Induction of immune tolerance to a transplantation carbohydrate antigen by gene therapy with autologous lymphocytes transduced with adenovirus containing the corresponding glycosyltransferase gene.

Induction of tolerance to transplantation carbohydrate antigens is of clinical significance in recipients of ABO-incompatible allografts, or of xenografts. The experimental animal model used for studying such tolerance was that of alpha1,3galactosyltransferase (alpha1,3GT) knockout (KO) mice, which lacks the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) and which can produce the anti-Gal antibody against it. In contrast, wild-type (WT) mice synthesize the alpha-gal epitope and are immunotolerant to it. KO lymphocytes transduced in vitro with adenovirus containing the alpha1,3GT gene (AdalphaGT) express alpha-gal epitopes. Administration of such lymphocytes into KO mice resulted in tolerization of naïve and memory anti-Gal B cells. Mice tolerized by AdalphaGT transduced lymphocytes failed to produce anti-Gal following immunizations with pig kidney membranes (PKM) expressing multiple alpha-gal epitopes. This tolerance was perpetuated by transplanted syngeneic WT mouse hearts expressing alpha-gal epitopes. Transplanted WT hearts survived in the tolerized KO mice for at least 100 days, despite repeated PKM immunizations. Control mice receiving lymphocytes transduced with adenovirus lacking the alpha1,3GT gene were not tolerized, but produced anti-Gal and rejected transplanted WT hearts. This study suggests that autologous lymphocytes transduced with adenovirus containing A or B transferase genes may induce a similar tolerance to blood group antigens in humans.

The α-Gal epitope (Galα1-3Galß1-4GlcNAc-R) in xenotransplantation.

Many patients with failing organs (e.g., heart, liver or kidneys), do not receive the needed organ because of an insufficient number of organ donors. Pig xenografts have been considered as an alternative source of organs for transplantation. The major obstacle currently known to prevent pig to human xenotransplantation is the interaction between the human natural anti-Gal antibody and the α-gal epitope (Galα1-3Galß1-4GlcNAc-R), abundantly expressed on pig cells. This short review describes the characteristics of anti-Gal and of the alpha-gal epitope, their role in inducing xenograft rejection and some experimental approaches for preventing this rejection.

Preparation of autologous leukemia and lymphoma vaccines expressing alpha-gal epitopes.

This study describes a novel method for increasing the immunogenicity of autologous tumor vaccines in leukemia and lymphoma patients by exploiting the natural anti-Gal antibody for in situ targeting of the vaccinating cells to antigen-presenting cells (APCs). Incubation of leukemia or lymphoma cells with neuraminidase and recombinant alpha 1,3-galactosyltransferase results in the synthesis of many alpha-gal epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) on their cell membranes. Vaccination with such processed tumor cells results in the binding of the natural anti-Gal immunoglobulin G (IgG) antibody to these epitopes and opsonization of these cells for effective phagocytosis by APCs, such as dendritic cells and macrophages. These APCs may transport the vaccine to adjacent draining lymph nodes for subsequent effective processing and presentation of tumor-associated antigens (TAA) peptides to activate TAA-specific helper and cytotoxic T cells. Once the TAA-specific cytotoxic T cells are activated, they can leave the lymph node, circulate in the body, and seek metastatic cells expressing TAA to destroy them. Alternatively, activated helper T cells may provide the help required for B cells to produce antibodies to TAA on the leukemia or lymphoma cells. Because every patient receives his or her own TAA within the vaccinating cells, such vaccines are customized for the patient. These autologous tumor vaccines may be used as an adjuvant treatment that complements currently used treatment regimens by providing the immune system with an additional opportunity to be exposed effectively to autologous TAA.

Synthesis of alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) on human tumor cells by recombinant alpha1,3galactosyltransferase produced in Pichia pastoris.

