Production of Homogeneous ADCs with Combination Warheads and Bispecifics using an Open Cell Free System Aaron K. Sato VP, Research Sutro Biopharma, Inc.
What do you do? • “Aaron works with proteins” 2
Three Simple Concepts • Sutro can: – Express antibodies and antibody fragments in a cell free system, – Reformat these fragments in a whole host of different bispecific frameworks, and – Produce homogenous site-specific antibody drug conjugates with single or dual warheads. 3
Expression of antibodies and fragments using a bacterial extract system • Case Study: Expression of marketed antibody, Trastuzumab • Case Study: Expression of panel of antibody fragments (Fab & scFv) 4
OCFS: Open Cell Free Synthesis A bacterial in vitro transcription translation (IVTT) system. We have precise control over the process of protein synthesis! Ideal redox, DNA, pH, temp, time, excipient concentrations o Accessory proteins and chaperones o Scalability can be maintained from small to large scale o Speed: faster production & cycle times o Expansion of the genetic code to non-natural AA’s o 5
OCFS: Engineered Extract (E.coli) DNA input Hours Assembled IgG + Energy + • AA’s, Metabolites • RNAP Ribosome • Chaperones • Redox, Excipients (Catalyst) Multi-domain eukaryotic proteins 6 6
Scalable Cell Free Synthesis: Utilizing a Cheap Energy Source Oxidative Phosphorylation TCA Cycle Jewett et al. , (2008) Molec Sys Biol Glutamate Exogenous glutamate addition Inverted membrane vesicles • Generates NADH reducing • Form during cell extract prep equivalents • House electron transport chain • Drives NTP production from NMPs 7
OCFS: High Titers at Multiple Scales 8
IgG Production in the Endoplasmic Reticulum Extremely high concs. of chaperones / foldases o • Antibody producing cells can have 10-100X increased chaperone up-regulation • 2 mM Protein Disulfide Isomerase, 1% of total cell protein • Antibodies require disulfide and proline isomerization to achieve correct quaternary structure Far more oxidizing environment vs cytosol o • GSSG: GSH ratios are 1:30 – 1:100 in the cytosol • GSSG: GSH are 1:3 in the ER 9
Chaperone families involved in IgG folding and assembly Cytoplasmic Hsp70 Disulfide Isomerase Translation HC Proline Isomerase LC Oxidizing environment in the ER ER QC Adapted from Feige, et al. (2009) Trends in Biochemical Sciences 35: 189-198 10
Sutro Engineered Strains: 10-20 g/L Chaperone Expression ELISA Titer 11
Chaperone Engineered Extracts Improve IgG Expression by >50x 2 nd Gen 1 st Gen 3 rd Gen 4 th Gen 4 th Gen DNA ratio A A A A B Assembled IgG ~1 gram / Liter IgG obtained in a 12 hour reaction 12
Aglycosylated Trastuzumab CF is Comparable to Commercial Material FACS Analysis Yin et al, (2012) mAbs 13
Production of Antibodies and Fragments Other examples Growth factors Immunotoxins Proteases Fab Knottins (ScFv-Fc) 2 IgG 1 Cellulases Hydrogenases Fab’ 2 ScFv BiTE Typical Expression Levels: • 0.25 -1 Gram / Liter (Pre-Optimized) • 6-8 Hour Reaction • 30% Extract 14
Fab Example: α IL-13R CF Fab Comparable to CHO Fab PRODUCED PURIFIED TESTED 400 k a (1/Ms) k d *(1/s) K D (M) 60 uL 1.9E+8 3.6E-4 1.9E-12 300 300 mL [Fab], mg/L 4.8 E -4 (CHO) * 200 100 0 0 2 4 6 8 Time, hr * J. Immunol. Methods, 2002,322:94-103 15
scFv Example: α IL23 CF scFv Production PRODUCED PURIFIED TESTED 16 Yin et al, (2012) mAbs
Q & A • Can your system produce glycosylated proteins? • Do your antibodies have favorable pharmacokinetics in vivo ? • Do your antibodies have ADCC activity, i.e. Fc γ R binding? 17
Reformatting targets into bispecific constructs & testing for binding and functionality • Case Study: Show that reformatting scFv (from other sources and ribosome display) into bispecific antibodies is quick and easy using our system. 18
Variety of Different Formats • Biology may dictate the best format to use • Is there a one size fits all format? • Ideally, it would be good to have a variety of formats available Kontermann, Mabs, 2012
MT103 Example α -CD19 Source: Micromet AG/ AMGEN 20
MT-103 CF : Pre-Optimized Expression Standard Expression Conditions 1-7 C14 Incorporation 97 69 55.4 MT103 CF 46 Initial Expression Level 0.4 gram / Liter Soluble 0.5 gram / Liter Total 30 (6 hours Expression) (30% Extract) 12 Minimal: • Proteolysis • Mixed Multimers • Truncation Products 21
