Eran ella The Chemistry Behind Antibody- Conjugation Antibody- Conjugation (ADC) Dominant Investigator - Taking the advantage of the specificity of mab (monoclonal antybodies) to deliver potent cytotoxic drug selectively to antigen-expressing tumor cells. Chemistry allows to use highly potent drugs that could not be used alone. - o. of publication with the concept "antibody drug conjugated" over ther years: - Peter D. enter,.d. - B.A Biotechnology (Berkeley),.D. Chemistry (University of Illinois), Postdoctoral in the Max-Planck Institute for Experimental Medicine. Worked at Cytokine etworks, istol-myers-quibb esearch Institute. Current position: Vice president, eattle Genetic. (Development of potent drugs, novel linker systems and conjugation methodology) enior editor of Molecular Cancer Therapeutics Affilate Professor (University of Washington) Antibody- Conjugates - inical eports (clinicaltrials.gov 2012) Antibody- Conjugates - istory Agensys - 3 ADCs in ase I (auristatine) - carcinoma and prostate cancer Bayer ealthcare - 2 ADCs in ase I (Auristatine/maytansinoid) Paul Erlich "Magic Bullet" 2 2 C 2 C 2 MT-Ab oncovalent linked ADC tested in animal models umanized mabs reported First FDA approved ADC Mylotarg Mylotarg withdrown from market Kadcyla approved Biogen - 1 ADC in ase I (DM4) - breast cancer Biotest - 1 ADC in ase II (DM4) - myeloma BM - 1 ADC in ase I (duocarmycin) - non-odgkin's lymphoma Celldex - 1 ADC in ase II (auristatine) Immunogen - 2 ADCs in ase I (DM1 and DM4) Pfizer - 1 ADC in ase III - non-odgkin's lymphoma 1913 1958 1972 1988 1993 Progenics - 1 ADC in ase I (auristatine) - prostate cancer oche - 1 ADC in ase III and 2 ADCs in ase I (DM1) - breast cancer 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 Discov. Today, 2013 1975 Covalent linked ADC tested in animal models ADC with calicheamicin 2013 2011 Adcetris approved anofi - 2 ADCs in ase II (DM4) - lymphoma and leukemia eattle Genetics - 1 ADC in ase III and 1 ADC in ase I (auristatine) - non-osdgkin's lymphoma and carcinoma 1
Eran ella The Chemistry Behind Antibody- Conjugation Mode of Action General Linkers - Use of highly potent drugs (Especially negative-charged drugs) - Ab can be also anti-cancer - is stable and usually inactive until it reaches the cancer cells ADC with a specific drug is usually more potent than the drug alone 2 2 amide C isothiocyanate thiourea haloacetyl thioether ImmunoGen Inc. Antibody- Conjugates - Components & Demands Attachment ite Linker maleimide -table -low hydrolysis thioether imple Conjugation Antibody: 1. Targets a well-characterized antigen with high tumor expression 2. Maintain all of it's properties upon conjugation to the drug 3. Minimal non specific binding Attechment site: 1. Typically through cysteine or lysine residues on the Ab 2. Variable drug:ab ratio or site-selective conjugation maleimidocaproyl Linker: 1. eavable or non cleavable 2. table with selective release of the drug : 1. ighly potent (usually 2-4 drugs per mab) 2. on-immunogenic 3. Could be linked to the Ab 4. Defined mechanism of action Current pinion in Chemical Biology, 2010, 14, 529 mercaptoacetamidocaproyl 2
