anti-alphaGal Antibodies Research Articles

BINDING OF HUMAN NATURAL ANTIBODIES TO NON??Gal XENOANTIGENS ON PORCINE ERYTHROCYTES1

Xenotransplantation is considered one of possible solutions for the serious shortage of organs and cells in transplantation. Although the alphaGal epitope (Gal alpha1,3Gal beta1,4GlcNAc-R) has been identified as being a major xenoantigen responsible for hyperacute rejection, the removal of anti-alphaGal antibody alone from human serum is insufficient to circumvent antibody-mediated immune responses. We report here the characterization of xenoreactive human natural antibodies against antigens without the alphaGal epitope (nonalphaGal xenoantigens) on porcine erythrocytes using flow cytometry and the evidence for their involvement in complement-mediated hemolysis. Furthermore, a novel protein of 45-kDa has been isolated from the porcine erythrocyte membrane as a major protein antigen recognized by human anti-nonalphaGal. The data presented here will add to our knowledge of xenoantigens on porcine red cells and be important for developing strategies to produce modified red cells immunologically compatible to humans.

Naturally acquired anti-αGal antibodies in a murine allograft model similar to delayed xenograft rejection

Antibodies directed against galactose-alpha1,3-galactose (alphaGal) are believed to play an important role in the pathogenesis of delayed xenograft rejection (DXR). This study was designed to determine whether alpha1,3-galactosyltransferase-deficient (Gal KO) mice can naturally acquire a sufficient anti-alphaGal titre to cause the delayed type rejection of alphaGal-expressing hearts. Gal KO mice of various ages were assessed for anti-alphaGal antibody levels. alphaGal-expressing hearts were transplanted heterotopically into these mice and monitored daily. Rejecting and surviving hearts were evaluated histologically. In Gal KO mice greater than 6-month-old, 64% had an anti-alphaGal antibody titre above the background level. When wild-type alphaGal-expressing hearts were transplanted into this group, 45% of grafts rejected within 5 to 13 days. Histological examination of the rejected hearts displayed marked tissue damage and an inflammatory infiltrate of predominantly macrophage/monocytes. Surviving grafts showed preserved morphology. Like humans, Gal KO mice naturally develop anti-alphaGal antibodies with age. The titre in these mice was sufficient to cause a "delayed-type" rejection of a significant proportion of alphaGal-expressing cardiac grafts. This model thus provides an opportunity to investigate the role of naturally acquired anti-alphaGal antibodies in the pathogenesis of DXR.

Different responses of human anti-HLA and anti-alphagal antibody to long-term intravenous immunoglobulin therapy.

Concentrated human immunoglobulin (IVIG) has been administered intravenously in the treatment of autoimmune disorders and to reduce anti-HLA antibodies in highly sensitized patients awaiting organ transplantation. It has also been shown, in experimental animals, to prevent the hyperacute rejection of discordant xenografts, possibly by anticomplement activity. The aim of the present study was to assess the effect of IVIG therapy on both acquired anti-HLA antibodies and natural antigalactose alpha1-3 galactose (alphaGal) antibodies in five patients awaiting heart transplantation. Five patients placed on mechanical circulatory support who had developed high HLA panel-reactive antibodies (PRA) or in whom the percentage of PRA was increasing rapidly were treated weekly with 500 mg/kg IVIG, which contained 1% of anti-alphaGal IgG. Levels of PRA, anti-alphaGal IgG and IgM, and serum cytotoxicity to pig cells were measured before, during, and after therapy. PRA percentages in the five patients were initially 85%, 53%, 23%, 19% and 19% (mean 39%). Mean PRA fell by 66% after 3 months of therapy (to a mean PRA of 14%), and by 96% after 6 months therapy (to a mean PRA of 2%). Anti-alphaGal antibody levels and serum cytotoxicity to pig aortic endothelial cells did not change significantly. These results confirm the effectiveness of IVIG therapy in reducing PRA in HLA highly sensitized patients. It is likely that IVIG does not contain the relevant anti-HLA antibody, resulting in an accelerated catabolism of native alloantibodies. However, as IVIG contains a normal level of anti-alphaGal IgG, catabolism of anti-alphaGal IgG is not modified, as it is being continuously replaced. To achieve a decrease in the anti-alphaGal IgG level it would be necessary to use IVIG depleted of this antibody.

Serum cytotoxicity to pig cells and anti-alphaGal antibody level and specificity in humans and baboons.

