MUCI. Forty-one patients with metastatic or locally advanced carcinoma of the breast (trial 1), colon (trial 2), and various adenocarcinomas (trial 3) received increasing doses of M-FP (1 to 300 microg). The immunizations were given at weekly intervals (weeks 1 to 3) and repeated in weeks 7 to 9. Cyclophosphamide (to increase cellular immunity) was given on weeks 1 and 4. M-FP was given intramuscularly in trial 1 and intraperitoneally in trial 2. No toxic effects occurred, and delayed-type hypersensitivity responses were present only as a microscopic lymphocytic infiltration. Overall, approximately 60% of the patients had high-titer MUC1 immunoglobulin G1 antibody responses, with the intraperitoneal route yielding approximately 10-fold higher responses. Cellular responses (proliferation, cytotoxic T cells, or CD8 T cells secreting tumor necrosis factor-alpha alphand interferon-gamma in response to MUC1 stimulation in vitro) were found in 28% of the patients, which was similar to that seen without cyclophosphamide. In most patients, disease progressed, but in five it remained stable. In addition, there were no objective responses. M-FP is not toxic and induces immune responses that were amplified by the intraperitoneal route of immunization. Cyclophosphamide was of no benefit.

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for golgi localization.

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of alpha1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the alpha1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of alpha1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser(5). Mutagenesis of Ser(5) to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of alpha1,2-fucosyltransferase.

Breast cancer in mice: effect of murine MUC-1 immunization on tumor incidence in C3H/HeOuj mice.

Mucin-1 (MUC-1), which is overexpressed in more than 90% of human breast cancers, is a potential target for immunotherapy. To develop a mouse model appropriate for the immunotherapy of human cancer, mouse mucin-1 (muc-1) fusion protein, containing ten tandem repeats, was made and used to immunize C3H/HeOuj mice, which supposedly have a high incidence of breast cancer. C3H/HeOuj mice were injected eight times with 5 microg oxidized mannan muc-1-glutathione-S-transferase (MMFP) with or without cyclophosphamide, which is used to increase cellular immunity. At 80 age weeks, only 12.1% (4 of 33) mice of the untreated C3H/HeOuj mice had mammary tumors. The reason for the low incidence of breast cancer in these mice is not known, but all the mammary tumors were MUC-1+ breast adenocarcinomas and were transplantable to C3H/HeOuj mice. The incidence was 11.4% (4 of 35) in mice injected with MMFP: 38.2% (13 of 34) in mice given cyclophosphamide; and 14.3% (2 of 14) in mice treated with glutathione-S-transferase. That is, cyclophosphamide increased the incidence of mammary tumors, and metastases were found in only these mice. Fewer tumors (6 of 34 or 17.6% compared with 13 of 34 or 38.2% with cyclophosphamide only) occurred in the group immunized with MMFP and cyclophosphamide. Mice immunized with MMFP had high levels of muc-1 antibodies and cellular immune responses (the frequency of the precursor of the cytotoxic Tlymphocyte cell was 1 of 40,000 to 1 of 100,000), which were not found in control groups. The occurrence of muc-1 immunity, particularly the presence of large amounts of anti-mucin-1 antibodies, had no effect on tumor incidence. Thus, the immunization with murine muc-1 reduced the tumor incidence in only cyclophosphamide-treated mice and led to strong muc-1 antibody production and to cellular responses. These findings have implications for human tumor immunotherapy in which strong antibody and weak cellular responses are to be expected and, indeed, have been found.

Genetic engineering for xenotransplantation.

Xenotransplantation is being pursued vigorously to solve the shortage of allogeneic donor organs. Experimental studies of the major xenoantigen (Gal) and of complement regulation enable model xenografts to survive hyperacute rejection. When the Gal antigen is removed or reduced and complement activation is controlled, the major barriers to xenograft survival include unregulated coagulation within the graft and cellular reactions involving macrophages, neutrophils, natural killer (NK) cells, and T lymphocytes. Unlike allografts, where specific immune responses are the sole barrier to graft survival, molecular differences between xenograft and recipient that affect normal receptor-ligand interactions (largely active at the cell surface and which may not be immunogenic), are also involved in xenograft failure. Transgenic strategies provide the best options to control antigen expression, complement activation, and coagulation. Although the Gal antigen can be eliminated by gene knockout in mice, that outcome has only become a possibility in pigs due to the recent cloning of pigs after nuclear transfer. Instead, the use of transgenic glycosyl transferase enzymes and glycosidases, which generate alternative terminal carbohydrates on glycolipids and glycoproteins, has reduced antigen in experimental models. As a result, novel strategies are being tested to seek the most effective solution. Transgenic pigs expressing human complement-regulating proteins (DAF/CD55, MCP/CD46, or CD59) have revealed that disordered regulation of the coagulation system requires attention. There will undoubtedly be other molecular incompatibilities that need addressing. Xenotransplantation, however, offers hope as a therapeutic solution and provides much information about homeostatic mechanisms.

Role of the mannose receptor in the immune response.

