Rudolf O. Duthaler

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

Preformed and elicited Abs against the Galalpha1,3Gal terminating carbohydrate chains (alphaGal Abs) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. alphaGal Abs are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of alphaGal Abs might be achieved in vivo by systemic administration of nonimmunogenic polyvalent alphaGal structures with high avidity for alphaGal Abs. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for alphaGal binding by alphaGal IgM (IC(50), 43 nM) and IgG (IC(50), 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 alphaGal Abs and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating alphaGal Abs and cytotoxicity over several months. Studies with [(14)C]GAS914 in rhesus monkeys and Gal(-/-) mice indicate that GAS914 binds to circulating alphaGal Abs and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment alphaGal Ab titers never exceeded pretreatment levels and no sensitization to either alphaGal 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 alphaGal Abs.

Novel glycodendrimers self-assemble to nanoparticles which function as polyvalent ligands in vitro and in vivo

The recognition of oligosaccharides by proteins represents the basis of many biologically important events.[1] Individual protein±carbohydrate interactions are generally weak (KD1⁄4 10 3±10 4m 1).[2] To overcome this, such processes often involve polyvalent binding, which is characterized by the simultaneous contact of multiple ligands (oligosaccharides) on one biological entity to multiple receptors (proteins) on another.[3] Polyvalent carbohydrate±protein interactions occur frequently in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions rendering it difficult to control them with individual small molecules.[4] Therefore, complex macromolecules have been used as polyvalent antagonists, however, both characterization and preparation of these nonuniform entities is demanding.[4] Here we present an alternative concept for the polyvalent presentation of ligands based on the supramolecular chemistry[5] of small molecules that fulfil single-molecule entity criteria (Figure 1). Novel dendrons capped with carbohydrate ligands (glycodendrimers[6]) were found to self-assemble to noncovalent nanoparticles which function as polyvalent ligands. We demonstrate that these particles–not the individual molecules–efficiently inhibit polyvalent interactions, such as IgM binding (IgM1⁄4 immunoglobulin), to the aGal-epitope[7] (a-d-Gal-(1!3)-b-d-Gal(1!4)-d-GlcNAc), both in vitro and in vivo. As self-assembly is dynamic, optimization of size and shape of the polyvalent ligand could occur utilizing the receptor as a template. Dendrimer cores were prepared by a convergent TMoutsidein∫ approach[8] based on a single building block 1a which was obtained frommethyl 3,5-diaminobenzoate and 4-(tert-butoxycarbonylaminomethyl)benzoic acid (Scheme 1a). Selective deprotection furnished 1b and 1c (first-generation dendrimer core, two end-groups). A one-pot procedure comprising coupling of 1c (1 equiv) and 1b (0.5 equiv) followed by methyl ester cleavage gave 2c (second-generation dendrimer core, four end groups).[9] The third-generation dendrimer 3c (eight end groups) was obtained from 2c (1 equiv) and 1b (0.5 equiv).[9] Applying the same procedure repetitively gave dendrimers with up to 64 end groups (4c, fourth generation, 16 end groups; 5c, fifth generation, 32 end groups; 6c, sixth generation, 64 end groups). Dendrimers 1c±6c were deprotected (!1d±6d) and transformed into their chloroacetamide derivatives (1e±6e) to allow subsequent introduction of thiolated oligosaccharides such as aGal-SH[10] and Lac-SH (Figure 2b) furnishing water-soluble glycodendrimers 1 f±6 f and 3g which were purified by ultrafiltration. Compound 7, which is similar to 2 f but contains butylene chains instead of the disubstituted aromatic rings, was also prepared (Scheme 1a). The integrity of all compounds was established by 1H NMR spectroscopy. Accordingly, the firstto third-generation dendrimers exist as single molecules (purity > 95%). The fourthto sixth-generation dendrimers possibly contain minute quantities of smaller fragments. The 500 MHz 1H NMR spectra of compound 3 f in [D6]DMSO demonstrates the remarkable purity of these compounds (Figure 2). The first indication that our glycodendrimers were aggregating in water came from 1H NMR spectroscopy of 2 f in D2O. At ambient temperature, we observed very broad signals which sharpened at elevated temperatures. The aggregation was quantified using multiangle light scattering (MALS; Table 1). The first-generation dendrimer 1 f forms small aggregates (50 kDa) whereas 2 f forms large particles of 7000 kDa (more than 1500 individual molecules per particle). Interestingly, the particle weight obtained for 3 f±6 f drops (2200 to 200 kDa) with increasing mass of the individual molecule. The root-mean-square radii of the particles formed by 2 f±6 f showed the same trend (for 2 f, 3 f, and 4 f 49, 34, and 12 nm, respectively; for 5 f and 6 f the radii were below the detection limit of 10 nm). Core-modified second-generation glycodendrimer 7 (4 iaGal), which is of comparable size and lipophilicity as 2 f but contains butylene chains instead of the disubstituted aromatic rings, does not form aggregates. The third-generation compounds 3g (8 iLac; 1900 kDa) and 3 f ZUSCHRIFTEN

