ADC Case Study-Custom Synthesis of ADC Linker-payload SET

1. ADC Case Study Custom Synthesis of ADC Linker-payload SET Introduction The warhead of an antibody-drug conjugate (ADC) is comprised of a cytotoxic payload drug and a molecular linker that covalently bridges the antibody and the payload. With an extensive library of payload drugs and linkers, plus years of experience in synthetic and conjugation chemistry, Creative Biolabs is dedicated to helping our clients design and prepare highly customized linker and drug-linker complexes for the creation of ADCs using our featured “DrugLnk” services. From conventional payload-linkers bearing auristatin or maytansinoid derivatives to more innovated warheads such as topoisomerase inhibitors, we have created many unique compounds tailored to clients’ special projects. Presented here is a case study for the synthesis of two customized payload-linker complexes via the “DrugLnk” organic synthesis service. SN38 is the payload of choice and two cleavable linkers are used to formulate the SN38-linker complexes. For the protection of custom IP, the reaction conditions, catalysts, as well as solvents are omitted from the synthesis routes. SN38: a novel payload for ADC development SN38 is an antineoplastic drug. It is the active metabolite of irinotecan (an analog of camptothecin - a topoisomerase I inhibitor) but is 1000 times more active than irinotecan. So far, two ADCs bearing SN38 as payload, both developed by Immunomedics, have entered clinical trial stage. CBL has extensive experience with SN38-related payload-linker synthesis. The successful cases include the synthesis of Mc-vc-PAB-SN38, a relatively simple synthesis involving 3 steps, and that of CL2A-SN38 (proprietary for Immunomed- ics), a much more complex construct that requires 10-step synthesis. SN38 ADCs in clinical trials SN38 (irinotecan prodrug) Sacituzumab govitecan (also known as IMMU-132) IgG1 TROP2 CL2A-SN38 TNBC (phase III) NCT02574455 Immunomedics (licensed to Seattle Genetics) Metastatic CRC (phase II) NCT01915472 Immunomedics IgG1 CEACAM5 CL2A-SN38 Labetuzumab govitecan (also known as IMMU-130) Case 1: Mc-vc-PAB-SN38 OH Synthesis of Mc-vc-PAB-SN38: O N Ret. Time: 1.68 ES-API Positive O O O O H N O N N H N H 496.2 O Reactions for each step were carried out in optimized conditions with suitable solvents and catalysts, if necessary. The yields of each step were recorded and reported, and the products from each step were characterized by LC-MS to ensure the correct MW. The final product: Mc-vc-PAB-SN38 was characterized by both LC-MS and 1HNMR to assess purity and validate structure. Synthesis lead time was 3 weeks. 1. O 17500 O O + 15000 N Molecular peak [M+H] 12500 NH O 10000 O NH2 7500 2. 5000 Mc Di-pep�de vc Self-elimina�on PAB 2500 130.2 186.2 991.0 0 Compound specifics: 200 400 600 800 1000 1200 1400 m/z 3. 1. 2. 3. Chemical formula: C51H58N8O13 MW: 991.05 Elemental analysis: C-61.82; H-5.86; N-11.31; O-21.01 2. Structure confirmation: compound structure validated by 1HNMR 4. 10.342 10.008 -0.001 8.089 8.075 8.066 8.048 7.811 7.793 7.606 7.589 7.435 7.417 7.412 7.307 7.290 6.996 6.944 5.504 5.320 5.310 5.099 5.071 4.193 3.450 3.376 3.361 3.347 3.104 3.089 2.635 2.500 2.423 2.362 2.182 2.167 2.153 2.140 2.126 1.491 1.476 1.458 1.311 1.297 1.281 1.195 1.180 1.165 0.908 0.894 0.879 0.852 0.839 0.821 0.808 Synthesis route design: Based on the structure of Mc-vc-PAB-SN38, a 3-step synthesis route was designed. Results OH O N O O O O H N Mc-vc-PAB-SN38 was successfully synthesized in 3 weeks. Final product is characterized as: O N N H N H O O O O N NH O O NH2 Chemical Formula:C51H58N8O13 ExactMass:990.41 1. Purity: > 95% by LC-MS Proton peaks are labeled and integrated for structure identification 6.16 8.56 1.77 0.76 5.94 5.23 1.72 5.06 *ADC1 A, ELSD 