Removal and/or "neutralization" of anti-Gal alpha1-3Gal (alphaGal) antibodies can prevent or delay the hyperacute rejection of pig organs transplanted into primates. To determine variations in (1) cytotoxicity to pig kidney (PK15) cells, (2) anti-alphaGal antibody level, and (3) specificity in adult human (n=46) and baboon (n=38) sera. Cytotoxicity to PK15 cells was determined by adding rabbit complement to heat-inactivated serum, using a two-color fluorescent dye to distinguish live and dead cells. Anti-alphaGal antibody level was determined by ELISA using alphaGal trisaccharide type 2-BSA glycoconjugate as antigen target. Specificity determined by ELISA using four different alphaGal-BSA glycoconjugates: (disaccharide, trisaccharides type 2 and 6, and pentasaccharide). Cytotoxicity of human AB sera varied from 30-100% PK15 relative cell damage (%RCD), although that of baboon sera of all blood groups varied from 35-100% RCD. In human AB sera, anti-alphaGal antibody level (at a dilution of 1:80) varied from undetectable to 0.75 (OD at 405 nm), although in baboon sera of all blood groups, anti-alphaGal antibody level varied from undetectable to >2.0. There was no correlation between anti-alphaGal antibody level and serum cytotoxicity in either species. Specificity varied among individuals in both human and baboon sera. These studies have demonstrated (1) considerable variation in cytotoxicity and anti-alphaGal antibody level in human and baboon sera, but a lack of correlation between these two parameters; (2) considerable variation in the specificity of anti-alphaGal antibodies; (3) blood group B human and baboon sera have lower levels of anti-alphaGal antibodies; (4) no relation between blood group and specificity of anti-alphaGal antibodies. Although there are minor differences in the parameters measured, baboons would appear to be suitable surrogates for humans in the pig-to-primate xenograft model.

Lack of variation in alphaGal expression on lymphocytes in miniature swine of different genotypes.

Gal(alpha)1-3Gal epitopes (alphaGal) have been demonstrated to be present on tissues of all pig breeds tested to-date and are the major target for human anti-(alpha)galactosyl (alphaGal) antibodies. We investigated members of an MHC-inbred miniature swine herd to assess whether there was an association between genotype and expression of alphaGal. Identification of a low expressor genotype would potentially enable selective breeding of pigs that might prove beneficial as donors in clinical xenotransplantation. we measured alphaGal expression on various pig cells by use of fluorescent-activated cell sorter (FACS) using (i) purified human anti-alphaGal antibody and (ii) the isolectin GS-I-B4. Initial studies were on porcine peripheral blood mononuclear cells (PBMCs) and subsequent studies on lymphocytes, platelets, and T cell subsets (CD4+ and CD8+ cells). there was considerable day-to-day variation in alphaGal expression on PBMCs from the same pig. When only lymphocytes were examined, there was a high degree of reproducibility, and no significant difference in alphaGal expression was detected between representative pairs of animlas of three different genotypes. Purified anti-alphaGal antibody bound to different sites on the alphaGal epitope than did Griffonia (Bandeiraea) simplicifolia I-B4 (GS-I-B4). Lectin binding was significantly reduced in the absence of divalent cations. When CD4+ and CD8+ T cells were examined for alphaGal expression, two distinct populations of each type of cell were observed, with larger cells expressing a higher level of alphaGal. although the number of pigs of different genotypes studied was small, on the basis of this limited study, pigs of a low alphaGal expressor genotype that could be selectively bred for use in clinical xenotransplantation were not identified.

Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption.

