Site-Specific Protein Conjugation as an ADC Optimization Tool

Transcription

1 Site-Specific Protein Conjugation as an ADC ptimization Tool livier Laurent, Ph.D. Vice President, Ambrx AAPS 2014 Conference

2 Ambrx Quick Facts perations San Diego, California-based biopharmaceutical company founded in 2003 Proven Talent 70 employees with track record of success and past experience with major companies Rich pipeline Multiple partnered program in clinical stage, one approved drug Multiple preclinical internal program, one IND ready compound Intellectual Property Broad, enabling U.S. patents issued Capital $100M in private equity financing Multiple partners For the past 8 years, financed solely by product partnerships

3 Ambrx Pipeline Ambrx ADC and Bi-/Multi-Specific Product Candidates ARX788 HER2 ADC HER2+ Breast Cancer (1) PSMA ADC ncology (2) CD70 ADC ncology (3) Bi-specific Anti-CD3 X Folate (4) CD184-FK506 ADC (5) Multi-specific ADCs (6) Discovery Preclinical Phase 1 Phase 2 Phase 3 Marketed Ambrx Long-Acting Protein Product Candidates ARX328 Leptin Weight Management with partner Collaboration Product and Product Candidates ARX618 FGF21 Diabetes ARX720 Relaxin Heart Failure Imrestor Mastitis in Cattle (7) ADCs ncology ADCs ncology Bio-conjugates (8) (1) Ambrx owns the worldwide rights to ARX788, with the exception of the People s Republic of China, for which the rights to ARX788 have been licensed to Zhejiang Medicine Co., Ltd. (2) Potential indications include prostate cancer and glioblastoma multiforme. (3) Potential indications include renal cell carcinoma and nasopharyngeal carcinoma. (4) Potential indications include ovarian cancer. Ambrx owns the worldwide rights to this product candidate, with the exception of the People s Republic of China, for which the rights have been licensed to a collaborator. (5) Potential indications include rheumatoid arthritis, inflammatory bowel disease, and psoriasis. (6) Potential indications include hematologic and solid tumor cancers. (7) Under this collaboration, Elanco Animal Health, a division of Eli Lilly & Co., has multiple projects directed at metabolic, infectious and inflammatory diseases in livestock and companion animals in early-stage clinical development, but we do not expect any of these other product candidates to launch before (8) Potential indications include oncology and autoimmune /inflammatory diseases.

4 Maturing ADC Pipeline Hits 30 Noteworthy clinical data SGN-35: Approved August 2011 Pivotal data; 34% CR and 40% PR in patients with R/R Hodgkin lymphoma and 87% of patients had >50% tumor shrinkage in systemic anaplastic large cell lymphoma (ALCL). T-DM1: Approved March 2013 Ph III data; PFS of 9.6 vs. 6.4 months and median S of 30.9 vs for patients with advanced breast cancer, previously treated with Herceptin and taxanes. CMC-544: Ph I/II 60% RR in R/R follicular lymphomas and 20% RR in DLBCL; looks even better in combo with rituximab. ADCs against 24 different Targets are being evaluated for both blood cancers and solid tumors ADC in Development Approved Ph III Ph I - Iib Preclinical Nature Review Drug Discovery Vol What we see with this first crop of ADCs is a lot of opportunistic use of failed mabs Beverly Teicher, Chief of Molecular Pharmacology US National Cancer Institute

5 The biotech toolbox for bio-conjugation Enzymatic modifications Selective esters Native CYS reduction Partial reduction Polysaccharides modifications Chemical approaches C TER and N TER SMARTag (Redwood Bio) Thiomabs Non selective decoration Sortagging Transamination In vitro Translation (Sutro) ReCDE / EuCDE (Ambrx) Gene fusions Sequence modifications

6 In Vivo, Site Specific Incorporation of Unnatural Amino-Acids 1 2 E. Coli and CH Ambrx Components UAG 4 ReCDE EuCDE Expression system E. Coli CH Proteins produced Production scale hgh, leptin, INF, etc liters (CM) IgG 250 liters (Ambrx) 500 liters (CM) Titer > 5 g.l -1 > 1 g.l -1

