Xpress CF+ : A Cell-Free Platform for the Rapid Screening and Production of Homogeneous ADCs Alexander R. Steiner, M.S. Director, Protein Biochemistry Tuesday Feb 3 rd, 215
Making novel drugs is Pammolli et al., Nat. Rev. Drug Discov., vol. 1, no. 6, pp. 428 38, Jun. 211. hard!
Why is it so hard? What do we need to consider? Complexity is increasing: Are there simple targets/therapies left to be discovered? i.e. mab vs ADC or bsab Manufacturability Chemical stability Physical stability PTMs Productivity Safety Immunogenicity Immunotoxicology Pharmacology
How Can We De-risk Biopharmaceutical Development? Need to test for critical quality attributes early. Assess developability and manufacturability in early discovery. Create workflows that allow the production of therapeutic leads in a true scale-down fashion. Paul, et al. Nat. Rev. Drug Discov., vol. 9, no. 3, pp. 23 14, Mar. 21.
My Goals for today Introduce an E.coli derived cell-free in vitro transcription translation platform for the selection and screening of mabs called: Xpress CF. Show how Xpress CF+ can be efficiently deployed for the rapid research and development of ADC therapeutics.
Sutro s Xpress CF Platform: Cell-Free Synthetic Biology Input DNA Energy, metabolites, RNAPs, NMPs, accessory proteins* Ribosomes Protein *Incorporated into strain for commercial production Cell-free environment
Advantages of Sutro s Xpress CF Platform Protein production is separated from biomass, allowing Off-the-shelf protein production Rapid expression for short make/test cycle Expression of toxic proteins high titer expression at g/l scales Xpress CF TM is an open system that can be manipulated, allowing Easy incorporation of non-natural amino acids Biochemical optimization of proteins via additives An great platform for antibody discovery!
Engineered extracts for improved IgG folding IgG
Rapid Execution of Antibody Discovery Programs Discover novel antibody fragments using ribosome display and screening Express antibodies and fragments with Sutro protein synthesis system Reformat fragments in host of different bispecific and antibody frameworks Select the best lead candidate based on in vitro and in vivo activity
Screening workflow Throughput/week 1 s Colony picking DNA prep Cell-free reaction & ELISA-based ranking 1 s Flower Plate Expression Oasis 6 1 s ELISA and FACS binding Ligand Blocking Biacore kinetic analysis
Detailed characterization of variants HT purified protein Protein quantitation ELISA And Cell Binding Kinetic characterization Epitope binning Cell Killing (secondary)
KD (nm) Ribosome Display: Unique Platform for Antibody Discovery and Optimization Rd1 +Ag -Ag Rd2 +Ag -Ag Rd3 +Ag -Ag 35. 3. 25. 2. 15. 1. 5.. Parent New leads 5 1 15 2 Recovered protein (ug/ml) Improved clones Parent K D vs protein recovered Cell Binding ELISA
Xpress CF High Titers at Multiple Scales
Scalable and Efficient a [rhgm-csf], mg/l 8 6 4 2 25 ul 3 ml 4L 1L 2 4 6 Time, min Zawada et al, (211) Biotech. & Bioeng.