Xenotransplantation would provide a solution to the current shortage of organs for transplantation. Our group has been successful in inducing tolerance in mice and monkey models of allogeneic transplantation. The present study attempts to extend the same tolerance-inducing regimen to a pig-to-baboon organ transplantation model. Nine baboons underwent a conditioning regimen (consisting of nonmyeloablative or myeloablative whole body and thymic irradiation, splenectomy, antithymocyte globulin, pharmacologic immunosuppression and porcine bone marrow transplantation [BMTx]), which has previously been demonstrated to induce donor-specific allograft tolerance in monkeys. In addition, immunoadsorption of anti-alphaGal antibody (Ab) was performed. Four of the nine baboons received pig kidney transplants (KTx), and one also underwent repeat transplantation with an SLA-matched kidney. Two received heterotopic pig heart transplants (HTx). Three baboons underwent conditioning without organ transplantation for long-term studies of natural Ab kinetics. In the three baboons that received the conditioning regimen without an organ transplant, immunoadsorption reduced Ab by approximately 90%, but recovery of Ab to pretreatment level or higher occurred within 7 days. In contrast, the level of Ab remained low after organ transplant. No Ab to pig antigens other than alphaGal was detected in any baboon before or after BMTx, KTx, or HTx. No graft succumbed to hyperacute rejection. KTx function began to deteriorate within 3-6 days, with oliguria and hematuria progressing to anuria, and the kidneys were excised after 3, 6, 9, 11, and 14 days, respectively. One HTx ceased functioning at 8 days; the second baboon died with a contracting HTx at 15 days. Features of coagulopathy and thrombocytopenia developed in all six transplanted baboons (high D-dimer, prolonged prothrombin time and partial thromboplastin time, and falling fibrinogen) resulting in serious bleeding complications in two baboons, one of which died on day 9. Donor organs showed progressive acute humoral rejection with deposits of IgM, IgG, and complement; a focal mononuclear cellular infiltrate was also observed. The ureter was the earliest structure of the KTx affected by rejection, with progression to necrosis. This conditioning regimen prevented hyperacute rejection but was ineffective in preventing the return of Ab, which was associated with the development of acute humoral rejection with features of coagulopathy. No baboon developed anti-pig Ab other than alphaGal Ab. Further modifications of the protocol directed toward suppression of production of Ab are required to successfully induce tolerance to pig organs in baboons.

Pharmacologic immunosuppressive therapy and extracorporeal immunoadsorption in the suppression of anti-alphaGal antibody in the baboon.

The aim of this study was to deplete baboons of anti-(alpha)galactosyl (alphaGal] antibody and attempt to maintain depletion by pharmacologic immunosuppressive therapy (PI). In 12 experiments, involving nine baboons, repeated extracorporeal immunoadsorption (EIA) was carried out by plasma perfusion through immunoaffinity columns of synthetic alphaGal trisaccharide type 6. Five of the baboons were immunologically naive and four had undergone various procedures at least 6 months previously. All, however, had recovered lymphohematopoietic function and (with one exception) had levels of anti-alphaGal antibody within the normal range. Eleven protocols included continuous i.v. cyclosporine (to maintain whole blood levels of approximately 1,600 ng/ml). In addition, in ten protocols, the baboon received one or more of the following drugs: cyclophosphamide (1-20 mg/kg/day), mycophenolate mofetil (70-700 mg/ kg/day), brequinar sodium (1-12 mg/kg/day), prednisolone (1 mg/kg/day), melphalan (0.15-0.6 mg/kg/day), methylprednisolone (125 mg/day x3), and antilymphocyte globulin (ATG) (50 mg/kg/day x3). EIA was carried out on 1-9 occasions in each study and was temporarily successful in removing all antibody. When no PI was administered, antibody returned close to pre-EIA levels within 48 hr. Cyclosporine alone delayed the rate of antibody return only slightly. While EIA was continuing on a daily or alternate day schedule, antibody levels (both IgM and IgG) were maintained at 20-45% of pre-EIA levels. Once EIA was discontinued but PI maintained, IgM rose to 40-90% and IgG to 30-60% of pre-EIA levels. In vitro testing demonstrated significant cytotoxicity to pig cells at these antibody levels. We conclude that i) EIA utilizing columns of alphaGal trisaccharide is successful in temporarily depleting baboons of anti-alphaGal antibody, but ii) none of the PI regimens tested suppressed antibody production to levels which would be expected to prevent antibody-mediated rejection of pig xenografts. Additional strategies will therefore be required if xenotransplantation is to become a clinical reality.

Anti-Gal antibody-mediated allograft rejection in alpha1,3-galactosyltransferase gene knockout mice: a model of delayed xenograft rejection.