Eran ella The Chemistry Behind Antibody- Conjugation Thiolation eagents 2 = mab or 2-iminothiolane "Traut's eagent" 1g = 3.5 $ C 1g = 7.4 $ C 2 K 2 C 3 quant. a 3 3 Ac 2 Canadian J. rg. Chem., 1984, 62(3), 586 Chem. Abstr., 1968, 68 C Ac Toluene reflux - 2 35% /Me 67% 2 2 Et 3 TCEP MeC MM Me PABA PP Pyridine 2 p=7.4 2 Chem. Commun., 2013, 49, 8187 p=4.5 2 PABA 3
Eran ella The Chemistry Behind Antibody- Conjugation Bifunctional Linkers 2 Bioconjugate Chem., 2014, 25, 460 C 2 Piperidine/ Tol xone Misc. Linking with pecific elease Mechanism Piperidine J. Controlled elease, 2012, 161, 422 ydrazone (apid hydrolysis in the lysosome) B p=7.4 -Tol 2 - Disulfide (eduction in lysosome) 2 Mainly by Glutathione 2 Linking by ick bosutinib Val-Cit dipeptide (site-specific proteolysis) ometimes also e-lys copper-free click - developed by Bertozzi (2007) PA, 104(43), 16793 Can deconjugate (retro-michael) Can't deconjugate 3 JAC, 2013, 135, 12994 4
Eran ella The Chemistry Behind Antibody- Conjugation Traceless Linkers 2 C thiazolidine (ydrolysis) Precise Controll on Conjugation to ynthesize homogeneous ADCs Advantages: 1. Known mab: ratio. 2. mab stability remains 2 xime Ligation C 2 2 Ellman eagent 2,2'-dithiopyridine (eduction) C 2 ature Protocols, 2013, 8, 2079 Genetically encoded unnatural amino acid with ortogonal chemical reactivity chultz, PA, 2012, 92(3), 13 2 PB, T, 30 min 1. TFAA 2. EDC, Bt 3 2 = auristatine 3. K 2 C 3 / Me 2 3 ick-like reaction for Tyrosine erceptin 3 3 1. PP, Et 3 2. EtC 2 GD 2 DTT (dithiothreitol) (eland's eagent) C P C C TCEP Et B, Pyridine Barbas, JAC, 2010, 132, 1523 3 3 K 2 C 3 / Me 3 3 3 3 5
Eran ella The Chemistry Behind Antibody- Conjugation pacers elf-immolative pacers (Cont.) n n -C 2 elf-immolative pacers Examples -C 2 2 2 Y Y Y -C 2 6
Eran ella The Chemistry Behind Antibody- Conjugation Adcetris (entuximab Vedotin) mab - entuximab, directs to protein CD30 (expressed in odgkin's lymphoma and large-cell lymphoma) Attachment ite - thiomaleimido caproyl Linker - Cathepsin (protease) cleavable linker (Val-Cit) pacer - PABA - Monomethyl auristatine E (MMAE) 2 MMAE - - antimitotic drug (inhibits mitosis, cell division), from the marine shell less Dolabella Auricularia - Blocking the polymerisation of tubulin - 200 times more potent than vinblastine Dolastatine 7
Eran ella The Chemistry Behind Antibody- Conjugation Adcetris (entuximab Vedotin) Pettit, JAC, 1989, 111(14), 5463 Dolastatine Cbz a, C 3 I TF 83% Cbz B 3, TF 95% Cbz 3 -Pyridine, DM 78% Cbz LDA, TF -78 o 87% total 33% separated Cbz (3,4, 5) C 3 2, BF 3 etherate 67% Cbz Me 2, Pd/C 63% Me Boc 1. B 3 - TF 2. modified wern 75% (2 steps) Boc LDA, Mg 2 47% after separation Boc Me 3 BF 4 DCM Boc Me K t Bu -20 o 57% Boc Me 1. 2 /Pd 2. TFA TFA 2 Me Boc Boc 1. 2 2. Mn 2 Boc 8
