Production of Homogeneous ADCs with Combination Warheads and - - PowerPoint PPT Presentation

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”

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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.

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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)

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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
• Accessory proteins and chaperones
• Scalability can be maintained from small to large scale
• Speed: faster production & cycle times
• Expansion of the genetic code to non-natural AA’s

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OCFS: Engineered Extract (E.coli)

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DNA input +

Energy Ribosome (Catalyst)

Multi-domain eukaryotic proteins

Assembled IgG

+

• AA’s, Metabolites
• RNAP
• Chaperones
• Redox, Excipients

Hours

Scalable Cell Free Synthesis: Utilizing a Cheap Energy Source

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Glutamate

TCA Cycle

Exogenous glutamate addition

• Generates NADH reducing

equivalents

Oxidative Phosphorylation

Inverted membrane vesicles

• Form during cell extract prep
• House electron transport chain
• Drives NTP production from NMPs

Jewett et al., (2008) Molec Sys Biol

OCFS: High Titers at Multiple Scales

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IgG Production in the Endoplasmic Reticulum

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• Extremely high concs. of chaperones / foldases
• 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
• GSSG: GSH ratios are 1:30 – 1:100 in the cytosol
• GSSG: GSH are 1:3 in the ER

Chaperone families involved in IgG folding and assembly

Adapted from Feige, et al. (2009) Trends in Biochemical Sciences 35: 189-198

Hsp70 Proline Isomerase Disulfide Isomerase Oxidizing environment in the ER Cytoplasmic Translation ER QC HC LC

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Sutro Engineered Strains: 10-20 g/L Chaperone Expression

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ELISA Titer

Chaperone Engineered Extracts Improve IgG Expression by >50x

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1st Gen 2nd Gen 3rd Gen Assembled IgG A DNA ratio A A A B 4th Gen 4th Gen

~1 gram / Liter IgG obtained in a 12 hour reaction

Aglycosylated TrastuzumabCF is Comparable to Commercial Material

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FACS Analysis

Yin et al, (2012) mAbs

Production of Antibodies and Fragments

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BiTE

(ScFv-Fc)2

ScFv Fab

Fab’2 IgG1

Other examples  Growth factors  Immunotoxins  Proteases  Knottins  Cellulases  Hydrogenases

Typical Expression Levels:

• 0.25 -1 Gram / Liter (Pre-Optimized)
• 6-8 Hour Reaction
• 30% Extract

8 6 4 2 400 300 200 100

Time, hr [Fab], mg/L

60 uL 300 mL

PRODUCED PURIFIED TESTED

ka (1/Ms) kd *(1/s) KD (M) 1.9E+8 3.6E-4 1.9E-12

* J. Immunol. Methods, 2002,322:94-103

4.8 E-4 (CHO)*

Fab Example: αIL-13RCF Fab Comparable to CHO Fab

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Yin et al, (2012) mAbs PRODUCED PURIFIED TESTED

scFv Example: αIL23CF scFv Production

Q & A

• Can your system produce glycosylated proteins?
• Do your antibodies have favorable pharmacokinetics

in vivo?

• Do your antibodies have ADCC activity, i.e. FcγR

binding?

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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.

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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

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Source: Micromet AG/ AMGEN

α-CD19

MT-103CF: Pre-Optimized Expression

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97 69 55.4 30 12 46 MT103CF Standard Expression Conditions 1-7 Initial Expression Level 0.4 gram / Liter Soluble 0.5 gram / Liter Total (6 hours Expression) (30% Extract) Minimal:

• Proteolysis
• Mixed Multimers
• Truncation Products

C14 Incorporation

Large Scale Production of MT103CF

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• Single Column Purification using Protein L support
• Large Monomeric Peak on Analytical SEC Column

10 kD- 15 kD- 20 kD- 25 kD- 37 kD- 50 kD- 75 kD- 100 kD- 150 kD- 250 kD-

SDS-PAGE of MT103CF:

LC-MS of MT103CF

5 x10

1 2 3 4 5 Deconvoluted Mass (amu) 54160 54200 54240 54280 54320 54360 54400 54,217.72

54242.31 54277.12 54313.85

Counts

Predicted Mass: 54,218.14 (–7.5 ppm)

MT103CF S-S bonds are intact: Protease Digestion Analysis

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 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 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 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 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 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 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 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 Q QW SS N PL TF GA G TK LE LK H HH HH H

20-25 86-108 276-279 331-354 346-354 147-150 195-226 217-225 412-417 454-495 412-417 3.6 ppm