The key role of anti-galactose alpha1,3-galactose (anti-alphaGal) xenoantibodies in initiating hyperacute xenograft rejection has been clearly demonstrated using a variety of in vitro and in vivo approaches. However, the role of anti-alphaGal antibodies in mediating post-hyperacute rejection mechanisms, such as antibody-dependent cellular cytoxicity, remains to be determined, primarily because of the lack of a small animal model with which to study this phenomena. Hearts from wild-type mice were transplanted heterotopically into alpha1,3-galactosyltransferase knockout (Gal KO) mice, which like humans develop antibodies to the disaccharide galactose alpha1,3-galactose (Gal). At the time of rejection, hearts were examined histologically to determine the mechanism of rejection. Hearts from wild-type mice transplanted into high-titer anti-alphaGal recipients were rejected in 8-13 days. Histological examination demonstrated a cellular infiltrate consisting of macrophages (80-90%), natural killer cells (5-10%), and T cells (1-5%). In contrast, wild-type hearts transplanted into low anti-Gal titer recipients demonstrated prolonged (>90 day) survival. However, a significant proportion (30-40%) of these underwent a minor rejection episode between 10 and 13 days, but then recovered ("accommodated"). The results of this study suggest that the Gal KO mouse is a useful small animal vascularized allograft model, in which the role of anti-alphaGal antibody in graft rejection can be studied in isolation from other rejection mechanisms. The titer of anti-alphaGal antibody was found to be the critical determinant of rejection. The histopathological features of rejection in this model are very similar to other models of delayed xenograft rejection, in both the timing and composition of the cellular infiltrate. The Gal KO mouse therefore provides a new rodent model, which will aid in the identification of the distinct components involved in the pathogenesis of delayed xenograft rejection.

ANTI-Gal??1-3Gal ANTIBODY RESPONSE TO PORCINE BONE MARROW IN UNMODIFIED BABOONS AND BABOONS CONDITIONED FOR TOLERANCE INDUCTION1

Mixed lymphohematopoietic chimerism can provide an effective means of inducing longterm immunological tolerance and has been documented in a monkey allograft model. A conditioning regimen including nonmyeloablative or myeloablative irradiation and splenectomy has been used to induce chimerism in a pig-to-primate transplantation model. Since the presence of anti-Gal(alpha)1-3Gal (alphaGal) natural antibodies leads to the hyperacute rejection of pig organs transplanted into primates, extracorporeal immunoaffinity adsorption (EIA) of anti-alphaGal antibodies is also included in the regimen. The effect of the tolerance induction protocol on the anti-alphaGal antibody response has been assessed. Anti-alphaGal antibody was measured after the EIA of plasma through an alphaGal immunoaffinity column in baseline studies involving two unmodified baboons, one splenectomized baboon, and one baboon that received a challenge with porcine bone marrow (BM), and in three groups of baboons (n=2 in each group) that received different conditioning regimens for tolerance induction. Group 1 received a nonmyeloablative conditioning regimen without porcine BM transplantation. Group 2 received nonmyeloablative conditioning with pig BM transplantation and pig cytokine therapy. Group 3 received myeloablative conditioning, an autologous BM transplant (with BM depleted of CD2+ or CD2+/CD20+ cells), and pig BM transplantation. In the baseline studies, a single EIA of anti-alphaGal antibodies in an unmodified animal initially depleted anti-alphaGal antibody, followed by a mild rebound. Nonmyeloablative conditioning (group 1) in the absence of pig cell exposure reduced the rate of anti-alphaGal antibody return. Pig BM cells markedly stimulated anti-alphaGal antibody production in an unmodified baboon (alphaGal IgM and IgG levels increased 40- and 220-fold, respectively). This response was significantly reduced (to an only 2- to 5.5-fold increase of IgM and IgG) in baboons undergoing nonmyeloablative conditioning (group 2). A myeloablative conditioning regimen (group 3) prevented the antibody response to pig BM, with the reduction in response being greater in the baboon that received autologous BM depleted of both CD2+ and CD20+ cells. No new antibody directed against pig non-aGal antigens was detected in any baboon during the 1 month follow-up period. (i) EIA of anti-alphaGal antibody in unmodified baboons results in a transient depletion followed by a mild rebound of antibody; (ii) exposure to pig BM cells results in a substantial increase in anti-alphaGal antibody production; (iii) a nonmyeloablative conditioning regimen reduces the rate of antibody return and (iv) markedly reduces the response to pig BM cells; (v) the anti-alphaGal response is completely suppressed by a myeloablative regimen if CD2+ and CD20+ cells are eliminated from the autologous BM inoculum. Furthermore, (vi) challenge with pig BM cells appears to stimulate only an anti-alphaGal antibody response without the development of other (non-alphaGal) anti-pig antibodies. We conclude that regimens used for T-cell tolerance induction can be beneficial in reducing the anti-alphaGal antibody response to porcine BM.

Intravenous infusion of Galalpha1-3Gal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts.