The mannose receptor (MR) recognizes a range of carbohydrates present on the surface and cell walls of micro-organisms. The MR is primarily expressed on macrophages and dendritic cells and is involved in MR-mediated endocytosis and phagocytosis. In addition, the MR plays a key role in host defense and provides a link between innate and adaptive immunity. Herein, we will review the role of the MR in innate host defense as well as the recent evidence for its role in the adaptive response, for both humoral and cellular immune responses.

Development of a fecal occult blood test using a monoclonal antibody to haptoglobin.

Monoclonal antibodies (mAbs) were produced to human haptoglobin by immunising with fecal extracts from patients with colon cancer. An enzyme-linked immunosorbent assay was developed with one of the mAbs (FE14.1), and its ability to diagnose colorectal carcinoma evaluated. Patients with colorectal cancer were positive (43/46 = 93.5%) compared to normal individuals (4/211 = 1.9%). The assay has a specificity 93.5% and sensitivity 98.1% and has several advantages over current fecal occult blood tests. The test is potentially useful for bowel cancer diagnosis and to quantitate the level of haptoglobin in other body fluids such as urine and in effusions.

Prolonged survival of baboon renal allografts using idarubicin-conjugated anti-CD4 monoclonal antibodies.

Tolerance to organ allografts in rodents and pigs can be easily achieved. However, tolerance induction in a large primate model has been more elusive. In this study, we have used an anti-CD4, murine monoclonal antibody as a carrier for the cytotoxic drug idarubicin (IDA) to delete or inactivate alloreactive T-cells responding to a renal allograft in a baboon transplant model. Fourteen Chacma baboons weighing between 15-25 kg received heterotopic renal allografts. Recipient and donor pairs were selected on the basis of ABO compatibility. Seven animals were given no immunosuppression and served as the control group. The remaining 7 animals received anti-CD4 IDA. The first 2 animals in this group received 2 mg IVI intraoperatively and three doses at 48-h intervals thereafter. The last 5 animals received a larger dose of 1 mg/kg, starting 24 h preoperatively and again on postoperative days 2 and 5. The untreated animals promptly rejected their allografts with a mean survival of 10 days. The survival of the 2 animals treated with 2 mg anti-CD4 IDA was 7 days each. However, the animals treated with 1 mg/kg anti-CD4 IDA survived 7, 18, 20, 40 and > 40 days. Peritransplant administration of anti-CD4 IDA prolonged renal allograft survival in a large primate model. This unique immunoconjugate has the potential of tolerance induction.

Selection and characterization of MUC1-specific CD8+ T cells from MUC1 transgenic mice immunized with dendritic-carcinoma fusion cells.

Mice transgenic for the human MUC1 carcinoma-associated antigen (MUC1.Tg) are tolerant to immunization with MUC1 antigen. Recent studies, however, have demonstrated that immunization of MUC1.Tg mice with fusions of MUC1-positive tumour and dendritic cells (FC/MUC1) reverses MUC1 unresponsiveness and results in rejection of established MUC1-positive pulmonary metastases. Here we demonstrate that lymph node cells from MUC1.Tg mice immunized with the FC/MUC1 fusion cells proliferate in response to MUC1 antigen by a mechanism dependent on the function of CD4, major histocompatibility complex (MHC) class II, B7-1, B7-2, CD28, CD40 and CD40 ligand. The findings demonstrate that stimulation of lymph node cells with MUC1 results in selection of MUC1-specific CD8+ T cells. We show that the CD8+ T cells exhibit MUC1-specific cytotoxic T lymphocyte (CTL) activity by recognition of MUC1 peptides presented in the context of MHC class I molecules Kb and Db. The MUC1-specific CD8+ T cells also exhibit antitumour activity against MUC1-positive metastases, but with no apparent reactivity against normal tissues. These results indicate that immunization of MUC1.Tg mice with FC/MUC1 reverses immunological unresponsiveness to MUC1 by presentation of MUC1 peptides in the presence of costimulatory signals and generates MHC-restricted MUC1-specific CD8+ T cells.

Fucosyl transferase (H) transgenic heart transplants to Gal-/- mice.