This study describes the processing of human tumor cells or cell membranes to express alpha-gal epitopes (Galalpha1-3Gal-beta1-4GlcNAc-R) by the use of New World monkey (marmoset) recombinant alpha1,3galactosyltransferase (ralpha1,3GT), produced in the yeast Pichia pastoris. Such tumor cells and membranes may serve, in cancer patients, as autologous tumor vaccines that are targeted in vivo to antigen-presenting cells by the anti-Gal antibody. This ralpha1,3GT lacks transmembrane and cytoplasmic domains, ensuring its solubility without detergent. It is effectively produced in P. pastoris under constitutive expression of the P(GAP) promoter and is secreted into the culture medium in a soluble, truncated form fused to a (His)(6) tag. This tag enables the simple affinity purification of ralpha1,3GT on a nickel-Sepharose column and elution with imidazole. The purified enzyme appears in SDS-PAGE as two bands with the size of 40 and 41 kDa and displays the same acceptor specificity as the mammalian native enzyme. ralpha1,3GT is very effective in synthesizing alpha-gal epitopes on membrane-bound carbohydrate chains and displays a specific activity of 1.2 nM membrane bound alpha-gal epitopes/min/mg. Incubation of very large amounts of human acute myeloid leukemia cells (1 x 10(9 )cells) with neuraminidase, ralpha1,3GT, and UDP-Gal resulted in the synthesis of approximately 6 x 10(6 )alpha-gal epitopes per cell. Effective synthesis of alpha-gal epitopes could be achieved also with as much as 2 g cell membranes prepared from the tumor of a patient with ovarian carcinoma. These data imply that ralpha1,3GT produced in P. pastoris is suitable for the synthesis of alpha-gal epitopes on bulk amounts of tumor cells or cell membranes required for the preparation of autologous tumor vaccines.

On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis.

This review focuses on the recent advances in investigations of the role of cell surface carbohydrates in tumor metastasis. It also summarizes the results of extensive studies of endogenous lectins, their structure, carbohydrate specificity and biological functions with the major emphasis on the significance of lectin-cell surface carbohydrate interactions in a metastatic process. Numerous data demonstrate that malignant transformation is associated with various and complex alterations in the glycosylation process. Some of these changes might provide a selective advantage for tumor cells during their progression to more invasive and metastatic phenotype. Cell glycosylation depends on the expression and function of various glycosyltransferases and glycosidases. Recently, transfection of genes encoding various glysosyltransferases gene in sense and antisense orientation helped to bring direct evidence that changes in cell surface carbohydrates are important for the metastatic behavior of tumor cells. Cell surface carbohydrates affect tumor cell interactions with normal cells or with the extracellular matrix during metastatic spread and growth. These interactions can be mediated via tumor cell carbohydrates and their binding proteins known as endogenous lectins. The family of the discovered endogenous lectins is rapidly expanding. The number of C-type lectins has reached 50 and at least 10 galectins have been identified. The biological significance of the endogenous lectins and their possible role in tumor growth and metastasis formation has started to unravel. Some lectins recognize the 'foreign' patterns of cell surface carbohydrates expressed by microorganisms and tumor cells, and play a role in innate and adaptive immunity. It was shown that lectins affect tumor cell survival, adhesion to the endothelium or extracellular matrix, as well as tumor vascularization and other processes that are crucial for metastatic spread and growth.

Genes coding evolutionary novel anti-carbohydrate antibodies: studies on anti-Gal production in alpha 1,3galactosyltransferase knock out mice.

This study analyzes the gene repertoire coding for antibodies to an evolutionary novel immunogenic carbohydrate antigen in mice. The alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R) is an autoantigen, abundantly expressed in wild type mice, but absent in alpha 1,3galactosyltransferase knock-out (KO) mice, where it can induce the production of the anti-Gal antibody. Hybridoma clones secreting anti-Gal were isolated from different mice and their immunoglobulin genes were analyzed. All anti-Gal clones were found to be encoded by the heavy chain gene VH22.1 and light chain gene VK5.1. Moreover, one 'forbidden' anti-Gal clone, produced in a wild type mouse, was also encoded by VH 22.1 and VK 5.1. The genes coding for the different anti-Gal clones were found to contain somatic mutations and different CDR3 domains. These data imply that a highly restricted gene usage combined with junctional diversity and somatic mutations can generate new antibodies that have not been produced in the course of the evolution of a species.

Differential immune responses to alpha-gal epitopes on xenografts and allografts: implications for accommodation in xenotransplantation.

Xenograft recipients produce large amounts of high-affinity anti-Gal IgG in response to Galalpha1-3Galbeta1- 4GlcNAc-R (alpha-gal) epitopes on the graft. In contrast, ABO-mismatched allograft recipients undergo "accommodation," a state of very weak immune response to ABO antigens. These differences in anti-carbohydrate immune response were studied in alpha1,3galactosyltransferase knock-out mice. Pig kidney membranes administered to these mice elicited extensive production of anti-Gal IgG, whereas allogeneic kidney membranes expressing alpha-gal epitopes elicited only a weak anti-Gal IgM response. Anti-Gal IgG response to xenograft membranes depended on helper T cell activation and was inhibited by anti-CD40L antibody. These T cells were activated by xenopeptides and not by alpha-gal epitopes. Moreover, allogeneic cell membranes manipulated to express xenoproteins also induced anti-Gal IgG response. Xenoglycoproteins with alpha-gal epitopes are processed by anti-Gal B cells. Xenopeptides presented by these cells activate a large repertoire of helper T cells required for the differentiation of anti-Gal B cells into cells secreting anti-Gal IgG. Alloglycoproteins with alpha- gal epitopes have very few immunogenic peptides and fail to activate helper T cells. Similarly, ineffective helper T-cell activation prevents a strong immune response to blood group antigens in ABO-mismatched allograft recipients, thus enabling the development of accommodation.