Hyperacute rejection (HAR) of pig-to-primate discordant xenografts is caused by the deposition of preexisting natural antibodies that recognize Galalpha1-3Gal (alphaGal)-terminating oligosaccharides on glycoproteins and glycolipids, followed by complement-mediated lysis of the graft's endothelium. In vitro, these natural xenoantibodies can be blocked by alphaGal-containing oligosaccharides. We undertook in vivo pig-to-baboon cardiac xenotransplantation experiments to evaluate free oligosaccharides as inhibitors of HAR. Initial 15-min intravenous infusions of alphaGal-oligosaccharides into baboons were used to measure pharmacokinetic parameters, and to assess the extent of neutralization of anti-alphaGal antibody activity. AlphaGal trisaccharide (Galalpha1-3Galbeta1-4GlcNAc) or pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc ) was administered at 0.5 mmol/kg into baboons. Next, two baboons that received porcine heterotopic heart xenografts were continuously infused with alphaGal pentasaccharide for 4-5 hr, maintaining the serum oligosaccharide concentration in the millimolar range. Pharmacokinetic analysis indicated that the oligosaccharides were rapidly cleared from the blood, with a serum half-life of 50 min. In the period during which blood oligosaccharide concentration was above 1 mM, as determined by high-pressure liquid chromatography, the serum cytotoxic activity against porcine cells was completely abolished. HAR of the xenograft was inhibited during the infusions, although there was some histological and immunohistological evidence of antibody-mediated injury on biopsies taken at the end of this period. Intravenous alphaGal oligosaccharides, by inhibiting anti-alphaGal antibody activity, delay but do not abolish the onset of HAR.

Sensitization of rhabdo-, lenti-, and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation.

Vesicular stomatitis virus, human immunodeficiency virus type 2, and human foamy virus, which were produced by cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars, were found to be less stable in human serum than those from alphaGal-negative cells, indicating that galactosyl(alpha1-3)galactosylation sensitizes these viruses as well as mammalian type C oncoviruses (Rother et al., J. Exp. Med. 182:1345-1355, 1995; Takeuchi et al., Nature (London) 379:85-88, 1996) to complement killing via natural anti-alphaGal antibodies. Thus, virus killing mediated by anti-alphaGal antibodies may play a role as a barrier to animal-to-human infection of various enveloped viruses. Virus vectors for human in vivo gene therapy based on the viruses mentioned above should be produced from alphaGal-negative cells.

Relationship between ABO blood group and levels of Gal alpha,3Galactose-reactive human immunoglobulin G.

The terminal Gal alpha1,3Galactose (alphaGal) determinant is present on all porcine glycoproteins and glycolipids, but is not expressed by human cells. Consequently human sera contain anti-alphaGal natural antibodies. The human blood group B antigen [Gal alpha1,3(Fuc1,2)Galactose] is differentiated from the alphaGal epitope by the presence of a fucosyl group. To determine whether the expression of the B antigen has any effect on the level of alphaGal-reactive natural antibodies, equal numbers (n=12) of A, B, AB, and O serum samples were evaluated by ELISA and flow cytometry. A significant reduction in IgG alphaGal reactivity was observed with serum samples from B antigen-expressing donors (B, AB) relative to non-B antigen-expressing donors (A, O). These results are consistent with the possibility that anti-alphaGal antibodies in non-B antigen-expressing individuals include a subset that is reactive with the structurally related B antigen and that this subset is absent in B and AB individuals.

In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal(alpha)1-3Gal column.