Eran ella The Chemistry Behind Antibody- Conjugation Adcetris (entuximab Vedotin) Tetrahedron, 2007, 63, 6115 Dolastatine Boc C ()-Boc-Isoleucine 1. CDI 2. Mg( 2 CC 2 CEt) 2 62% Boc Et u 2 [()-YP) 2, 12bar, 50 o, 24h 100% conversion 92:8 Boc Et 1. LMD. MPA, MeTf 2. aq. a / Et 60% (2 steps) Boc Me P 2 P 2 Boc Boc Et Boc Me ()-Boc-Proline Me Pyridine 77% Me Li 56% 1. Ipc 2 B 2. a 3. 2 2 60% 99% ee P 3 P 3, DEAD 3 1. P 3, 4 2. DIBC 44% >99%ee Boc 9
Eran ella The Chemistry Behind Antibody- Conjugation Kadcyla (Trastuzumab emtansine) mab - Trastuzumab, targets E2 receptor, also stop cell groth alone Attachment ite - MCC (succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate) - Mertansine (3.5 units/mab in avarage) Mertansine (DM1) - Thiol derivative of Maytansine - Antimitotic Me Me Me LiAl 4 TF 88% Me Me 1. MeC, K 2 C 3 2. a/me 90% Me Me 1. Ms, Et3 2. ai 94% Me Me CEt I 2, C 4 LDA 50% 1.DP, pts 2. Buli 3. Me Cu Me Li Cu TP Me I Me Me Li Cu 90% TP Me Me TP 1. pts/me 2. Mn 2 95% Me Me 2 LDA, TM 1. sec-buli Me Me 73% TM 2. Me Me Corey JAC, 1978, 100(9), 2916 Tel. Lett. 1978, 12, 1051 JAC, 1980, 102(4), 1439 3. Ac/aAc 10
Eran ella The Chemistry Behind Antibody- Conjugation Kadcyla (Trastuzumab emtansine) Ac Ac Ac 1. ame/me 2. gac 75% Acg Me 1. a 2. ab4 3. 99% Me 1. Trt, Pyridine 2. a (4 eq.) 3. isopropylbenzene sulfonyl imidazole 75% Trt Me Me MeLi, CuI Trt Me C 3 / 96% BF 3 Et 2 86% Et MEM 99% MEM MEM Me Me 1. nbuli 2. a, MeI Me Me Me MEM 1. C 3 Li 2. 4 3. Pb(Ac) 4 Me Me MEM Li TM then Pyridinium chloride 82% Me Me MEM 1. Me P Me Li 2. Bu 4 3. Mes 2 4. 10% 2 4 Me Me Me 1. PP, Pyridine 2. 4 / t-butanol 3. g 2, CaC 3 76% Corey JAC, 1978, 100(9), 2916 Tel. Lett. 1978, 12, 1051 JAC, 1980, 102(4), 1439 Me Me (-)--Methylmeysenine 11
Eran ella The Chemistry Behind Antibody- Conjugation Mylotarg (Gentuzumab ozogamicin) mab - Gentuzumab (antibody to CD33 expressed in leukemia cells) Linker - includes ydrazone and stable - linkage - Calicheamicin Calicheamicin - from Micromonospora echinospora, Texas, 1980 - Causes DA strand scission C 2 Me C 2 Me C 2 Me initiation Bergman Cycloaromatization DA ugar ugar ugar C 2 Me DA diradical 2 DA scission ugar 12
Eran ella The Chemistry Behind Antibody- Conjugation C 2 Me Et 3 i Bz Pd C-C acetyilide-aldehyde condensation tetronic acid ethylene glycol pts 60% DIBAL 84% MEM s-buli MEM Li Ipc 2 BMe MEM BIpc 2 (Z) 2 2, a MEM 1. TB, Im 2. C, Pyr TB Bz MEM 1. TBAF 2. wern 3. 2 Bz MEM a Bz MEM Bz MEM isoxazoline 51% after separation 1. ame 2. Cr 3, 2 4 3. TM 4. Ac 2 5. Zn 2 Li TM Ac 1. wern 2. P 3 =CC 2 Me TM Ac C 2 Me icolaou, JAC, 1992, 114, 10082 13
Eran ella The Chemistry Behind Antibody- Conjugation C 2 Me Et 3 i Pd C-C Bz TM Ac C 2 Me acetyilide-aldehyde condensation 1. K 2 C 3 /Me 2. TE triflate 3. TM Pd(P 3 ) 4, CuI 91% Et 3 i TM C 2 Me 1. Mo(C) 6 2. K 2 C 3 82% Et 3 i 2 C C 2 Me Pyridine Me 2 Et 3 i C 2 Me 1. i 2 2. Ac 2 Et 3 i C 2 Me th C KMD -90 o 48% Et 3 i C 2 Me th 1. Ms 2. i 2, Pyr Et 3 i th 1. Me 2 (99%) 2. Triphosgene, Pyr/Me (82%) 3. DIBAL, ab 4 (82%) 4. C, Pyr (84%) Et 3 i C 2 Me Bz icolaou, JAC, 1992, 114, 10082 14