Differential expression of alpha-GAL epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) on pig and mouse organs.

BACKGROUND: Expression of the alpha-gal epitope in mice can be completely eliminated by disruption of the alpha1,3 galactosyltransferase gene. As an initial step for assessing the feasibility of this approach in the pig, it was of interest to compare the expression of alpha-gal epitopes in pig and mouse organs. METHODS: Membranes from pig and mouse organ homogenates were analyzed for alpha-gal epitope expression by Western blots, enzyme-linked immunosorbent assay (ELISA), immunostaining of tissues, and ELISA inhibition assay. RESULTS: Immunostaining of Western blots with human anti-Gal detected alpha-gal epitopes on glycoproteins from pig organs but not on glycoproteins from the corresponding mouse organs. ELISA with membrane homogenates and immunostaining of tissue sections demonstrated a much higher binding of human anti-Gal to alpha-gal epitopes on pig membranes than on mouse membranes. ELISA inhibition assay with monoclonal anti-Gal indicated that alpha-gal epitope expression in pig organs is up to 500-fold higher than in mouse organs. CONCLUSION: Expression of alpha-gal epitopes in pig organs is many fold higher than in mouse organs. The abundance of these epitopes in pigs raises the question of whether pigs can properly develop without expression of alpha-gal epitopes.

alpha-galactosyl epitopes on glycoproteins of porcine renal extracellular matrix.

BACKGROUND: The pig is the donor animal of choice for human xenotransplantation. In the most relevant pig-to-baboon model, pig organs transplanted into baboons are hyperacutely rejected by natural xenoantibodies, which mainly bind to alpha-galactosyl (alphaGal) epitopes expressed at the surface of endothelial cells. Recent advances in controlling hyperacute rejection have led to improved survival of these xenografts, and it is now important to identify alphaGal binding sites in other cells and tissues that may be subject to immunologic attack. To this end, we have studied whether alphaGal antibodies bind to glycated proteins of the extracellular matrix in the kidney and other organs most likely to be used for human xenotransplantation. METHODS: High-titer anti-alphaGal antibodies, similar to human natural xenoantibodies, were prepared in baboons, and their reactivity with components of pig extracellular matrix was tested by serology and immunohistology. RESULTS: The antibodies recognized epitopes of immobilized murine, bovine or porcine thyroglobulin, laminin, heparan sulfate proteoglycans, and fibronectin. In sections of pig tissue, the antibodies bound to endothelial and certain epithelial cells, as shown in previous studies, and also to mesenchymal cells, basement membranes, and extracellular matrices, in which they colocalized with matrix glycoproteins, especially laminin and heparan sulfate proteoglycans. CONCLUSIONS: These results suggest that when pig xenografts can be made to survive for prolonged periods, the reactivity of alphaGal antibody with matrix molecules can induce basement membrane and matrix lesions similar to those induced in laboratory animals by antilaminin and antiheparan sulfate proteoglycans antibodies.

Alpha-galactosyl antibody redistributes alpha-galactosyl at the surface of pig blood and endothelial cells.

The interaction of antibodies with cell surface antigens may induce redistribution of immune complexes, followed by antigen depletion, with increased resistance to injurious effect of antibody and complement (antigenic modulation). Human natural antibodies to Gal alpha 1,3Gal beta 1,4GlcNAc-R (alpha Gal) epitopes expressed at the surface of pig cells are a major obstacle to xenotransplantation. Recent studies have shown that these antibodies do not modulate alpha Gal, but the morphological consequences of the antigen-antibody interaction are unknown. Pig blood and endothelial cells, were exposed to baboon alpha-Gal antibodies, and studied by immunofluorescence and phase contrast microscopy, flow cytometry, and inhibition enzyme-linked immunosorbent assay. In cells studied at 4 degrees C or fixed, alpha Gal was diffusely expressed at the surface. After cross-linking at 37 degrees C, antigenic modulation did not occur, but granular redistribution of alpha Gal immune complexes was seen in all cell types. In other systems a similar redistribution is known to induce perturbation of the plasma membrane/cytoskeletal structure with changes in adhesive properties, gene regulation, and T cell activation, which could be important if pig xenografts will be made to survive for prolonged periods.

