Removal of anti-Galα1,3Gal xenoantibodies with an injectable polymer

Preformed and elicited Ab’s against the Galα1,3Gal terminating carbohydrate chains (αGal Ab’s) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. αGal Ab’s are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of αGal Ab’s might be achieved in vivo by systemic administration of nonimmunogenic polyvalent αGal structures with high avidity for αGal Ab’s. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for αGal binding by αGal IgM (IC₅₀, 43 nM) and IgG (IC₅₀, 28 nM) in vitro. Injections of GAS914 in cynomolgus monkeys, at the dose of 1 mg/kg, resulted in the immediate decrease of more than 90% of circulating αGal Ab’s and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating αGal Ab’s and cytotoxicity over several months. Studies with [¹⁴C]GAS914 in rhesus monkeys and Gal^–/– mice indicate that GAS914 binds to circulating αGal Ab’s and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment αGal Ab titers never exceeded pretreatment levels and no sensitization to either αGal or the polylysine backbone has been observed. Furthermore there was no apparent acute or chronic toxicity associated with GAS914 treatment in primates. We conclude that GAS914 may be used therapeutically for the specific removal of αGal Ab’s.

The most immediate hurdle in pig-to-primate transplantation is the presence of naturally occurring Ab’s against the Linear B trisaccharide (Galα1,3Galβ1,4GlcNAc) that is expressed on pig but not on the tissues of man and Old World monkeys (ref. 1, for review see ref. 2). Galα1,3Gal terminating carbohydrate chains (αGal) Ab’s destroy pig organs via activation of complement, macrophage, and NK cell recruitment and endothelial cell activation (2, 3). When transplanted into primates, transgenic pig organs expressing human complement inhibitors resist hyperacute rejection (4, 5) but succumb to acute vascular rejection principally caused by induced αGal Ab’s (6). It is now accepted that anti-complement strategies are not sufficient to protect the xenograft from acute vascular rejection and that removal of the αGal barrier will be necessary to achieve extended xenograft survival. Pending the generation of αGal-deficient pigs (7), elimination of αGal Ab’s is a promising method for overcoming this barrier.

In α1,3-galactosyltransferase–deficient (Gal^–/–) mice, αGal Ab’s are produced by a B-1b–like splenic cell population and not by conventional B2-derived plasma cells (8). The analogous population in human and nonhuman primates is not responsive to conventional immunosuppression, cyclophosphamide, or treatment with B cell–depleting Ab’s (9, 10), and their phenotype remains elusive. In the absence of effective pharmacological control of Ab synthesis by these cells, extracorporeal immunoabsorption has been examined as a means of αGal Ab depletion (11). Extracorporeal immunoabsorption reduces circulating αGal Ab’s before transplant, but the procedure is associated with significant morbidity in humans and mortality in nonhuman primates (6). A soluble inhibitor that can bind and clear αGal Ab’s systemically might compete better with the xenograft for binding of Ab’s and prove more effective in protecting it from humoral damage. Such an inhibitor should bind to αGal Ab’s at least as tightly as xenogeneic tissue antigens; it should be nonimmunogenic; and the resulting immune complexes should be rapidly cleared from the circulation to avoid immune-complex deposition disease.

αGal Ab’s have high avidity for multivalent antigens, glycoproteins, and glycolipids on cell surface membranes, but low affinity for single oligosaccharides (12). Inhibition with monovalent compounds is therefore inefficient. This is particularly relevant for αGal IgMs that have only millimolar affinity for monomeric αGal oligosaccharides, but account for most of the anti-pig cytotoxicity in primate serum. We have prepared a series of linear polymers designed to provide flexible backbones for the multivalent presentation of Linear B monomers and maximize avidity to αGal Ab (IgG and IgM). Avidity, measured on a per monomer basis, was increased by the length of the backbone, and was also influenced by the loading of the carbohydrate monomer and choice of spacer (13). Maximal increase in avidity relative to the monomer was achieved with GAS914, a linear polylysine backbone with an average length of 1,000 lysines and with 25% of side chains conjugated to Linear B trisaccharide. In vitro, GAS914 is a potent inhibitor of the binding of both αGal IgG and IgM as well as the complement-dependent hemolysis of pig erythrocytes by human serum (Figure 1).

