Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates.

Most chemical techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.

Synthesis of phosphoantigens: scalable accesses to enantiomers of BrHPP and studies on N-HDMAPP synthesis.

Phosphoantigens enable the access to a new anti-tumoral and anti-infectious therapeutic pathway, based on innate immunity through the selective activation of Tγ9δ2 lymphocytes. The first proof of concept of this new immunotherapy approach was demonstrated with the synthetic phosphoantigen named bromohydrin pyrophosphate (BrHPP, IPH 1101) which was administrated in racemic form to about 200 patients in six clinical trials with good safety and promising early signals of efficacy in type C viral hepatitis and follicular non-Hodgkin's lymphoma. Enantiopure samples of BrHPP in gram scale are required for further studies on structure-bioactivity relationship. Thus we developed two complementary synthetic pathways, the first using transformation of a chiral compound and the second involving asymmetric synthesis starting from a prochiral building-block. The synthesis of a second-generation phosphoantigen, N-HDMAPP, which bears a phosphoramidate moiety, was also investigated.

Synthesis and biological activity of phosphonate analogues and geometric isomers of the highly potent phosphoantigen (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate.

Gammadelta-T-lymphocytes contribute to innate immunity and are selectively activated by nonpeptide phosphorylated molecules (so-called phosphoantigens) produced by organisms responsible for causing a broad range of infectious diseases. gammadelta-T-cells are also activated by synthetic phosphoantigens and are cytotoxic to tumor cells. Here we report the synthesis, NMR characterization, and comparative biological evaluation of new pyrophosphate, phosphonate, and pyrophosphonate monoesters whose structures correspond to isosteric analogues and stereoisomers of the highly potent isoprenoid metabolite ( E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate called HDMAPP (hydroxy-dimethyl-allyl pyrophosphate). Both pyrophosphate and pyrophosphonate series elicit promising gammadelta-T-cell stimulatory responses in vitro, the pyrophosphonate ester (C-HDMAPP) being by far more stable than its parent pyrophosphate ester (HDMAPP) with improved ADMET properties and a similar pharmacodynamic profile based on in vivo studies in nonhuman primate. In both series, we found that E-stereoisomers are the most active derivatives and that Z-stereoisomers show very marginal bioactivity levels. These results indicate that the use of bioisosteric analogues of HDMAPP may represent promising new leads for immunotherapy.