Interaction of baboon anti-alpha-galactosyl antibody with pig tissues.

As barriers to xenotransplantation are surmounted, such as suppression of hyperacute rejection allowing improved graft survival, it becomes important to define longer-term host-xenograft interactions. To this end we have prepared in baboons high titer anti-alpha-Galactosyl (alphaGal) and anti-porcine aortic endothelial cell antibodies, similar to human natural xenoantibodies and reactive with epitopes of thyroglobulin, laminin, and heparan sulfate proteoglycans. When injected into pigs with a protocol similar to that used in the rat to show the nephritogenic potential of heterologous anti-laminin and anti-heparan sulfate proteoglycan antibodies, baboon immunoglobulins bound first to renal vascular endothelium, and later to interstitial cells, especially fibroblasts and macrophages, and to antigens in basement membranes and extracellular matrix, where they colocalized with laminin- and heparan sulfate proteoglycan-antibodies, and with bound Griffonia simplicifolia B4. A similar binding was observed in other organs. The pigs did not develop an acute complement-dependent inflammation, but rather chronic lesions of the basement membranes and the extracellular matrix. Incubation of renal fibroblasts with baboon anti-alpha-Galactosyl antibodies resulted in increased synthesis of transforming growth factor-beta and collagen, suggesting a possible basis for the fibrotic response. The results demonstrate that in this experimental model a consequence of alphaGal antibody interaction with porcine tissues, is immunoreactivity with alphaGal on matrix molecules and interstitial cells, priming mechanisms leading to fibrosis resembling that in chronic allograft rejection. The possibility that similar lesions may develop in long-surviving pig xenografts is discussed.

Increased immunogenicity of tumor vaccines complexed with anti-Gal: studies in knockout mice for alpha1,3galactosyltransferase.

A major prerequisite for the success of tumor vaccines is their effective uptake by antigen-presenting cells (APCs) and transport of these APCs to the draining lymph nodes, where the processed and presented tumor-associated antigens activate tumor-specific naive T cells. We previously suggested that the immunogenicity of autologus tumor vaccines in humans may be augmented by engineering vaccinating tumor cell membranes to express alpha-galactosyl (alpha-gal) epitopes (i.e., Galalpha1,3Galbeta1,4GlcNAc-R). Subsequent in situ binding of natural anti-Gal IgG molecules to these epitopes would result in the formation of immune complexes that target tumor vaccines for uptake by APCs, via the interaction of the Fc portion of anti-Gal with Fcgamma receptors on APCs. This hypothesis was tested in a unique experimental animal model of knockout mice for alpha1,3galactosyltransferase (alpha1,3GT) and the mouse melanoma B16-BL6 (referred to here as BL6). Like humans, these mice lack alpha-gal epitopes and produce anti-GaL BL6 melanoma cels are highly tumorigenic, and like human tumor cells, they lack alpha-gal epitopes. Expression of alpha-gal epitopes on these melanoma cells was achieved by stable transfection with alpha,3GT cDNA. The transfected melanoma cells (termed BL6alphaGT) express approximately 2 x 10(6) alpha-gal epitopes per cell and readily form immune complexes with anti-Gal. Vaccination of the mice with 2 x 10(6) irradiated melanoma cells that express alpha-gal epitopes, followed by challenge with 0.5 x 10(6) live parental melanoma cells, resulted in protection for at least 2 months (i.e, no tumor growth) in one-third of the mice, whereas all mice immunized with irradiated parental melanoma cells developed tumors 21-26 days post-challenge. The proportion of protected mice doubled when the mice were immunized twice with irradiated melanoma cells expressing alpha-gal epitopes and challenged with 0.2 x 10(6) live BL6 cells. Histological studies on the developing tumors in challenged mice that were immunized with melanoma cells expressing alpha-gal epitopes demonstrated extensive infiltration of T lymphocytes and macrophages, whereas no mononuclear cell infiltrates were observed in tumors of mice immunized with parental tumor cells. Overall, these studies imply that immunization of alpha1,3GT knockout mice with BL6 melanoma cells that express alpha-gal epitopes elicits, in a proportion of the population, protective immune response against the same tumor lacking such epitopes. These studies further suggest that similar immunization of cancer patients with autologous tumor vaccines that are engineered to express alpha-gal epitopes may increase the immune response to autologous tumor-associated antigens and, thus, may elicit immune-mediated destruction of metastatic cells expressing these antigens.