Delphine Bregeon

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.

Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models

Antibody-drug conjugates (ADCs) have demonstrated clinical benefits that have led to the recent FDA approval of KADCYLA and ADCETRIS. Most ADCs that are currently in clinical use or development, including ADCETRIS, are produced by chemical conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, we describe the in vitro and in vivo characterization of four novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as ADCETRIS, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivatives of monomethyl auristatin E (all comprising a cleavable linker) to the glutamine at positions 295 and 297 of cAC10, thereby yielding homogeneous ADCs with a DAR of 4. In vitro cell toxicity experiments using two different CD30-positive cell lines (Karpas 299 and Raji-CD30(+)) revealed comparable EC50 values for ADCETRIS (1.8 ± 0.4 and 3.6 ± 0.6 ng/mL, respectively) and the four cAC10-based ADCs (2.0 ± 0.4 to 4.9 ± 1.0 ng/mL). Quantitative time-dependent in vivo biodistribution studies (3-96 h p.i.) in normal and xenografted (Karpas 299 cells) SCID mice were performed with a selected (125)I-radioiodinated cAC10 ADC and compared with that of (125)I-ADCETRIS. The chemo-enzymatically conjugated, radioiodinated ADC showed higher tumor uptake (17.84 ± 2.2% ID/g 24 h p.i.) than (125)I-ADCETRIS (10.5 ± 1.8% ID/g 24 h p.i.). Moreover, (125)I-ADCETRIS exhibited higher nontargeted liver and spleen uptake. In line with these results, the maximum tolerated dose of the BTG-coupled ADC (>60 mg/kg) was significantly higher than that of ADCETRIS (18 mg/kg) in rats. These results suggest that homogeneous ADCs display improved pharmacokinetics and better therapeutic indexes compared to those of chemically modified ADCs with variable DARs.

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-Hodgkins 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.

Abstract 2514: Towards homogenous adcs: A new site-specific antibody conjugation using bacterial transglutaminase (btg-adc)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose : Bacterial Transglutaminase (BTG) allows coupling on endogenous Q295 from an aglycosylated antibody. Here, the work relates to ADCs synthesis through BTG approach on mAbs with single point mutations and the study of stability, pharmacokinetics, as well as in vitro and in vivo efficacy of resulting BTG-ADCs. AdcetriS®, brentuximab vedotin, has been used as comparator, hence for consistency BTG-ADCs have been conjugated with the different linkage strategies (one step and two step) containing distally the same protease sensitive moiety compared to ADCETRIS®, i.e. valine-citrulline-PAB-MMAE. Methods : . For synthesis, a two-step process has been designed consisting of coupling first a linker and coming back with a toxin, and affording versatility and efficiency. The DAR stability was monitored over one week in human and cynomolgus plasma by affinity capture/LC/MS. The pharmacokinetic profile was studied in rat and monitored by affinity capture/LC/MS for conjugated antibody and ELISA for total antibody. In vitro efficacy of ADCs conjugated using BTG was compared side-by-side with ADCETRIS® on Karpas 299 cells. In vivo efficacy was studied in xenogenic nude mice engrafted with Karpas 299 in SC with BTG ADCs or ADCETRIS at doses of 0.6mg/kg. Results: BTG two-step process leads to ADCs with DAR of exactly 2.0 or 4.0 for antibodies with N297S or N297Q single point mutation respectively. The BTG ADCs were stable in human and cynomolgus plasma, with no degradation observed over 15 days in rats and furthermore with favorable total antibody clearance. In vitro efficacy for BTG ADCs DAR=4.0 was comparable to ADCETRIS (mean DAR=4) with respect to EC50 and plateau. In vivo efficacy for BTG ADC was demonstrated to be at least equivalent to ADCETRIS®. Conclusions: Pre-clinical proof of concept is demonstrated for BTG coupling. When considering versatility, speed and efficiency of the process, BTG-ADC coupling should provide a promising technology for the next generation of homogeneous ADCs. Citation Format: Florence Lhospice, Delphine Bregeon, Christian Belmant, Agnes Represa, Angelique Boedec, Yannis Morel, Patrick Dennler, Roger Schibli, Francois Romagne. Towards homogenous adcs: A new site-specific antibody conjugation using bacterial transglutaminase (btg-adc). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2514. doi:10.1158/1538-7445.AM2014-2514