atrc @ ucsf overview markscraikwells.pdf · existing antibodies . functional abs not available for...
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ATRC @ UCSF OVERVIEW
*Antibody technology research center
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Goals of the ATRC
Robust approaches for rAbs to the proteome – Initially focus (largely) on secreted proteins
• Single and multipass membrane proteins/proteases • PTMs (phospho-specific rAbs)
Technologies – Phage Ab – Automation and high throughput screening – In vitro antigen and Ab expression – Automatable cell selection
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Existing antibodies Functional Abs not available for >90% of proteome
Where available
– Half or more are not specific – Lot-to-lot and vendor-to-vendor variability – Relatively expensive – Not renewable (not cloned) – Typically IgG only
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Phage Ab based rAb generation – rAbs defined by their sequence – Forever renewable – Can be made as fragment or with any Fc (species/isotype)
A technology-driven extension and expansion of what we are doing at a cottage industry level
– Generating robust phage Ab libraries – Automation of rAb generation – Generating rAbs to challenging antigens – Providing technology training
ATRC Solution
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ATRC is funded by a P41 mechanism, that requires TRD, DBP, CSP
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TR&Ds • TR&D 1 (Marks):
– Next generation phage Ab libraries – Robust rAb generation to secreted and single and multipass membrane proteins
• TR&D 2 (Craik): – Generate secreted and type 2 TM serine proteases, receptors and intramembrane proteases – rAbs to protease targets including inhibitory rAbs
• TR&D 3 (Wells): – Phage libraries/technologies to generate rAbs to phosphorylated antigens, neo- and conformational-epitopes – Automation of selections
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Technologies: in vivo vs in vitro Abs Adapted from www.medscape.com
BINDING SELECTION
AMPLIFICATION
E.coli host
Washed Away
Fab-phage pool
Non-binding phage
Immobilized Antigen
DNA
Fab
1010
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Ab generation pipeline
Phage Selections
Antigen Production
Fab Validation
Competition ELISA
Sequencing & Expression Validation
3-4 Rounds M
amm
alia
n Ye
ast-
Disp
lay
Fab Selection
Antigen Presentation
Test to select Fab-Phage with good binding affinity
Antigen QC: SDS-PAGE; SEC rAb QC: Sequence, affinity, specificity
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TRD 1: Nature-inspired synthetic antibody libraries
Y-EphA2ECD
R0 R1 R2 R3
MDA-MB-231 (EphA2 +) cells > 30 unique antibodies
6 VH scaffolds
5 VL scaffolds
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Antibody fragments for nanoliposome targeting
Anti-EphA2 liposomal docetaxel MM-310 Completed manufacturing Phase 1 Q2 2016 Anti-EphA2 scFv
scFv D2-1A7
Anti-HER2 liposomal doxorubicin MM-302 Registration Phase 2 HER2+, progress on T DM-1 Anti-HER2 scFv
scFv F5
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rAbs to CXCR1 with functional activity
Immune phage library - GPCR DNA immunization
scFv 1C12 Select on
transfected CHO cells
B-arrestin assay for mAb biologic activity: inhibit IL-6 CXCR1 activation
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Ab Activity Based Probe to Matriptase Provides Diagnostic Information in Colon Cancer
HT29-Colon
PDX-Colon Schneider E, et al. J. Mol Bio 412 (2012) (2012 LeBeau AM, et al. PNAS 110 (2013)
Normal Adenoma Stage II Metastatic
Mat
ripta
se/H
AI-1
mRN
A Ex
pres
sion
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top view
side view
Dimeric Chloride Channel (Jan)
Conformationally Selective Fabs to Challenging Targets
Kim J et al. Nature (2015)
Nucleotide binding domains
Transmembrane domains
ABC Transporter (Chang)
TMPRSS6 membrane anchored serine protease (Bayer) TMPRSS6 TMPRSS6 + Fab
CBF-β
Vif EloC
EloB
3 Fabs
2 Fabs
HIV-host complexes: VCBC (Gross)
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Engineered Antibody Projects
Antibody-Qdot conjugates using the SpyTag/SpyCatcher system
Nanobodies selected against VGLUT2 reconstituted in nanodiscs
Labeling using LplA acceptor peptide (LAP) peptide or aldehyde peptide (CxPxR)
PDX-Colon
Drake CR et al. ACS Chem Biol. 2016 Mar 31.
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Many Thanks
Cheng Lab
Melody Lee
Gross Lab Robert Stroud
Jim Wells Kathrin Zuberbuhler Natalia Sevillano Jim Marks
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TRD3 capabilities
• Industrialized Selections • Mutant and PTM specific selections • How is the Surfaceome changed by RAS transformation? • Multiplexed antibody detection for proteomics
• BaNGS (Bar-coded antibodies NGS) • PhaNGS (Phage-antibodies NGS)
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Industrialized Antibody Pipeline
Hornsby et al 2015
~3000 Fab 346 TFs 211 Epigenetic Factors Ave affinity: 10-20nM Expression: 1-10 mg/mL
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Selection for high resolution antibodies
PTMs: Phosphorylation
Conformations: On/Off state
Mutations: KRAS
Phospho-specific
“Anchor”
Peptide Context “Reader Domain”
L3 H2 H3
Gao PNAS 2009 Thomson PNAS 2013
Koerber Nat Biotech 2013 P.Lee, K.Mou, unpublished
P. Marinec, Unpublished,
Fabon Faboff
“on” form
“off” form apo “on”
form
“off” form apo
KD(10-
9M) 2.5 >1000 330 99 4.7 17
kon(104
M-1s-1) 66 N.D. 0.8 1.6 135 55
koff(10-
3s-1) 1.7 N.D. 2.6 1.6 6.4 9.5
0.0
0.5
1.0
1.5
PSM-014
PSM-025
-10 -9 -8 -7 -6 -50
100
200
300
400Fabon
Fabcontrol
Faboff
Fab Conc (Log[M])
% C
hang
ein
Enz
yme
Velo
city
[Fab]2=[E]2
EC50=11.7±2.2nM
Ki = 0.92 uM
Gly12 Gly13 Gln61
A A E
C C H
D D K
R R L
V V P
S S R
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How oncogenes remodel cell surfaces
MCF10A-EV MCF10A-EV
“Normal” + oncogene (KRAS)
Transformed
Hanahan, D., & Weinberg, R. A. (2011). Cell
A Martinko J Blonder G Whiteley S. Bandopadhyay unpublished
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DNA-barcoding for multiplex detection BaNGS: Amy Weeks
PhaNGS: Sam Pollock Jason Moffat Dev Sidhu
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How can FNLCR help?
• Protein Antigens (100/yr) • Fc fusions • Membrane proteins and complexes
• IgG conversion, expression, distribution (100/yr) • Access to high priority targets and collaborations