Rapid Production of Biotherapeutics DAYS 2 4 6 8 Synthetic DNA Sutro Technology 5 µg protein HTS plate format 5 g protein 5-L reactor 1 g protein 1-L reactor
Antibody drug conjugates with Xpress CF+
Translation of nnaa-containing Proteins Enables Site-Specific Conjugation nnaa Input DNA NNN CF Engineered MJ TyrRS + M.j. trna RF1 Ribosomes Stop mrna NNN UAA Cell-free environment Protein
Sutro Azido nnaa IgG with DBCO-Based Linker-Warhead Azido nnaa Cu Free Click Conjugation Chemistry DBCO Linker-Peptide Warhead (MMAF)
Data Driven Design Production of Many Variants in Hours SP Number Position TAG site Mutate All Surface Sites Produce nnaa IgG Conjugate nnaa Purify Test Light Chain: 111 Sites Heavy Chain: 133 Sites Trastuzumab Model Case
Relative Cell Viability Mean Fluorescence Intensity Rapid Selection of Optimal Sites for Expression, Conjugation, Binding and Killing nnaa Incorporation and Expression SKBR3 Binding Assay Conjugated Variants Compared to Herceptin Conjugation Efficiency (Drug/MAb Ratio) DAR.4.7 1.1 MFI DAR 1.1 1.5 1.6 25 2 15 1 5 15 nm.1.1 1 1 1 1 Relative Cell Viability (Cellular ATP content, % of control) 1 5 Relative Cell Viability (Cellular ATP content, % of control) 15 1 5 nm Transform of Transform of Data 1 Data 1 Cell Killing Assay Herceptin Sutroceptin E293 K334 S136 ERB2 S136 ELISAERB2 E.1.1.1.1.1.1.1.1 1 1 ug/ml mg/ml ug/ml T359 K36 CF-Trastuzumab N361 N361 Q362 K37 Y373 S375 W381 S383 Pos N384 Cont ADC N384 S136, GY DAR=1.6 WT HIC T359 K36 Q362 K37 Y373 S375 W381 S383 S136, GY WT HIC
Top Sites: Nearly Complete Conjugation in Under 4hr with 2.5x molar Excess Warhead x1 5 1.2 1.1 1.9.8.7.6.5.4.3.2.1 HC(Site 1) DAR: 1.8 1 149553.94 147 1475 148 1485 149 1495 15 155 151 1515 152 Counts vs. Deconvoluted Mass (amu) 2 15933.5 14723.9 148173.8 149967.26 152132.8 x1 5 1.4 1.2 1.8.6.4.2 HC(Site 2) DAR: 1.95 149522.99 14778.73 14818.5 148668.6 15264.69 15878.41 148 1485 149 1495 15 155 151 1515 152 1525 Counts vs. Deconvoluted Mass (amu) 5 1.6 1.4 1.2 1.8 HC(Site 3) DAR: 1.96 15793.38 x1 5 1.4 1.2 1.8 LC(Site 4) DAR: 1.95 14957.78.6.6.4.4.2 145697.7 146826.54 148367.28 14941.37 151896.68.2 147629.94 14522.64 146532.75 148437.61 149372.7 145 146 147 148 149 15 151 152 153145 1455 146 1465 147 1475 148 1485 149 1495 15 Counts vs. Deconvoluted Mass (amu)
Single Site ADCs Shows Comparable Cell Killing Activity & Binding Affinities SKBR3 Cells Direct Binding Median Fluorescence Intensity (MFI) 15 1 5.1 1 1 1 1 nm 125 Herceptin TM HC Site 1, DAR 1.96 HC Site 4, DAR 1.97 HC Site 5, DAR 1.96 HC Site 6 DAR 1.96 SKBR3 3cells/well Day 5 Treatment Trastuzumab-CF AB4285 (MMAF) Sample SKBR3 binding SKBR3 Cell killing Kd, nm IC5, nm Herceptin TM 2. Not calculated HC Site 1 4.7.12 HC Site 4 5.6.12 HC Site 5 2.4.17 HC Site 6 7.3.19 Note; cell killing was tested on 3k cells/well, hence IC5 are in.1nm range Relative Cell Viability (Cellular ATP content, % of control) 1 75 5 25 Herceptin TM HC Site 1, DAR 1.96 HC Site 4, DAR 1.97 HC Site 5, DAR 1.97 HC Site 6, DAR 1.96 Trastuzumab-CF AB4285 (MMAF).1.1.1 1 1 nm