Figure 1

Structure of GAS 914 and inhibition of αGal IgG, IgM, and HAPAb. (a) Chemical structure of random copolymer with: n, average degree of polymerization; x, fraction of glycosylated monomer; 1 – x, fraction of thioglycerol-capped monomer. Arrows indicate the positions labeled with ¹⁴C for pharmacokinetic studies. (b) Atomic force microscopy of GAS914 shows a chain-like molecule with an average chain diameter of 4.0 ± 0.2 nm and average chain length of 79 ± 24 nm. (c) Inhibition curves of αGal IgG (IC₅₀ monomer = 54 μM, IC₅₀ GAS914 = 0.028 μM). (d) Inhibition curves of αGal IgG (IC₅₀ monomer = 332 μM, IC₅₀ GAS914 = 0.043 μM. (e) Inhibition curves of HAPAb (IC₅₀ monomer = 400 μM, IC₅₀ GAS914 = 0.004 μM. Concentrations shown are on a per oligosaccharide basis as indicated in Methods. Curves are representative of more than seven experiments performed on different days.

In the present study, we show that GAS914 is also highly efficacious in vivo in eliminating circulating αGal Ab’s in primates without side effects and with minimal complement activation and no immune response against either the carbohydrate or the backbone. This mode of antigen-specific treatment promises to protect pig organs transplanted into primates.

In vitro inhibition of αGal Ab’s and HAPAb. We have previously shown that multivalent presentation of carbohydrates on polylysine backbones potentiates the affinity of carbohydrate receptors (16). By optimizing the degree of polymerization (average number of lysines per polymer) and the carbohydrate density, we obtained GAS914. This compound is a flexible linear polymer and shows a dramatic fold increase of 1 × 10³ to 1 × 10⁴ in avidity with both the IgG and IgM classes of αGal Ab’s compared with the monomer. This increased affinity is also reflected in a potent inhibition of HAPAb (Figure 1, c and e). The values reported in Figure 1 are on a per carbohydrate basis. That is, the concentrations are based on the equivalent weight of Linear B trisaccharide and not on the molecular weight of the polymer and thus directly relate to the avidity between Ab’s and the carbohydrate hapten. Polylysine backbone with no carbohydrate or with lactosamine attached inhibits neither αGal Ab’s nor anti-pig cytotoxicity up to concentrations of 1 mM (data not shown). To our knowledge, GAS914 is the most potent inhibitor of αGal Ab’s reported to date. Previously published polyacrylamide polymers conjugated with αGal moieties have reported in vitro affinity to αGal IgM that is similar to GAS914, but have lower affinity to αGal IgG, and no in vivo data have been reported for this class of compounds (17). However, recently reported polyethylene glycol αGal conjugates lead to good in vivo reduction of αGal IgM Ab’s at 50 mg/kg (18).

Removal of αGal Ab’s in vivo. Although GAS914 is an excellent in vitro inhibitor of αGal Ab’s, it was unclear whether it could be used safely in vivo due to potential risk factors of immunogenicity and immune complex formation. Indeed αGal-HSA was found to be a potent immunogen in cynomolgus monkeys (R.G. Warner, unpublished data). To avoid such problems, we initially injected smaller polylysine derivatives (average degree of polymerization = 250; see Figure 1a) into cynomolgus monkeys. Although the smaller polylysine conjugates are good in vitro inhibitors of αGal Ab’s, they are ineffective in vivo, resulting in the incomplete removal of circulating αGal Ab’s and HAPAb (data not shown).