• 1.6 ppm, 3.6 ppm

1.2 ppm, 5.3 ppm

• 10.1 ppm,

1.6 ppm

Trypsin Trypsin+GluC Double digest

Sutro Biopharma

MT103CF Selectively Binds to CD19+ and CD3+ Cells

B-cells T-Cells

Mean Fluorescence Intensity (MFI)

Dreier et al, IJC 2002

• Expressed and purifed in Sutro CF System
• Flow-cytometric analysis of binding to human CD3+ and CD19+ blood cells

KD Dreier (nM) KD 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) CF MT-103 (ug/ml)

MT103CF binding to Nalm-6 cells

MT103CF: comparable affinity to HEK and CHO MT103

Kd CF-MT103 1.5 HEK MT-103 2.0 CHO MT-103 3.9

Comparing MT103 Cell killing Activity

CF MT103 HEK MT103 CHO MT103 CD8+ T-cell EC50 (pg/ml) EC50 (pg/ml) EC50 (pg/ml) donor 1 3.7 3.5 2.7 donor 2 3.0 1.4 1.6

Donor 1 Donor 2

Vh Vl

BiTE

CH2 CH3 CH2 CH3

scFv-Fc

CH2 CH3 CH2 CH3 Vh Vl Vh Vl CH2 CH3 CH2 CH3

Tree Scorpion scFv

Fc Fc Fc

Examples of Alternative Scaffolds Tested at Sutro

Q & A

• Do all bispecific scaffolds express and fold well in

your system?

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Hypothetical Example: T-cell Recruiting Bispecific Campaign

ScFv

• 10 scFv against Target A, e.g. CD19
• 10 scFv against Target B, e.g. CD3

Bispecific

• BiTE (A+B): 10 x 10 matrix
• scFv(A+B)-kih-Fc: 10 x 10 matrix
• scFv(A+B)-HSA: 10 x 10 matrix

Test

• FACS Binding Assay – Test for Dual Target Binding
• T-cell Killing Assay

Lead

• Scale up and Test in Tumor Model

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Hypothetical Example: Multiple Target Bispecific Campaign

ScFv

• 6 different Tumor Antigens (TA1 – 6): One scFv against each
• TA1, TA2, TA3, TA4, TA5, TA6
• Each of these scFvs bind TA receptors and block their ligand

Bispecific

• Make all bispecific combinations of each in BiTE format
• TA1+TA2, TA1+TA3, TA1+TA4, TA1+TA5, TA1+TA6 …

Test

• In vitro functional assays
• In vivo efficacy models

Lead

• Scale ups top combinations and move towards non-GLP toxicology …

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Let biology drive the decision process on leads to move forward!

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

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Antibody Drug Conjugates and the Founding Fathers …

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

# drugs / Ab # drugs / Ab # drugs / Ab 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
• rthogonal chemistries regulates number and location
• f drugs attached to Mab

Translation of nnAA-Containing Proteins Enables Site-Specific Conjugation

input (DNA)

nnAA Ribosome

(Catalyst)

cell-free extract (E. coli)

NNN

mRNA

NNN UAA

Stop

tRNA

Data driven design: Production of many variants in hours

• Mutate Sites in IgG: Choose nnAA

sites using rational design, or just make all of them!

• Produce nnAA IgG: Incorporate

nnAA at 100’s of chosen sites

• Conjugate: Conjugate nnAA with

appropriate chemistry

• Purify: Separate conjugated IgG

away from unincorporated linker- warhead

• Test: Assay conjugated IgG’s for

binding and cell killing

Surface Scan Trastuzumab Model Case

nnAA TAG IgG Screen

Light Chain: 111 Sites Heavy Chain: 133 Sites

SP Number

Position

TAG site

Sutro nnAA IgG with AB3627 Linker+Warhead

Azido nnAA Cu Free Click Conjugation Chemistry DBCO Linker-Warhead

R R

Rapid Selection of Optimal Sites for Expression, Conjugation, Binding and Killing

SKBR3 Binding Assay Conjugated Variants Compared to Herceptin

Mean Fluorescence Intensity

CF-Trastuzumab Pos Cont ADC DAR=1.6

ug/mL

nM

Cell Killing Assay

Relative Cell Viability nnAA Incorporation Efficiency

nnAA Incorporation and Expression Conjugation Efficiency (Drug/MAb Ratio)