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts

Abstract:  Background:  In pig‐to‐nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay‐accelerating factor (hDAF), the main type of rejection is antibody‐mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement‐regulatory function of the transgene, neutralization of natural antibodies to Galα1–3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10).

Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: Influence of pre-existing anti-pig antibodies and prevention by the αGal glycoconjugate GAS914

Abstract:  Background:  Our introductory pig‐to‐cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay‐accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti‐pig antibodies. We evaluated the efficacy of GAS914, a Galα1–3Gal trisaccharide linked to a poly‐l‐lysine backbone, in inhibition of HAR.

Stereoselective transformations mediated by chiral monocyclo-pentadienyl titanium, zirconium, and hafnium complexes

The chiral cyclopentadienyldialkoxytitanium chlorides 3 and 4 with ligands deri- ved from (D)-glucose and tartaric acid afford highly stereoselective reagents for the addition of nucleophiles to aldehydes. The methyltitanium derivatives of 4 are unreactive, but interesting reagents result from mixtures of the chloride 4 and different organometallics before the trans- metalation occurs. Most successful for the methyl transfer are, however, the Cp*Zr- and Cp*Hf-analogs of the titanacycle 4. While the cyclic complex 4 is especially suited for the al- lyltitanation of aldehydes, the diacetoneglucose system 3 is the best choice for aldol reactions. The stereochemical course of these reactions can be rationalized by six-membered cyclic tran- sition states with either chair-conformation in the case of allyltitanations or boat-confoxmation for the aldolizations. Interactions of the alkoxy ligand(s) with the cyclopentadienyl group seem to be crucial for good stereoselectivity. The mechanism of the often perfect enantiofacial diffe- rentiations is, however, still unclear and might be due to stereoelectronic effects. These rea- gents have been successfully applied for the synthesis of C(2)-modified pentoses from glycer- aldehyde.

Reduction of anti-Galα1,3Gal antibodies by infusion of types 2 and 6 gal trisaccharides conjugated to poly-l-lysine

Abstract:  To investigate the specificity of anti‐Galα1,3Gal (Gal) antibodies (Abs) with respect to Gal oligosaccharides of types 2 and 6, eight baboons received an intravenous infusion of either a poly‐l‐lysine conjugate of Gal type 2 (n = 5) or type 6 (n = 3), followed 48 h later by the alternative Gal type 6 or 2 conjugate, respectively. IgM Abs reactive to Gal type 2 were depleted by 80 to 89% by either Gal conjugate. IgM reactive to Gal type 6 was less efficiently depleted by the Gal type 2 conjugate (57% depletion) than the Gal type 6 (82% depletion). Gal‐reactive IgG was depleted more slowly and less efficiently by either glycoconjugate (initially by only 28 to 54%). Our results indicate that the Gal type 6 conjugate depletes most anti‐Gal IgM, but the Gal type 2 conjugate is less efficient in depleting anti‐Gal IgM reactive with type 6. There remain small fractions of antibody that are unadsorbed, particularly of IgG, probably due to their low affinity and distribution in both the intra‐ and extra‐vascular compartments.

Depletion of anti-Gal antibodies by the intravenous infusion of Gal type 2 and 6 glycoconjugates in baboons

Abstract: Background:  Natural anti‐Gal antibodies (NAb) to Gal epitopes play a key role in the rejection of pig cells or organs transplanted into primates. We have investigated the effect on NAb return after extracorporeal immunoadsorption (EIA) of the continuous intravenous (i.v.) infusion of (i) bovine serum albumin conjugated to Gal type 6 oligosaccharides (BSA‐Gal) or (ii) a poly l‐lysine backbone conjugated to Gal type 2 or 6 oligosaccharides (PLL‐Gal).

Failure to deplete anti-Galalpha1-3Gal antibodies after pig-to-baboon organ xenotransplantation by immunoaffinity columns containing multiple Galalpha1-3Gal oligosaccharides.

Abstract:  Background:  The impact of anti‐Galα1‐3Gal (αGal) antibodies on the acute humoral xenograft rejection (AHXR) of pig organs transplanted in baboons is unclear.