In subsequent experiments, intravenous injections of 1 mg/kg GAS914 were administered to a group of four cynomolgus monkeys on days 0, 3, and 6. Ab and biochemical analyses were performed until day 28. GAS914 injections resulted in an immediate disappearance of circulating αGal Ab’s and HAPAb; these began to return slowly starting 12 hours after injection and reached approximately 30% of pretreatment values by day 3 after injection (Figure 2). Repeated injections with GAS914 further lowered the circulating αGal titers with equal efficiency. Circulating titers slowly rose after the third injection but by day 28 were still below pretreatment levels (Figure 2). A similar response was observed when the same group of animals was injected intravenously with 1 mg/kg on days 105, 108, and 111 after the first GAS914 injection to test for sensitization (Figure 2). Surprisingly, GAS914 injections did not elicit an immune response against αGal. In fact, both αGal and HAPAb titers showed a sustained decrease throughout and remained below pretreatment levels at the end of the observation period. Sera from this study were analyzed on ELISA plates coated with control thioglycerol-capped polylysine (Figure 1a, x = 0). No change in the signal against control polylysine was observed throughout the observation period, indicating that no anti-backbone Ab’s were elicited by GAS914 injections.

Figure 2

Cynomolgus monkeys (n = 4) received intravenous injections of 1 mg/kg GAS914 on days 0, 3, 6, 105, 108, and 111 (indicated by arrows). The animals were bled at the indicated times and serum was assayed for anti-αGal Ab’s and anti-pig hemolytic activity. IgM, IgG, and HAPAb titers shown are relative to human standard. Symbols represent titers of the same animal in all three panels.

In a control study, two cynomolgus monkeys received 1 mg/kg intravenously of a thioglycerol-capped polylysine (Figure 1a, x = 0) on days 0, 3, and 6 and were followed for 28 days. No change in the circulating levels of αGal Ab’s or HAPAb was observed over the treatment period, indicating that the polylysine backbone itself does not cause Ab depletion. Similar to the GAS914 study, no Ab’s against the backbone were detected by ELISA during the control study.

No clinically adverse effects were observed during treatment with GAS914 and during the subsequent observation period. Serum urea, creatinine, and blood phosphate remained normal throughout the observation period. Similarly, serum ALT and AST also remained within normal ranges, with the exception of a single animal that had mildly elevated baseline levels of ALT (50 mU/ml) and displayed consistently elevated ALT values (80 mU/ml) through-out treatment. All other biochemical markers were normal in this animal. Since clinical signs of im-mune complex–mediated glomerulonephritis might include proteinuria, urine was collected daily on days –1 to 28 and 105–130. Urinary volume and protein levels were always within normal range for all animals. Histological examination of kidney, spleen, and liver sections collected at necropsy (day 138) showed no significant pathological findings.

Levels of the anaphylatoxin C3a-desArg were followed during the second set of GAS914 injections (days 105–132) as a measure of systemic complement activation. In two of the four animals (the two with the highest levels of αGal IgM), small increases in C3a levels appeared immediately after the first GAS914 injection, but the levels returned to baseline within 12–24 hours. Subsequent injections were associated with much smaller increases in C3a levels. C3a production was also measured as a percentage of total C3 (measured after ex vivo complement activation with cobra venom factor). It was calculated that the highest activation lev-els represented less than 0.5% activation of the total C3 available.

Long-term αGal Ab suppression in baboons using GAS914. In order to achieve extended xenograft survival, it is anticipated that effective long-term treatment will be required after transplantation until either the αGal response of the recipient is neutralized or the transplanted organ accommodates to its new environment (6). To determine the ability of GAS914 to control αGal Ab levels chronically, we treated a group of three baboons with repeated GAS914 injections over 3 months. Animals received a single intravenous injection of GAS914 (5 mg/kg during the initial 3 weeks and 1 mg/kg thereafter), when HAPAb titers reached 15% of the human standard level. Circulating αGal Ab and HAPAb titers for a single representative animal over the complete observation period are shown in Figure 3. Injections of GAS914 in baboons showed an efficacy similar to that obtained in cynomolgus monkeys, leading to rapid elimination of circulating αGal Ab’s followed by a slow return. Initially, injections every 2–3 days were required to keep circulating HAPAb within the target level. After the initial few weeks of continuous treatment, less frequent injections (once a week) sufficed to maintain both αGal Ab and HAPAb levels below target levels. One month after treatment was discontinued, a reduction of αGal Ab’s and HAPAb persisted, with levels below 40% of the human standard (Figure 3).