Large Scale Production of MT103 CF SDS-PAGE of MT103 CF : 250 kD- 150 kD- 100 kD- 75 kD- 50 kD- 37 kD- 25 kD- 20 kD- 15 kD- 10 kD- • Single Column Purification using Protein L support • Large Monomeric Peak on Analytical SEC Column 22
LC-MS of MT103 CF Predicted Mass: 54,218.14 (–7.5 ppm) 5 x10 54,217.72 5 4 Counts 3 2 54242.31 1 54277.12 54313.85 0 54160 54200 54240 54280 54320 54360 54400 Deconvoluted Mass (amu)
MT103 CF S-S bonds are intact: Protease Digestion Analysis 3.6 ppm M DI QL TQ S PA SL A VS LG Q RA TI SCK AS Q SV DY DG DS YL N WY QQ IP GQ PP K LL IY DA SN L V 60 20-25 S G IP PR F SG SG S GT DF TL NI H PV EK VD AA T YH CQ QS TE DP W TF GG GT KL E IK GG GG SG G G 120 86-108 -1.6 ppm, 3.6 ppm G S GG GG SQ V QL QQ S GA EL VR P GS SV KI SC K AS G YA FS SY WM N WV KQ RP GQ GL E WI GQ IW P 180 147-150 GD G DT NY N GK FK GK A TL TA DE S SS TA YM QL SS L AS ED SA VY F C A RR ET T TV GR YY YA MD Y 240 195-226 217-225 W GQ GT TV T VS SG G GG SD IK L QQ SG AE LA RP G AS VK MS C KT SG YT F TR YT MH WV KQ R PG QG 300 276-279 1.2 ppm, 5.3 ppm L EW I GY IN P SR GY TN Y NQ KF KD KA T LT TD KS SS T AY MQ LS SL TS E DS AV YYC A RY YD DH Y 360 331-354 346-354 C L DY WG Q GT TL TV S SV EG GS G GS GG SG GS G GV DD IQ LT QS P AI MS AS PG EK VTM TCRA S S 420 -10.1 ppm, 412-417 1.6 ppm SV SY MN W YQ QK S GT SP KR W IY DT SK VA SG V PY RF SG SG SG T SY SL TI SS M EA ED AA TY Y C 480 454-495 Q QW SS N PL TF GA G TK LE LK H HH HH H Trypsin Trypsin+GluC Double digest 412-417
MT103 CF Selectively Binds to CD19+ and CD3+ Cells o Expressed and purifed in Sutro CF System o Flow-cytometric analysis of binding to human CD3+ and CD19+ blood cells Sutro Biopharma Dreier et al, IJC 2002 Mean Fluorescence Intensity (MFI) B-cells T-Cells 0 CF MT-103 (ug/ml) K D Dreier (nM) K D Sutro (nM) CD19 + Nalm-6 cells 1.5 1.9 CD3 + T- cells 260 236 Sutro BiTe binding is comparable to Micromet published data (Dreier 2002)
MT103 CF binding to Nalm-6 cells Kd CF-MT103 1.5 HEK MT-103 2.0 CHO MT-103 3.9 MT103 CF : comparable affinity to HEK and CHO MT103
Comparing MT103 Cell killing Activity Donor 1 Donor 2 CF MT103 HEK MT103 CHO MT103 CD8+ T-cell EC 50 (pg/ml) EC 50 (pg/ml) EC 50 (pg/ml) donor 1 3.7 3.5 2.7 donor 2 3.0 1.4 1.6
Examples of Alternative Scaffolds Tested at Sutro CH2 CH2 Fc CH3 CH3 CH2 CH2 CH2 CH2 Fc Fc V h V l CH3 CH3 CH3 CH3 V l V h V h V l scFv BiTE scFv-Fc Tree Scorpion
Q & A • Do all bispecific scaffolds express and fold well in your system? 29
Hypothetical Example: T-cell Recruiting Bispecific Campaign • 10 scFv against Target A, e.g. CD19 • 10 scFv against Target B, e.g. CD3 ScFv • BiTE (A+B): 10 x 10 matrix • scFv(A+B)-kih-Fc: 10 x 10 matrix Bispecific • scFv(A+B)-HSA: 10 x 10 matrix • FACS Binding Assay – Test for Dual Target Binding • T-cell Killing Assay Test • Scale up and Test in Tumor Model Lead 30
Hypothetical Example: Multiple Target Bispecific Campaign • 6 different Tumor Antigens (TA1 – 6): One scFv against each • TA1, TA2, TA3, TA4, TA5, TA6 ScFv • Each of these scFvs bind TA receptors and block their ligand • Make all bispecific combinations of each in BiTE format • TA1+TA2, TA1+TA3, TA1+TA4, TA1+TA5, TA1+TA6 … Bispecific • In vitro functional assays • In vivo efficacy models Test • Scale ups top combinations and move towards non-GLP toxicology … Lead Let biology drive the decision process on leads to move forward! 31
Produce homogenous site-specific antibody drug conjugates with single or dual warheads • Case Study: Show that we can make single site-specific homogeneous antibody drug conjugates • Case Study: Show that we can make dual site-specific homogeneous antibody drug conjugates with two different warheads 32
Antibody Drug Conjugates and the Founding Fathers … # drugs / Ab # drugs / Ab # drugs / Ab 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Random Lysine Conjugation Cysteine Conjugation Site Specific Cys Conjugation Heterogeneity translates to poor PK, stability and efficacy
Antibody Drug Conjugates and the Generation X Era … The Non-Natural Amino Acid Advantage 1. Controlled stability: nnAA chemical space provides alternatives to cysteine or lysine for creating stable MAb~drug junction 2. Homogeneity: site-specific conjugation using orthogonal chemistries regulates number and location of drugs attached to Mab
Translation of nnAA-Containing Proteins Enables Site-Specific Conjugation input nnAA (DNA) NNN tRNA Stop mRNA NNN UAA Ribosome (Catalyst) cell-free extract ( E. coli )
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