The major role of anti-alphaGal antibodies in the hyperacute rejection of pig organs by humans and baboons has been clearly demonstrated. Spacered alpha-galactose disaccharide (Gal(alpha1)-3Gal) hapten was produced by chemical synthesis and covalently attached to a flexible, hydrophilic polymer (PAA), which in turn was covalently coupled to macroporous glass beads, forming an immunoadsorbent that is mechanically and chemically stable and can be sterilized. The extracorporeal immunoadsorption (EIA) of anti-alphaGal antibodies using this column has been investigated in vivo in 3 baboons. In Baboon 1 (which had hyperacutely rejected a pig heart transplant 4 months previously, was not splenectomized, and did not receive any pharmacologic immunosuppression) the levels of anti-alphaGal antibody and antipig IgM and IgG, as well as serum cytotoxicity, fell significantly after each of 3 EIAs but were not eliminated. Serum cytotoxicity, antipig immunoglobulin and anti-alphaGal antibody rose steeply within 24 hr of the final EIA, suggesting that the return of cytotoxicity was associated with anti-alphaGa1 antibody. In Baboons 2 and 3 (which were immunologically naive and splenectomized, and received triple drug immunosuppressive therapy) serum cytotoxicity was totally eliminated and anti-alphaGal antibody and antipig IgM and IgG levels were greatly reduced by courses of EIA. In Baboon 2, cytotoxicity and all antibody levels remained negligible for approximately one week after the final (fourth) daily EIA. In Baboon 3, cytotoxicity and antibody levels were maintained low by intermittent EIA (over a period of 13 days) for almost 3 weeks, although antipig IgM began to rebound 4 days after the final EIA. We conclude that, in an immunosuppressed, splenectomized baboon, repeated EIA using a specific alphaGal disaccharide column will reduce antipig and anti-alphaGal antibody levels and serum cytotoxicity significantly for several days. This reduction in cytotoxicity will almost certainly be sufficient to delay the hyperacute rejection of a transplanted pig organ, but further studies are required to investigate whether it will be sufficient to allow accommodation to develop.

Glycans derived from porcine stomach mucin are effective inhibitors of natural anti-alpha-galactosyl antibodies in vitro and after intravenous infusion in baboons.

The current shortage of donor organs has stimulated investigation of pig-to-human xenotransplantation as a practical alternative to allotransplantation. However, a major obstacle to this xenotransplantation is hyperacute rejection, which is believed to be initiated by the interaction of natural anti-alpha-galactosyl (alphaGal) antibodies with alphaGal epitopes on pig vascular endothelium. Previously, we reported that neutral oligosaccharides derived from porcine stomach mucin (PSM) are effective inhibitors of human anti-alphaGal IgG in vitro. We now report that O-glycans derived from PSM by beta-elimination (PSMO) reduce the cytotoxicity of both baboon and human sera to pig kidney (PK15) cells in vitro. Crude PSM had some inhibitory effect in vitro, but PSMO were more than 100 times more potent. Moreover, 1 microg/ml of beta-eliminated PSMO that bound to an immunoaffinity column of anti-alphaGal antibodies were four times more efficient than total PSMO in protecting PK15 cells from the cytotoxic effect of baboon or human sera. Blood recovered from baboons after intravenous infusion of PMSO also showed significant protection of PK15 cells. We conclude that PSMO eluted from an anti-alphaGal immunoaffinity column demonstrate potent inhibitory effects against baboon and human serum cytotoxicity to PK15 cells in vitro and when administered intravenously. PSM may provide a cheap and readily available source of glycans that will be of therapeutic value in the prevention of hyperacute rejection.

Monoclonal antiidiotypic antibodies neutralize cytotoxic effects of anti-alphaGal antibodies.

The aims of this study were to produce and characterize mouse monoclonal antiidiotypic antibodies (AIAs) that specifically bind human antipig (anti-alphaGal) antibodies and to select those AIAs that neutralize the cytotoxicity of human or baboon serum to pig (PK15) cells. Mice were immunized with human anti-pig antibodies, and hybridomas were produced using conventional techniques. From a total of 480 clones, 11 produced AIAs that bound with high affinity to human anti-alpha Gal IgG and F(ab')2 fragments, and individually reduced serum cytotoxicity to pig cells by 40 - 90%. Seven of the AIAs also bound to human peripheral B lymphocytes (that express the same idiotypes as the antibodies produce). Several combinations of two or three AIAs provided 100% protection of PK15 cells. Selected AIAs injected intravenously into baboons reduced the cytotoxicity of subsequently drawn sera by 50--80% for >48 hr. The cytotoxicity studies also indicated that there are at least two dominant idiotypes expressed in the human anti-alphaGal population. We conclude that AIAs against anti-alphaGal antibodies could be successfully used in 1) preoperative characterization of a recipient's anti-alphaGal profile, (2) prevention of hyperacute rejection of a pig organ, and (3) specific immunosuppression through elimination of anti-alphaGal-producing B lymphocytes.

The reducing end of alpha Gal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera.

Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl-alpha-Gal gave weak inhibition, Gal alpha 1-3Gal substantially inhibited the reaction (400muM), and Gal alpha 1-3Gal beta 1-4GLcNAc was ten times more efficient (30 muM). The modification from alpha to beta anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-alphaGal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Gal alpha 1-3Gal beta 1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.