Camille Martin, Inmaculada Vieitez, Anaïs Mirault, Stéphanie Letast, Caroline Denevault-Sabourin, Renaud Respaud, Nicolas Joubert,* Marie-Claude Viaud-Massuard.
UMR 7292 Equipe 4
GICC UMR 7292 CNRS - Université de Tours, Equipe 4 Innovation Moléculaire et Thérapeutique, UFR Sciences Pharmaceutiques, 31 Avenue Monge, 37200 TOURS. Contact:
[email protected]
ADC: Antibody-Drug Conjugate
Bioconjugation Heterogeneity
State of the Art
Following the visionary concept of the magic bullet introduced by Paul Ehrlich in 1906, developing new chemotherapeutic drugs showing superior efficacy with reduced toxicity is a continuing challenging endeavor. To answer that quest, selective delivery of a cytotoxic payload by an antibody-drug conjugate (ADC) is becoming an increasingly important area of research, with over 30 ADC in clinical trials in 2014.1 ADC results from the grafting of a powerful cytotoxic agent on a therapeutic antibody (MAb) through a judiciously constructed spacer arm (linker). From antibody …
More than three decades of ADC development in oncology led to FDAapproved Brentuximab Vedotin (Adcetris®) in August 2011 against various lymphomas and to Trastuzumab Emtansine (Kadcyla®) in February 2013 against HER2+ breast cancer. Adcetris® (Seattle Genetics) anti-CD30 Average DAR 4.0
To overcome this heterogeneity in ADC production, upstream modifications of the MAb are necessary (including mutagenesis using natural or unnatural amino-acids at strategic positions, or modification of the sugar moieties) during its bioproduction, which then guarantees efficient and regiospecific modifications to build an homogeneous ADC.2 Indeed, pioneer work of Jununtula in 2008 opened the way of various MAb modifications to reach that challenging goal including mutagenesis to introduce cysteine residues or use of 6-thiofucose among others. Thiomab® (HC-A114C variant, Genentech) anti-MUC16
Relative abundance
Average DAR 2.0 0
1
2 3 4 5 6 7 Drug-to-antibody ratio
8
9
Relative abundance
gicc.cnrs.univ-tours.fr gicc.cnrs.univ-tours.fr
New Chemical Tools for the Modular Construction of Homogeneous Antibody-Drug Conjugates.
Kadcyla® (Immunogen) anti-HER2 Relative abundance
Average DAR 3.5
… to ADC
0
Analogous approaches C239 / anti-CD70 0
1
2 3 4 5 6 7 Drug-to-antibody ratio
8
1
2 3 4 5 6 7 Drug-to-antibody ratio
8
9
Fucose 6-SH / anti-CD70 or anti-CD30
9
Despite their growing success, each ADC is a heterogeneous mixture, which is a noteworthy drawback leading to analytical and process challenges, and difficulties to anticipate the biological responses induced by this complex mixture. Similarly, lack of reproducibility of the labeling fluorescent kits (various number of modified lysines, random locations) may impact on the biological properties of the studied MAb and the objective interpretation of the data.
However satisfying and efficient these methods are, they require a heavy amount of efforts including a transposition for each antibody of interest.
Unoptimized therapeutic index of ADC Reproducibility issues from labeling kits
Efficient Targeted Therapy
Our Homogeneous ADC
Our Original Modular Methodology We aimed to tune a new chemical technology to achieve similar results but with a broader spectrum of application to virtually all IgG.3 We took Adcetris as a starting model ADC. We chose to keep the cathepsin B sensitive trigger, among with the very toxic dolastatin derivative called MMAE (MonoMethyl Auristatin E). However, as we focus our attention on solid tumors, including various types of breast cancers, we therefore chose to first optimize trastuzumab (anti-HER2), by a new method of chemical biology. It will also allow us to compare our ADC against T-DM1, the only FDAapproved ADC against metastatic breast cancer.