Figure 3

Baboons (n = 3) received intravenous injections of GAS914 at the times indicated by arrows. Heavy arrows indicate injection of 5 mg/kg and light arrows indicate injection of 1 mg/kg. Anti-αGal IgM (circles), IgG (triangles), and HAPAb (squares) titers relative to human standard are shown (see Methods). Data from a single baboon are shown; the other two animals had a similar response.

In vivo half-life of GAS914. For pharmacokinetic studies, GAS914 was labeled with ¹⁴C (see Figure 1a) to allow detection of the backbone even if the carbohydrate is metabolized. The half-life of [¹⁴C]GAS914 was determined after a single intravenous injection in normal mice and rats (species with no circulating αGal Ab’s). In both species, radioactivity was eliminated in a biphasic manner. In the first phase, more than 95% of radioactivity was eliminated with extremely rapid kinetics from systemic circulation (half life of first elimination phase = 3.7 ± 0.2 min). From 30 minutes onward, a much slower multiexponential elimination was evident (half life of terminal elimination phase = 30 ± 7 h). Whole-body autoradioluminograms of rats at different times showed that radioactivity was quickly taken up by the liver and then excreted primarily through the kidneys. Analysis of blood samples at various times by PAGE suggests that GAS914 is metabolized by the liver and recycled into the circulation as two fragments of lower molecular weight. The GAS914 fragments are then taken up by the kidney where they are further metabolized and excreted in urine.

In view of the short circulating half-life of GAS914 in rodents with no αGal Ab’s, it was important to determine the pharmacokinetics of this compound in the presence of circulating αGal Ab’s. Immunized Gal^–/– mice with titers of circulating αGal Ab (both IgM and IgG) comparable to those of primates were injected intravenously with 1 mg/kg [¹⁴C]GAS914. The same short circulating half-life and essentially identical excretion kinetics were observed in these animals as in normal mice, suggesting that circulating αGal Ab’s do not substantially alter GAS914 metabolism. Whole-body autoradioluminograms (Figure 4) show that, as in the wild-type mice and rats, the liver is the primary target organ and the kidney is the secondary target organ. In strong contrast to the complete clearance of radioactivity from all tissues of normal mice and rats, significant levels of GAS914-associated radioactivity accumulated in the spleen and lymph nodes of immunized Gal^–/– mice and were also evident in the whole-body autoradioluminograms (Figure 4). Immunohistochemical analysis of tissues from the Gal^–/– mice showed that GAS914 is associated with B cells in the spleens of these animals.

Figure 4

Whole-body autoradioluminographs of Gal^–/– mice immunized with rabbit erythrocytes and injected with 1 mg/kg intravenous [¹⁴C]GAS914. The bright areas indicate GAS914 or its metabolites. Times indicated are after injection.

[¹⁴C]GAS914 injected intravenously in rhesus monkeys was effective in clearing αGal Ab’s and had a circulating half-life of 32 ± 12 minutes (Figure 5a), which is comparable to that observed in rodents. Circulating αGal IgM was undetectable by ELISA at 5 minutes after [¹⁴C]GAS914 injection (Figure 5a). Distribution and excretion of radioactivity in rhesus monkeys was the same as observed in Gal^–/– mice. At 72 hours after injection, radioactivity was essentially cleared from all tissues, with the exception of the spleen and lymph nodes, where small amounts of the labeled compound were still detected (Figure 5b). Immunohistochemistry of tissues from animals receiving GAS914 injections using an Ab recognizing the polylysine backbone confirmed the presence of GAS914 in spleen and lymph nodes but its absence from all other tissues examined, such as kidney, liver, lung, heart, gut, and skin. Double labeling with anti-GAS914 Ab and anti–T cell or anti–B cell Ab’s revealed that GAS914 immunoreactivity is associated with the B cell area in lymph nodes (Figure 6).

Figure 5

Three rhesus monkeys received a single intravenous injection of 1 mg/kg [¹⁴C]GAS914. Blood was collected at the indicated times. (a) Radioactivity (squares) in whole blood disappeared rapidly from circulation in a biphasic manner. The proportion of radioactivity in plasma versus whole blood was constant (90%), suggesting that GAS914 is not significantly taken up by blood cells (data not shown). Almost no anti-αGal IgM was detected in serum by ELISA after the first bleeding at 5 minutes after injection. There was a slow return of anti-αGal IgM (circles) starting approximately 12 hours after injection. (b) Tissue concentration of total radioactivity 72 hours after administration of 1 mg/kg [¹⁴C]GAS914.