4.61 4.25 2.10 2.02 1.68 1.59 1.57 0.83 0.71 0.72 Purity calculated based on peak areas mAu 1.675 780 0 0.5 1 1.5 2 2.5 min* 2 0 10 8 6 4 PPM Case 2: CL2A-SN38 Synthesis route design: Results OH Based on the structure of CL2A-SN38, a 10-step synthesis route was designed. CL2A-SN38 was successfully synthesized in 10 weeks. Final product is characterized as: H2 N N O *DAD1 A, Sig=214,4 Ref=off OH H N N H O Purity calculated based on peak areas N 1.437 mAU O MMTNH MMTNH MMTNH H2N O O 0 NH2 O O O O O O 0 0.5 1 1.5 2 2.5 m 52% 91% 88% O O NH Fmoc OH OH H2N NH Fmoc HN HN FmocNH ES-API Positive OH OH O 1 2 4 3 N H O O O 494. 4 n OTBS N N 250 00 N O H N MMTNH N OTBS O N n=7 200 00 MMTNH O O O NH HN N Cl OH 741. 0 N O 70% O O O H N O 100 00 O Compound specifics: O 5 150 00 NH HN O N3 O N O 7 n O O O O O H N 62% H2O O N3 O 537. 2 O 50 00 O 751. 8 n 1. 2. 3. Chemical formula: C73H97N11O21 MW: 1479.7 Elemental analysis: C-59.88; H-6.63; O-22.97; N-10.52 O 0 6 OH 40 0 60 0 80 0 100 0 120 0 140 0 160 0 180 0 m/ z H2N N steps O 2. Structure confirmation: compound structure validated by 1HNMR O NH HN O N O O O O H N 8.099 8.077 8.039 8.020 7.978 7.794 7.537 7.513 7.494 7.473 7.457 7.284 7.263 7.239 7.219 7.151 6.685 5.974 5.724 5.682 5.372 5.330 5.301 5.292 5.261 5.177 4.933 4.903 4.556 4.533 4.516 4.501 4.488 4.130 4.099 4.056 3.882 3.870 3.813 3.622 3.617 3.609 3.582 3.514 3.496 3.479 3.461 3.367 3.333 3.316 3.071 3.063 3.053 2.992 2.193 2.174 2.133 2.115 2.098 2.080 1.856 1.824 1.734 1.704 1.699 1.671 1.642 1.485 1.425 1.400 1.354 1.335 1.316 1.290 O CL2A-SN38 structure breakdown: O O N O O n O NN N HN O TM n = 7 O OH 7 10 H2N HO N O O Cl HN HN O O OH N O O Cl O O O O N Cl O O O H O Cl Synthesis of CL2A-SN38: H N O N Cathepsin B cleavable peptide when AA=Phe { O O Short PEG for solubility [ ] O SN-38 O O O 1. Reactions for each step were carried out in optimized conditions with suitable solvents and catalysts, if necessary. The yields of each step were recorded and reported, and the products from each step were characterized by LC-MS to ensure the correct MW. The final product: CL2A-SN38 was characterized by both LC-MS and 1HNMR to assess purity and validate structure. Synthesis lead time was 10 weeks. O O O { O O O H N N N H N N [AA]-Lys- H N CH2O O O O O N pH-dependent cleavage site 7 O 2. 35.41 O O N N Proton peaks are labeled and integrated for structure identification 0.86 1.13 1.84 2.00 0.98 2.35 1.00 2.03 1.13 1.83 2.28 9.04 8.23 6.88 5.19 4.25 CL2-SN-38: AA = Phe CL2A-SN-38: AA=None 3.35 3.16 N 2.41 2.25 2.19 2.02 1.96 O O N H 3. hRS7 N 10 8 6 4 2 0 PPM O NMRB003, CDCl3, 3 1 . 0 0 4 : 1 F 0 3 g z : X E USER: nmrB601 -- DATE: Fri Jan 13 02:21:33 2017 8 6 7 2 3 : d 1 S T P 4. 2 0 . 1 : 2 F 0 0 8 : 1 W S 1 : W P 2 4 2 2 : 1 F O 4 . 7 6 4 8 : A N u 8 . 3 e s c e s 0 . 1 : D P c L B 0 . 0 : Nuts - $pdata Conclusions References Using the "DrugLnk" custom synthesis service, Creative Biolabs successfully synthesized two 1. Ramesh, M., Ahlawat, P., Srinivas, N.R. Biomed Chromatogr. 2010, 24: 104-123. 2. Cardillo, T.M., Govindan, S.V., Sharkey, R.M., et al. Bioconjug Chem. 2015, 26: 919-931. 3. Sharkey, R.M., Govindan, S.V., Cardillo, T.M., et al. Mol Cancer Ther. 2012,11: 224-234. 4. Jain, N., Smith, S.W., Ghone, S., et al. Pharm Res. 2015, 32: 3526-3540. 1. SN38-linker complexes. "DrugLnk" custom synthesis service is well-suited for simple synthesis tasks with only a few 2. steps and complex organic synthesis with multiple steps (in this case, a 10-step synthesis was Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Tel: 1-631-871-5806 Fax: 1-631-207-8356 Email: info@creative-biolabs.com Web: https://www.creative-biolabs.com/adc successfully demonstrated). The compounds prepared by the "DrugLnk" custom synthesis service are correct in structure 3. and show excellent purity.