Site of Conjugation and Killing Activity With Different Cytotoxin Warheads Trastuzumab-CF site specific ADC cell killing activity on SKBR3 cells 12 MMAF SKBR3 Cell Killing 1K cells/well 5-day Treatment 12 vc-mmae SKBR3 Cell Killing 1K cells/well 5-day Treatment Relative Cell Viability (Cellular ATP content, % of control) Relative Cell Viability (Cellular ATP content, % of control) 1 8 6 4 2 12 1.1.1 1 1 1 8 6 4 2 SKBR3 Cell Killing nm 1K cells/well 5-day Treatment GEMCITABINE.1.1 1 1 1 nm Site 1 MMAF ADC Site 2 MMAF ADC Site 4 MMAF ADC Site 5 MMAF ADC Site 6 MMAF ADC Site 1 MMAF ADC AB4285 MMAF only Relative Cell Viability (Cellular ATP content, % of control) Relative Cell Viability (Cellular ATP content, % of control) Site 1 Gemcitabine ADC Site 2 Gemcitabine ADC Site 4 Gemcitabine ADC Site 5 Gemcitabine ADC Site 6 Gemcitabine ADC Site 1 Gemcitabine ADC SC18 Gemcitabine only 1 8 6 4 2.1 SKBR3.1 Cell Killing 1 1K 1 cells/well 1 5-day Treatment nm 12 1 8 6 4 2 DM1.1.1 1 1 1 nm Site 1 MMAE ADC Site 2 MMAE ADC Site 4 MMAE ADC Site 5 MMAE ADC Site 6 MMAE ADC Site 1 MMAE ADC AB4546 MMAE only Site 1 DM1 ADC Site 2 DM1 ADC Site 4 DM1 ADC Site 5 DM1 ADC Site 6 DM1 ADC Site 1 DM1 ADC DM1 only HC SIte 1 HC Site 4 HC Site 5 HC Site 6 LC Site 2 LC SIte 1 Free drug
Single Site ADCs Show Comparable PK Properties Trastuzumab-CF AB4285 (MMAF) ADC PK 1 Plasma concentration (µg/ml) 1 1.1.1 2-3mg/kg Beige nude xids 5 1 15 2 25 3 HC Site 1, DAR 1.96 HC Site 4, DAR 1.97 HC Site 5, DAR 1.97 HC Site 6, DAR 1.96 Trastuzumab-CF AB4285 (MMAF) Time (days) Parameter Trastuzumab-CF AB4285 (MMAF) HC Site 1 HC Site 4 HC-Site 5 HC Site 6 AUC (µg*h/ml) 1333 5191 8121 342 AUC last (µg*h/ml) 9417 6312 96 371 CL [ml/h/kg].3.32.4.54 C (µg/ml) 45 35 73 35 Half-life [d] 8 11.9 8 8.3
Best Single Site ADCs Show Differential Efficacy (single 15 mg/kg IV dose) Tumor Volume, mm3 16 14 12 1 8 6 4 2 1 2 3 4 5 6 7 Days Free Drug,.54mg/kg Dose equivalence at DAR of 4. Vehicle HC Site 1, 15mg/kg HC Site 1, DAR 1.84 HC Site 4, DAR 1.97 HC Site 5, DAR 1.97 HC Site 6, DAR 1.96 Unconjugated control 15mg/kg Trastuzumab-CF AB4285 (MMAF) Herceptin TM, 1x 3mg/kg (t =), 3x15mg/kg (weekly) All MMAF ADC treated groups vs. vehicle p <.1**** up to d45 All treatments are single dose i.v. @ t= Multi-doses of Herceptin TM dosed i.p No significant weight loss observed in all treatment groups
Concluding Remarks To balance complexity and risk in drug discovery, potential leads need to be tested and evaluated in light of developability and manufacturability considerations. Xpress CF offers the ability to produce proteins rapidly in a scalable manner, thereby allowing for fast make/test cycles fail fast, re-engineer, re-test.
Speed to the Clinic Test fast, fail fast, adjust fast. Tom Peters