Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985.

Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.

Use of tetravalent galabiose for inhibition of streptococcus suis serotype 2 infection in a mouse model.

Streptococcus suis is an important swine pathogen associated with a variety of infections such as meningitis, arthritis and septicemia. The bacterium is zoonotic and has been found to cause meningitis especially in humans occupationally exposed to infected pigs. Since adhesion is a prerequisite for colonization and subsequent infection, anti-adhesion treatment seems a natural alternative to traditional treatment with antibiotics. In order to optimize the inhibitory potency a multivalency approach was taken in the inhibitor design. A synthetic tetravalent galabiose compound was chosen which had previously shown promising anti-adhesion effects with S. suis in vitro. The aim of this study was to evaluate the in vivo effects of the compound using an infection peritonitis mouse model. As such S. suis serotype 2 infection and treatment were tested in vivo and the effects were compared to the effect of treatment with penicillin.

Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14.

Synthetic overlapping oligosaccharide fragments of Streptococcus pneumoniae serotype 14 capsular polysaccharide (Pn14PS), [6)-[beta-D-Galp-(1-->4)-]beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->](n), were conjugated to CRM(197) protein and injected into mice to determine the smallest immunogenic structure. The resulting antibodies were then tested for Pn14PS specificity and for their capacity to promote the phagocytosis of S. pneumoniae type 14 bacteria. Earlier studies have reported that the oligosaccharide corresponding to one structural repeating unit of Pn14PS, i.e., Gal-Glc-(Gal-)GlcNAc, induces a specific antibody response to Pn14PS. The broader study described here, which evaluated 16 oligosaccharides, showed that the branched trisaccharide element Glc-(Gal-)GlcNAc is essential in inducing Pn14PS-specific antibodies and that the neighboring galactose unit at the nonreducing end contributes clearly to the immunogenicity of the epitope. Only the oligosaccharide conjugates that produce antibodies recognizing Pn14PS were capable of promoting the phagocytosis of S. pneumoniae type 14. In conclusion, the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc may be a serious candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by S. pneumoniae type 14.

Inhibition of P-fimbriated Escherichia coli adhesion by multivalent galabiose derivatives studied by a live-bacteria application of surface plasmon resonance.

OBJECTIVES: Uropathogenic P-fimbriated Escherichia coli adheres to host cells by specific adhesins recognizing galabiose (Galalpha1-4Gal)-containing structures on cell surfaces. In search of agents inhibiting this first step of infection, the inhibition potency of a set of synthetic mono- and multivalent galabiose compounds was evaluated. In order to mimic the flow conditions of natural infections, a live-bacteria application of surface plasmon resonance (SPR) was established. METHODS AND RESULTS: For the measurement of the binding of E. coli to a surface containing galabiose, live bacteria were injected over the flow cell, and the inhibition of adhesion caused by the galabiose inhibitors was recorded. Quantitative binding data were recorded in real-time for each inhibitor. The results were compared with those of conventional static haemagglutination and ELISA-based cell adhesion assays. Compared with the Gram-positive Streptococcus suis bacteria, which also bind to galabiose and whose binding inhibition is strongly dependent on the multivalency of the inhibitor, E. coli inhibition was only moderately affected by the valency. However, a novel octavalent compound was found to be the most effective inhibitor of E. coli PapG(J96) adhesion, with an IC50 value of 2 microM. CONCLUSIONS: Measurement of bacterial adhesion by SPR is an efficient way to characterize the adhesion of whole bacterial cells and allows the characterization of the inhibitory potency of adhesion inhibitors under dynamic flow conditions. Under these conditions, multivalency increases the anti-adhesion potency of galabiose-based inhibitors of P-fimbriated E. coli adhesion and provides a promising approach for the design of high-affinity anti-adhesion agents.

Probing the substrate specificity of four different sialyltransferases using synthetic beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH(2))7CH3 analogues general activating effect of replacing N-acetylglucosamine by N-propionylglucosamine.

The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminal D-GlcN were tested for their influence on the alpha-2,3- and alpha-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function.

Inhibition of Streptococcus suis adhesion by dendritic galabiose compounds at low nanomolar concentration.

A series of mono-, di-, and tetravalent galabiose (Galalpha1-4Gal) compounds were synthesized in good yields by coupling of a general carboxylic acid-bearing sugar building block to dendritic scaffolds based on the 3,5-di-(2-aminoethoxy)benzoic acid branching unit. Furthermore, a poly(amidoamine)- (PAMAM-) based dendritic galabioside was synthesized containing eight galabiose units. All galabiosides were tested in a hemagglutination assay and a surface plasmon resonance (SPR) competition assay in order to establish their potency in the binding to the bacterial Gram-positive pathogen Streptococcus suis. A monovalent galabioside containing a short spacer was used as a reference compound in all the assays. Variations in the scaffold as well as in the spacer arms were introduced to determine their influence on the inhibition. The best inhibitor of hemagglutination was an octavalent galabioside with a minimal inhibitory concentration (MIC) of 0.3 nM, to the best of our knowledge the first example of inhibition of bacterial binding by a soluble carbohydrate at a subnanomolar concentration.

Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14.

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14.

The chemo-enzymatic synthesis is described of beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->O(CH(2))(6)NH(2) (1), beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (2), beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (3), and beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (4), representing fragments of the repeating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5-8 were synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk beta-1,4-galactosyltransferase as a catalyst. The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal models.