Figure 6

Cynomolgus monkeys received daily intravenous GAS914 for 3 days (a and b) or 4 weeks (c and d) and were sacrificed 72 hours after the last injection. (a) Spleen of animal receiving 0.1 mg/kg GAS914 and (b) kidney of animal receiving 1 mg/kg GAS914. Both sections were stained with anti-GAS914 Ab (brown). Note the presence of GAS914 staining in the spleen and absence in the kidney. (c and d) Bronchial lymph node of animal receiving 1 mg/kg stained with anti-GAS914 Ab (red) and (c) anti–B cell Ab (brown) or (d) anti–T cell Ab (brown).

The high levels of αGal Ab’s in primates (19) are a formidable barrier for pig organ transplantation into these species. Our results demonstrate that GAS914 can lead to rapid and long-lasting antigen-specific removal of αGal Ab’s from circulation without activation of B cells. The GAS914 polymer has a rapid uptake and metabolism accompanied by very low levels of complement activation. The physical and pharmacokinetic properties of the GAS914 polymer are likely to be important contributors to the in vivo efficacy, low toxicity, and lack of immune response to this compound. Lack of complement activation may be influenced by the shape, structural features such as lack of lipophilicity and charge, and size of the polylysine derivative, or may result from the short circulating half-life. Lack of immunogenicity may be the result of low complement activation, short half-life, or unproductive engagement with B cell receptors.

In αGal knockout mice, production of elicited αGal Ab’s is T cell dependent (20). In this system, xenopeptides are highly efficient in eliciting T cell help, whereas allopeptides bearing αGal residues are ineffective (21). GAS914 my act as an allopeptide that fails to elicit T cell help. In the primate setting, elicited αGal Ab’s are also T cell dependent, as evidenced by the efficacy of anti-CD154 treatment to inhibit elicited Ab’s (22). However, production of αGal Ab’s from existing plasma cells is not inhibited by anti-CD154 treatment and is very rapid after immunoabsorption. GAS914 has a dual function since it not only fails to elicit new αGal Ab’s but also substantially limits new production of circulating αGal Ab’s.

Continuous infusions of oligosaccharide mono-mers at very high doses have been used to inhibit circulating αGal Ab’s in pig-to-primate xenotrans-plantation, but have provided only incomplete protection against vascular rejection (23, 24). In contrast to this approach, small dosages of GAS914 efficiently eliminate αGal Ab’s from circulation. An important advantage of GAS914 is that it is a well-defined polymer that can be synthesized in large amounts with restricted physicochemical variability, there-by offering a relevant therapeutic option for xenotransplantation.

In considering the development of antigen-specific inhibitors of xenoreactive Ab’s, the carbohydrate specificity is crucial. The great majority of naturally occurring primate anti-pig Ab’s bind to carbohydrate structures terminating with αGal (25, 26), but have variable specificities for di-, tri- or pentasaccharides and also for type II (Galα1,3Galβ1,4GlcNAc) versus type VI (Galα1,3Galβ1,4Glc) core structures (27). We synthesized GAS914 with the Linear B type II trisaccharide, and therefore it binds and eliminates αGal Ab’s with affinity for this carbohydrate. In numerous in vitro studies with human, cynomolgus monkey, and baboon sera, we find that GAS914 consistently eliminates more than 80% of natural anti-pig Ab’s. In humans, natural Ab’s against porcine antigens are mainly against αGal, and there is no evidence to suggest that new anti-carbohydrate specificities emerge in the immediate posttransplant period (28). We therefore expect that in animals where GAS914 achieves good pretransplant removal of circulating αGal Ab’s, it will continue to control these Ab’s effectively after transplant. This was recently demonstrated in a life-supporting pig kidney to baboon model (29) and in several ongoing solid organ transplantation models (R. Mañez et al., R. Morris et al., unpublished results). Even with GAS914 treatment, however, exposure to pig organs leads to the development of elicited Ab’s against non-αGal antigens. This has been well described in several nonhuman primate models.