BACKGROUND: We have previously described the rejection of Gal+ mouse hearts by mice lacking Gala(1,3)Gal (Gal-/-) and demonstrated this to be a model of xenogeneic hyperacute rejection (HAR) which would occur in pig-to-human/primate xenotransplantation, where Gal+ antibody (Ab) and complement (C') mediate HAR. To reduce the amount of Gal present we used fucosyl transferase (H) as a transgene, H transferase competes for the same substrate as Gal transferase and reduces Gal expression by >90%. METHODS: Gal-/- mice received a heart graft from C57BL/6 Gal+ or H transgenic mice and additional Gal Ab and C' provided; HAR was monitored by direct observation for up to 90 min, or by palpation thereafter. When grafts were rejected they were examined macro- and microscopically. RESULTS: H transgenic mice were used as donors to Gal-/- mice; it was found that: 1) C57BL/6 or H transgenic hearts were not rejected by Gal-/- recipients within 90 min in the absence of additional Gal Ab. 2) If additional Gal Ab and C' were provided as fresh normal human serum (NHS), Gal+ (C57BL/6) grafts were rejected by Gal-/- mice in approximately 34 min, whereas H transgenic hearts mostly lasted up to 17 hr, but were then rejected. The histological appearances showed features of both Arthus and Shwartzmann phenomena. 3) Mice hyperimmunized with Gal with anti-Gal titers of >1:20,000, rejected Gal+ grafts in 31 min; the survival was prolonged to 75 min with the H transgenic hearts. CONCLUSION: The presence of the H transgene in donor hearts transplanted to naive Gal-/- mice delays the onset of HAR, but rejection ultimately occurs; if the mice are hyperimmune earlier rejection occurs. The expression of the H transgene alone is insufficient to avoid HAR in the Gal-/- mouse model; the presence of other transgenes and techniques will be required to give an appropriate increase in survival of pig-to-human/primate grafts.

Immunotherapy with mannan-MUC1 and IL-12 in MUC1 transgenic mice.

Mice immunised with oxidised mannan-MUC1 fusion protein (M-FP) develop MHC restricted CD8(+) cytotoxic T cells. We now demonstrate that in MUC1/HLA-A2 transgenic mice, IL-12 gives enhanced CTL, CTLp and tumor protection. CTLp in MUC1 transgenic mice with M-FP were 1/55,000, and with IL-12, this increased to 1/19,000, with improved tumor protection. Thus, IL-12 is important for effective CTL responses to MUC1 in transgenic mice.

Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway.

Antigens such as MUC1 coupled to oxidized mannan lead to rapid and efficient MHC class I presentation to CD8+ cells and a preferential T1 response; after reduction there is class II presentation and a T2 immune response. We now show that the selective advantage of the oxidized mannan-MUC1 is due to the presence of aldehydes and not Schiff bases, and that oxidized mannan-MUC1 binds to the mannose and not scavenger receptors and is internalized and presented by MHC class I molecules 1,000 times more efficiently than when reduced. After internalization there is rapid access to the class I pathway via endosomes but not lysosomes, proteasomal processing and transport to the endoplasmic reticulum, Golgi apparatus and cell surface. Aldehydes cause rapid entry into the class I pathway, and can therefore direct the subsequent immune response.

A role for IL-5 in the induction of cytotoxic T lymphocytes in vivo.

IL-5 is generally regarded as a Th2 cytokine involved in eosinophil maturation and function and in B cell growth and antibody production, but without any well-established effects on T cells. Early reports suggested that IL-5 could stimulate the production of cytotoxic T lymphocytes (CTL) in vitro, but no evidence has been obtained to date for such a role in studies with IL-5-deficient (IL-5-/-) mice. Here we demonstrate that when oxidized mannan MUC1 fusion protein (M-FP) is used as an antigen in mice, IL-5 is required for the optimal generation of the CTL response. IL-5 was as effective as IL-2 for the induction of CTL from spleen cells in vitro and both CD4+ and CD8+ T cells from M-FP-immunized animals could be shown to secrete IL-5 in culture. In IL-5-/- mice, CTLp frequency was greatly diminished resulting in the inability to reject MUC1- tumors. Clearly, IL-5 is produced by functional T cells, especially the Tc1 type, after M-FP immunization and is required for an optimal CTL response to this antigen.

Ex vivo targeting of the macrophage mannose receptor generates anti-tumor CTL responses.

MUC1 is highly expressed in adenocarcinomas and is a possible target for immunotherapy. In mice, oxidized mannan linked to MUC1 (M-FP), given in vivo, induces potent MHC-restricted CTL and tumor protection. Because of the resistance of cancer patients to immunization, ex vivo immunization of macrophage/dendritic cells was examined using oxidized mannan MUC1 to target the mannose receptor and the MHC Class I antigen presentation pathway. Here, we show that murine mannose receptor (MR) bearing macrophages derived from peritoneal exudate cells (PEC) and cultured ex vivo with M-FP can, after adoptive transfer, efficiently present MUC1 to T cells, leading to the generation of high frequency of CTL and protection from tumor challenge. Mice immunized once with syngeneic PEC pulsed with M-FP elicit a similar CTLp frequency to that obtained with three in vivo immunizations. Targeting the MR is crucial to obtain high frequency CTL, and without oxidation the CTLp frequency was low. GM-CSF is important, as GM-CSF o/o mice gave reduced responses, a deficiency corrected by in vivo GM-CSF. In addition, the treatment of macrophages ex vivo with GM-CSF gave enhanced responses and treating mice with GM-CSF prior to M-FP immunizations also enhanced cellular responses. M-FP targets the MR and ensures rapid passage of peptides to Class I molecules, and can also directly stimulate in vitro IL-12 production by macrophages. While many studies are now focussing on dendritic cells, in this study the cells involved were adherent F4/80+ 33D1- macrophages. The findings could be of benefit for the immunization of patients with cancer.