7 Natural side chains for conjugations H H H H 2 N H N H H HS H 2 N H 2 N NH H N 2 NH N H N NH S

8 Conjugation chemistry in use at Ambrx Amino Acids (Conjugation sites) Reactions paf H 2 N paf2 H NaCNBH 3 H 2 N H ph 4.0 H N N xime linkage H 2 N paf3 H 2 N Axa 9.2 H H N 3 N 3 ph 4 to 5 ph 4 to 9 H N H 2 N ph 8 N N N N N H 2 N H

9 Conjugation on LYS side chains Lu & al. J. Pharm. Sci available lysines

10 Conjugation on CYS side chains Bio-conj. Chem 2005, 16: (Seattle Genetics)

11 Ambrx Creates Novel and ptimized Bio-Conjugates We place conjugate(s) at specific sites on proteins Site-specific conjugation enables optimization which can unlock efficacy/activity previously limited by toxicities Enhance potency + decrease toxicity = wider therapeutic index Random Conjugation Site-Specific, ptimized Conjugation Ambrx optimization by Medicinal Chemistry for Proteins Toxic Dose Toxic Dose Effective Dose mg/kg = Therapeutic Index (TI) Effective Dose

12 DAR affects efficacy, safety and PK CYS Conjugation LYS Conjugation Clin Cancer Res ct 15;10(20): JC June 1, 2010 vol. 28 no Higher drug loading degrades PK (stability, hydrophobicity) Higher drug loading results in lower Therapeutic Index

13 Conjugation location is critical to ADC stability Intact ADC as a function of time LC-V205C-ADC HC-A114C-ADC Fc-S396C-ADC Genentech et. al., Nature Biotech Jan 2012

14 From hundred sites to 3 preferred locations Reversed phase HPLC of deglycosylated reduced and denatured ADC Jackson & al. (2014) PLoS NE 9: e83865

15 Representative reaction kinetic on site 1 ADC Reaction without Catalyst mau Toxin Toxin % Diconjugated DAR 0 DAR 1 DAR Time (hr) Toxin % Diconjugated ADC Reaction with Catalyst DAR 0 DAR 1 DAR Time (hr) min

16 Ambrx Technology: Homogenous Conjugates No1rm DAD1 A, Sig=214,4 Ref=600,50 (R:\2\DATA\SD091102A\SD091102A \2 TX B.D) DAD1 A, Sig=214,4 Ref=600,50 (R:\2\DATA\SD091102A\SD091102A \1 TX.D) Non-Reduced Unconjugated Blue Unconjugated Red Conjugated Monoconjugated Di-conjugated 200 Drug-linker 0 No1rm DAD1 A, Sig=214,4 Ref=600,50 (R:\2\DATA\SD091102A\SD091102A \4 TX B REDUCED.D) *DAD1 A, Sig=214,4 Ref=600,50 (R:\2\DATA\SD091102A\SD091102A \3 TX A REDUCED.D) min Reduced Light Chain Unconjugated Heavy Chain Conjugated Heavy Chain Blue Unconjugated Red Conjugated Drug-linker min Non-reduced profiles separate unconjugated, mono-, and di- conjugated antibody Reduced profiles show no change to light chain and clear separation of unconjugated and conjugated heavy chain No additional peaks are observed in NR and R profiles that correlate to >2 drug-linker conjugations per mab

17 Linkers and Conjugation Sites Affect Tendency to Aggregate Drug-Linker a 1200 Blue Pos5 Linker1a-Drug Red Pos5 Linker1b-Drug Green Pos5 Linker2a-2Drug % HMW Drug-Linker b Drug-Linker c a 1b 5 0 Site 15 Site 12 Site a Monomer Sample Name Drug-Linker % Monomer % HMW 1a Pos 5pAF 1b a Pos 1pAF 1a b a HMW Species Pos 3pAF 1a b a min

18 Linkers and Conjugation Site Affect Biophysical Properties mab 1 mab 2 1 Drug-Linker 6 Different Positions 1 Position 9 Different Drug-Linkers