Naive primates have small amounts of αGal Ab’s that are not cross-reactive with GAS914 and may expand after transplant. Such Ab’s may be removed with polylysine conjugates analogous to GAS914 but carrying different αGal structures. To this end, we have conjugated Linear B type VI to the polylysine backbone of GAS914 and have shown that this reagent is a similarly potent in vitro inhibitor that preferentially inhibits type VI–specific Ab’s. Ongoing studies with type II (GAS914) and type VI GAS914-like polymer conjugates in primates indicate a more complete removal of αGal Ab’s when the two injectable polymers are combined. The effect of this combination in pig-to-primate solid organ and bone marrow transplantation is under investigation.

Antigen arrays have been shown to eliminate Ab’s and lead to antigen-specific B cell unresponsiveness (30). There is evidence that they function by engaging the B cell receptor in a manner that generates apoptotic instead of activation signals (31–33). It has been suggested that the size and hapten valence of antigen arrays is important for eliciting an inhibitory instead of an activating response (30).

The GAS914 polymer was optimized for its ability to bind αGal Ab’s and not for its effect on B cells. We show here accumulation of GAS914 in the lymphoid organs of treated primates and Gal^–/– mice (Figure 4, Figure 5, and Figure 6), and suppression of circulating αGal Ab’s for periods far in excess of the period of time GAS914 is measurable in the blood. Simple removal of Ab’s is probably not the only effect of GAS914, since removal of αGal Ab’s by immunoabsorption leads to a quick rebound of circulating Ab’s as homeostatic mechanisms produce more Ab. We speculate that the GAS914 accumulated in the lymphoid organs is important for the long-term lowering of circulating αGal Ab’s. GAS914 may associate with B cells and temporarily inhibit αGal Ab production. Alternatively, GAS914 may absorb newly formed αGal Ab’s as they are produced in the lymphoid organs and prevent them from reaching the circulation. In agreement with the former hypothesis, GAS914 inhibits formation of plaques on pig erythrocyte–coated plates by splenocytes from immunized Gal^–/– mice, even when the splenocytes are washed after incubation with GAS914 so that no free GAS914 is present in the solution (A. Katopodis, unpublished results). In the mouse, the αGal IgM is produced by splenic B cells with αGal Ab receptors (8) to which GAS914 may bind in a manner that suppresses Ab production. However, the deposition of GAS914 in the lymphoid organs of Gal^–/– mice and primates is consistent with immune complexes in the network of follicular dendritic cells in secondary follicles and not direct association with B cells as such. Interestingly, immune complexes in secondary follicles are believed to be required for effective B cell stimulation and immuno-globulin class switch, but GAS914 is clearly nonimmunogenic. Additional polylysine conjugates of varying length and ligand density are currently being tested for their ability to elicit B cell hyporesponsiveness in Gal^–/– mice.

Many autoimmune diseases are the result of the generation of pathogenic Ab’s against specific antigens. Elimination of auto-Ab’s has been shown to be associated with disease remission (34). The polylysine backbone of GAS914 offers an excellent scaffold for antigen-specific therapies in Ab-mediated diseases where the immunogenic epitope is defined.

We thank Dario Anselmetti for atomic force microscopy images of GAS914 and Henk Schuurman for critically reading the manuscript.

Valerie F.J. Quesniaux’s present address is: Experimental and Molecular Genetics, Transgenose Institute, CNRS, Orleans, France.

Emanuele Cozzi’s present address is: Addenbrooke’s Dialysis Centre, University Hospital, Cambridge, United Kingdom.

Hugh F.S. Davies’ present address is: Department of Surgery, University of Cambridge, Cambridge, United Kingdom.

David White’s present address is : Robarts Research Institute, University of Western Ontario, London, Ontario.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: Galα1,3Gal terminating carbohydrate chains (αGal); α1,3-galactosyltransferase–deficient (Gal^–/–); hemolytic anti-pig Ab (HAPAb); alanine aminotransferase (ALT); aspartate aminotransferase (AST).

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