Morgane Sayes– Charette Group Literature Meeting 16-11-2016

Thomas H. Pillow Education Experiences 1996 – 2000 : BS Chemistry at Trinity University, San Antonio, Texas 2003 – 2009 : Ph.D, Organic Chemistry, Professor Paul A. Wender, Stanford University, California Experiences 2000 – 2003 : Associate Manufacturing Engineer at Sony Semiconductors 2009 – now : Chemistry Team Leader : Antibody-Drug Conjugates (ADCs) at Genentech, San Francisco, California

Genentech Areas of research Numbers Member of the Roche Group (March 2009) Leading biotechnology company : treatment of patients with life-threatening medical conditions Areas of research Oncology Immunology Infectious diseases Neuroscience Metabolism Ophtalmology Numbers 13.3 K employees 11.3 K patents received 35 medicines on the market 19 Antibody-Drug Conjugates (ADCs) https://www.gene.com

Monoclonal Antibodies Recognise and attach to specific proteins (antigen) produced by cancer cells Naked monoclonal antibodies Work by themselves Trigger the immune system Block molecules that stop the immune system Block signals telling cancer cells to divide Conjugated monoclonal antibodies Joined to a chemotherapy drug or to a radioactive particle : used as a CARRIER Reduce the damage to normal cells http://www.cancerresearchuk.org

Antibody-Drug Conjugates Multistep sequence of events Binding to the cell surface Endocytosis Trafficking to a lysosome Proteolytic degradation Diffusion of the released cytotoxic agent to the target Cell death Zhao, R. Y., Chari, R. V. J. et al., J. Med. Chem. 2011, 54, 3606-3623.

Targeted drug delivery Improvement of THERAPEUTIC INDEX and PHARMACOKINETICS of small molecule drugs Need of a STABLE but LABILE connection to the drug Chari, R. V. J., Miller, M. L., and Widdison, W. C., Angew. Chem. Int.Ed 2014, 53, 3796-3827.

Targeted drug delivery Active small molecule drug Antibody for specific recognition Cleavable linker (protease) Wu, A. M. and Senter, P. D., Nature biotechnology 2005, 23, 1137-1146.

Classification of linkers Cleavage by enzymes such as proteases expressed either intra- or extracellularly Best known example : cathepsin B in lysosomes Elimination linkers p-aminobenzyloxycarbonyl (PABC) 1,6-elimination Requirement of a PRIMARY or SECONDARY amine (formation of carbamate or amide) Cyclization linkers Kratz, F., Muller, I. A., Ryppa, C. and Warnecke, A., ChemMedChem 2008, 3, 20-53.

Challenges Introduction of charges PABCs used in half of the ADCs Lack of DIVERSITY in drug structures Too hydrophobic molecules AGGREGATION Introduction of charges GPTR : hydrophobic protein (tetramer in native state) T20 : HIV inhibitor peptide May : Maytansine derivative (cytotoxic agent) Adem, Y. T. et al., Biconjugate Chem. 2014, 25, 656-664. ; Zhao, R. Y. et al., J. Med. Chem. 2011, 54, 3606-3623. Xiao, J., Burn, A. and Tolbert, T. J., Bioconjugate Chem. 2008, 19, 1113-1118.

Tertiary and heteroaryl amines Anticancer

Tertiary and heteroaryl amines Antibacterial Expand the scope of conjugatable complex small-molecule drugs Generate a charged functional group Goals

Auristatins and Tubulysins Clinically validated mechanism of action but not sufficient activity at tolerated doses Auristatin and MMAE : same activity Adcetris (ADC) R = H : 100-fold loss of potency Conversion to a secondary amine NOT VIABLE

Concept Linker choice p-AminoBenzyl Quaternary amonium salt (PABQ) Protease cleavable trigger : Valine-citrulline dipeptide (ValCit)

Proof of concept Model of quaternary ammonium salt system PBS buffer pH 7, 37 °C, overnight STABLE Exposure to cathepsin B

Concise and broadly applicable synthesis Development of a SHORT synthesis of molecule containing a REACTIVE ELECTROPHILE Protecting group FREE synthesis Broad spectrum of NUCLEOPHILICITY (5 log variation in pKa) Starting point Benzyl bromide Methanesulfonate

Concise and broadly applicable synthesis Limited optimization Strong base and acid sensitive Temperature sensitive (> 40°C) Low solubility (DMF or DMSO needed) Best conditions Multigram scale

Stability and release of the drugs MRSA : Methicillin-Resistant S. aureus

Stability and release of the drugs PABQ-9a LC/MS chromatogram of reduced anti-MRSA-9c after digestion with cathepsin mixture Efficient IMMOLATION

In vivo stability and efficacy studies In vivo efficacy EFFICACY SELECTIVITY DAR : Drug to Antibody ratio Severe combined immunodefiency mice Mice bearing WSU-DLCL2 human NHL xenographts

Hydrophobicity and log D Correlation between AGGREGATION and LOG D Log D = log P at a precise pH (7,4) for a compound of a specific pKa MC-ValCit-PABC-MMAE : log D = 3,5 MC-ValCit-PABQ-AE : log D = 0,18 Resch-Genger, U. et al., Bioconjugate Chem. 2011, 22, 1298-1308. MC : Malemidocaproyl

Heteroaryl amine Efficient IMMOLATION

Disulfide conjugates Protease cleavage in cytosol Vlahov, I. et al., J. Org. Chem. 2007, 72, 5968-5972.

Disulfide conjugates Protease cleavage in cytosol Pillow, T. H. et al., Chem. Sci. 2016, ASAP.

Disulfide conjugates Protease cleavage in cytosol

Improved therapeutic activity of ADCs Carbamate linked conjugate Quaternary ammonium linked conjugate DNA31

Conclusion Increase the DIVERSITY of therapeutic agents EFFICIENT, protecting group FREE synthesis for modification of tertiary and heteroaryl amines STABILITY and generation of the FREE DRUG on TRIGGERING mechanisms IMPROVEMENT in ACTIVITY over conjugate connected to carbamate Possible application to INFECTIOUS DISEASES and INFLAMMATION