The story of the origins began with one question: how to stabilize the ADC while minimizing the number of species generated during the bioconjugation process started from a native MAb? Knowing that Adcetris is unstable due to the reduced interchain disulfide bridges, necessary to graft the drug on free sulfydryls, we wanted to shape a method allowing introduction of the drug while maintaining the tertiary structure of the native MAb AND avoiding the heterogeneity problems encountered during the manufacturing of both Adcetris® and Kadcyla®. To achieve this goal, we used a bifunctional linker, which extremities will serve two purposes: the first one had to rebuild the disulfide bridge which was preliminary reduced, while the second had to graft the molecule of interest (a drug or a fluorescent probe), as followed:
In contrary of biological methods leading to homogeneous ADC, our chosen retrosynthetic pathway allowed a particularly modular construction of the ADC, using interchangeable parts. This means that any drug could be chosen, any enzyme-sensitive trigger, any bioconjugation moiety and any antibody of interest. Concerning the bioconjugation head, we focused our attention on a particular maleimide skeleton able to undergo a bisfunctionalization by two sulfhydryls while incorporating a chemical linker. This linker later allowed introduction of the desired drug/fluorophore. We performed experiments of reduction/rebuilding of the disulfide bridge, with miscellaneous linkers bearing variable lengths or chemical natures (alkylated, pegylated, amino-acids) and using various protocols. Modifications of the MAbs were followed by RP-HPLC, and confirmed by mass spectroscopy analysis. Hundreds of attempts led to the optimized conditions (for all linkers), which are the only ones shown for clarity purposes.
Anti-CD4 Murin IgG2
RP-HPLC
Upstream modification / NOT modular needed for EVERY MAb
We developed an innovative method for bioconjugate chemistry,4 to achieve controlled and unambiguous bioconjugation of MMAE on potentially every native MAb of interest.5 For now, we have been able to functionalize either IgG1 or IgG2, human or murine isotypes.
anti-HER2
Average DAR 4.0 Relative abundance
Therefore, an ADC is a targeted therapy which tackles the challenge to harness both strength of the selectivity of the MAb for its target and high potency of the payload against the malignancy. Insuring the overall success of the ADC, all parts of the linker have an outmost importance to optimize safety, efficacy and therapeutic index.
0
1
2 3 4 5 6 7 Drug-to-antibody ratio
8
9
We also applied our strategy for the bioconjugation of fluorescent probes, to be used either to study antibody biological properties, to perform diagnosis or to undertake mechanism studies. anti-HER2 or anti-CD20
Our methodology was based on bifunctional linkers including original heterocycles as new chemical tools for the modular construction of homogeneous ADC. Biological evaluations of ADC and experiments involving labeled-MAb are currently performed and will be reported in due course.
Our Methodology Anti-HER2 Human IgG1
Example:
Maldi-TOF
HIC-HPLC
Deconvolution
Complementary analysis centro0
centro1
centro2
centro3
centro4
8%
0%
0%
3%
89%
centro 5
Centro6, 7, 8
Average DAR 4.0 Species DAR 4.0: 89%
1 single species, for targeted therapy or imaging / modular approach for all IgG
References: 1. a) Teicher, B. A. Curr. Opin. Oncol. 2014, 26, 476-483; b) Senter, P. D. et al. Annu. Rev. Med. 2013, 64, 15–29; 2. a) Mallet, W. et al. Nat. Biotechnol. 2008, 26, 925-932; b) Schultz, P. G. et al. Proc. Natl. Acad. Sci. 2012, 109, 16101-16106; 3. a) Webb, S. et al. Nat. Biotechnol. 2013, 31, 191-193; b) Million, R. P. et al. Nat. Rev. Drug Discov. 2013, 12, 259-260. 4. a) Joubert, N. et al. eBook Chapter. Ed. Future Science, 2015, accepted; b) Baslé, E. et al. Chem. Biol. 2010, 17, 213227; 5. a) Joubert, N. et al. Fr. Demande 2015FR1356837; b) Joubert, N. et al. PCT/FR2014/051802 WO2015004400.