19 Linker Stability Assessed in Serum Position 1 (Mouse) Position 2 (Mouse) % Free MMAD SC-D1 L-A L-A SHC-D1 L-B HC-D1 % Free MMAD L-A SC-D1 SHC-D1 L-A L-B HC-D1 Incubation Time (Hours) Incubation Time (Hours) Position 1 (Rat) Position 2 (Rat) % Free MMAD Incubation Time (Hours) L-A SC-D1 L-A SHC-D1 L-B HC-D1 % Free MMAD Incubation Time (Hours) L-A SC-D1 L-A SHC-D1 L-B HC-D1 Linker A Drug Linker B Drug Incubation in plasma at 37 o C with extraction and mass spec analysis

20 Conjugation site affects linker stability in vivo ELISA Total Mab Detection Protease DRUG ADC position 1 ADC position 2 Attached Drug Detection Payload transfer to HSA (Agensys collaboration, Jackson & al. (2014) PLoS NE 9:e83865 ADC position 1 ADC position 2 Study Male Sprague Dawley rats IV bolus at 1 mg/kg, n=5/dose group Sampling: at intervals from 3 hrs to 21 days

21 In Vivo PK and ADC Stability comparison PK Comparison of Ambrx vs. Conventional ADCs (Partner ADC Program)

22 Rodent Model of NCI-N87 Human Gastric Cancer Experimental Details: Ten animals per group 1, 3 or 5 mg/kg single dose I.V. Same mab Same Drug (Amberstatin 269) DAR=2 for Ambrx ADCs DAR= average of 4 for cysteine ADC Conclusion by Agensys: At 5 mg/kg dose, efficacy of Ambrx site-specific ADCs were statistically superior to the cysteine ADC (p value = )

23 Anti-Her2 ADC (AS132) versus Standards Conjugates vs Disulfide Cys vs Thiomab vs T-DM1 - Comparator ADCs produced by Agensys - DAR = Drug:mAb ratio - Amberstatin-132 toxin - MDAMB361DYT2 cell line - Dose = 1 mg/kg X 4 - Data generated in two separate studies using the same protocol

24 ARX788 efficacy compared to T-DM1 Xenograft studies replicated by partner JIMT-1 (breast) tumors NCI-N87 (gastric) tumors Tumor Volume (mm3) Vehicle Her2-AS269 1 mg/kg Her2-AS mg/kg T-DM1 1 mg/kg T-DM1 3.3 mg/kg Days after start of dosing Tumor Volume (mm3) vehicle Her2-AS269 1 mg/kg Her2-AS269 5 mg/kg T-DM1 1 mg/kg T-DM1 5 mg/kg Days after cell implantation Tumor Volume (mm3) SKV-3 ovarian Vehicle (PBS) IV qdx1 Her2-AS269 IV 1.67 mg/kg Her2-AS269 IV 5 mg/kg T-DM mg/kg T-DM1 5 mg/kg Days after start of dosing Tumor Volume (mm3) BT474-EEI tumors Days after cell implantation vehicle Her2-AS269 1 mg/kg Her2-AS269 3 mg/kg T-DM1 1 mg/kg T-DM1 3 mg/kg

25 ARX788: Differentiated Efficacy Profile BT474-EEI breast tumor cell xenograft studies Tumor volume post-treatment (mm 3 ) T-DM1 ARX788 Ambrx ADC Dose (mg/kg) Dose (mg/kg) Day 42 tumor study data from trastuzumab-dm1 publication Day 42 tumor data from Ambrx xenograft study

26 Defined Drug Loading Increases Stability AND Tolerability Collaboration with Agensys

27 Summary Heterogeneous conjugation degrades performance Stability and PK Safety Therapeutic Index Site specific conjugation can be used to optimize many ADC attributes Aggregation propensity Thermodynamic stability Half-life Potency and preclinical safety (rodents) Moving an ADC portfolio forward internally and with partners

28 Acknowledgements The scientists at Ambrx and at our partners