DAR: 0.4 0.7 1.1 DAR: 1.1 1.5 1.6

Site Dependent Impact on Cell Killing Observed

TAG Scanning: DAR vs. Cell Killing

Cell Killing DAR

Improving nnAA Incorporation Efficiency

•  nnAA Incorporation Efficiency
•  Expression/yield

RF1 Attenuation

aa nnAA- tRNA

Full length nnAA containing protein Truncated

Protease Sensitive RF1 is inactivated during extract production

• Abbrev. Sample

M Marker P Pellet L Lysate C Clarified 1 1 hour time point 2 2 hour time point 3 3 hour time point

De-compartmentalized Extract Cleaves RF1

HC Amber Codon Suppression

A121

WT 1 2 3 4 5 6 7 8 9 10 11 12 C

Novel nnAAs with Boosted Conjugation Kinetics

DBCO z

[Azido], mM 0 0.5 1 1.5 2 2.5 3 kobs, sec-1 0.002 0.004 0.006

pAzF pMeAzF AB 3562

7x

z Az- nnAA #1 Az-nnAA #2 Az-nnAA #3 Azido-nnAA DBCO

New Chemistry offers Improved Kinetics, Flexibility R R

Some sites are completely conjugated in under 4 hours!

[MAb] 5-20uM 5x M excess Linker/warhead Room temp.

Top Sites: Nearly Complete Conjugation

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x10 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 Counts vs. Deconvoluted Mass (amu) 147000 147500 148000 148500 149000 149500 150000 150500 151000 151500 152000

150933.50 149553.94 148173.08 147023.90 152132.08 149967.26

HC(Site 1) DAR: 1.80

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x10 0.2 0.4 0.6 0.8 1 1.2 1.4 Counts vs. Deconvoluted Mass (amu) 148000 148500 149000 149500 150000 150500 151000 151500 152000 152500 150878.41 149522.99 148668.60 148180.50 150264.69 147708.73

HC(Site 2) DAR: 1.95

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10 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Counts vs. Deconvoluted Mass (amu) 145000 146000 147000 148000 149000 150000 151000 152000 153000 150793.38 149410.37 151896.68 148367.28 146826.54 145697.07

HC(Site 3) DAR: 1.96

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x10 0.2 0.4 0.6 0.8 1 1.2 1.4 145000 145500 146000 146500 147000 147500 148000 148500 149000 149500 150000 149057.78 149372.07 147629.94 146532.75 148437.61 145220.64

LC(Site 4) DAR: 1.95

1 2

IgG HC AB3627

a5 mg/kg, Balb-c mice, Sutro b2mg/kg, Sprague-Dawley rats (US7994135B2, SeaGen patent 2011)`

CF-Trastuzumab Drug conjugate pharmacokinetics are in good agreement with Trastuzumab- MMAF conjugate literature Sutro Dataa SeaGen Datab CF-Trastuzumab Drug Conjugate Trastuzumab MMAF Conjugates AUCinf [day/µg/mL] 248 299 Clearance [mL/d/kg] 8.1 9 Half-life [d] 8 10

Pharmacokinetics of Cell Free ADCs are Comparable to Cell Derived ADCs

CF-Trastuzumab Drug Conjugate Trastuzumab MMAF Conjugates

Trastuzumab-vc-PAB-MMAF-TEG Total Ab Trastuzumab-vc-PAB-MMAF-TEG Conjugate Ab Trastuzumab-vc-PAB-MMAF Total Ab Trastuzumab-vc-PAB-MMAF Conjugated Ab

Mean -/+ SD plotted 3 mice per time point 2mg/kg, Dosed i.v.

Dose equivalence at DAR of 4.0

Best Single Site ADCs Show Differential Efficacy

KPL-4 breast

• rthotopic model

ADC

Combination Warhead ADCs

input (DNA)

nnAA1 Ribosome

(Catalyst)

cell-free extract (E. coli)

mRNA

NNN

NNN UAA

• tRNA1

NNN

NNN UAA

• tRNA2

nnAA2

Q & A

• Is the nnAA antibody used for your ADCs immunogenic?
• Can you incorporate two different nnAA into one chain of

the antibody, e.g. either HC or LC?

• Can this nnAA conjugation technology be used for other

types of molecules?

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Bringing it all together … Simple!

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Express antibodies and fragments with cell free protein synthesis

Reformat these fragments in a whole host of different bispecific frameworks Produce homogenous site- specific antibody drug conjugates with single or dual warheads

Choose the Bispecific or ADC Leads to Move Ahead to IND

OR

Speed to the Clinic …

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Offer: Contact Information

Aaron K. Sato, Ph.D. Sutro Biopharma Vice President of Research 310 Utah Avenue, Suite 150, South San Francisco, CA 94080 asato@sutrobio.com Direct: 650-676-4628 Cell: 650-438-2039 http://www.linkedin.com/in/aaronsato

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