Immunohistochemistry Antibody Validation Report for Anti-MIF Antibody (STJ94126)
APEXTM Antibody Discovery and Production Technology€¦ · B-cell immortalization using cell...
Transcript of APEXTM Antibody Discovery and Production Technology€¦ · B-cell immortalization using cell...
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APEXTM
Antibody Discovery and Production
Technology
Aridis Pharmaceuticals 1
Vu L. TruongPEP Talk Conf.
San Diego 24Jan20
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Aridis Pharmaceuticals 2
Outline
▪ Single cell cloning & mAb discovery
▪ B-cell immortalization using cell fusion
▪ Enhanced mAb production of established cell lines
• CRISPR-enabled activation of transcription regulatory genes
▪ mAb production CHO cell line technology
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Aridis Pharmaceuticals 3
Outline
▪ Single cell cloning & mAb discovery
▪ B-cell immortalization using cell fusion
▪ Enhanced mAb production of established cell lines
• CRISPR-enabled activation of transcription regulatory genes
▪ mAb production CHO cell line technology
▪ COMING SOON: Transposons-mediated gene amplification
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Aridis Pharmaceuticals 4
Rapid functional screening of entirerepertoire for potent, rare B-cell
Rapid production of mAb at high titer
Antibody Discovery and Production Challenges
Size of B-cell repertoire
~1x 106 or more unique cells
Immunized orConvalescent
Immunized
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Aridis Pharmaceuticals 5
Single Cell Screening & Clonal Selection Using NanoArrays
Key Features
▪ Enables comprehensive, functional screening at
single cell level of 106-107 cells simultaneously
▪ ~100,000x minimization of tissue culture wells.
0.5 to 3 million wells can be accommodated in a
96-well plate surface area
▪ Increase protein concentration by ~5-orders of
magnitude enables sub-picogram mAb detection
▪ Enables simultaneous single cell cloning and
plucking by microneedles for expansion
▪ Proteins, viruses, bacteria can be adsorbed as
antigen for detection of mAb
▪ Highest binders can be identified in ~1-2 days
APEXTM
Nanoculture Arrays
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Fabrication of Nanoculture Arrays
Add polymer tomaster mold
Silicon wafer
Add photoresistlayer & mask
UV
Peel
Key Features
▪ Simple, reproducible fabrication process
▪ Scalable, cost effective, no specialized companion
equipment
▪ Variety of dimensions, geometries, biocompatible polymer
materials with surface chemistries
▪ Variety of biomaterials can be adsorbed
Aridis Pharmaceuticals 6
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Aridis Pharmaceuticals 7
B-cell Repertoire Screening, Single Cell Analysis &
Single Cell Cloning with APEXTM Nanoarrays
Ag
B-cellsor CHO
Antibody
Fluorescently labeled detection antibody
Antigen(proteins, viruses, bacteria, etc.
100µm
30µm 30µm
75µm75µm
APEXTM Nanoarrays: A) Schematic top view of honeycomb shape arrays with no gaps in-between the wells, which allows seeding of all cells in solution. Packing density of 1.4 million wells can be accommodated per 96-well surface area (75um opening diameter). C) High content fluorescent imaging of B-cells secreting mAbs in nanoarrays
A) B) C)
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Versatile On-chip Assay Format for Single Cell Analysis
Live B-cell secreting Anti-RSV F-protein IgG (=antigen)
An
ti-I
gG
A
Antigen= Human AR-105 mAb
B
Top view of Nanoculture wells (A-D). Wells are embedded with antigen as indicated, followed by detection with fluorescence labeled secondary mAb
Sideview of Nanoculture wells
C
Antigen= adsorbed whole cell P. aeruginosa bacteria expressing alginate (detected w/ anti-alginate mAb)
Antigen= Adsorbed intact rotavirus expressing G1 protein (detected with anti-G1 mAb)
D
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Aridis Pharmaceuticals 9
APEXTM Technology Suite
▪ Single cell cloning & mAb discovery
▪ B-cell immortalization using cell fusion
▪ Enhanced mAb production of established cell lines
• CRISPR-enabled activation of transcription regulatory genes
▪ mAb production CHO cell line technology
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Aridis Pharmaceuticals 10
Expression Vector
Construction,Clonal
Selection
Upstream & Downstream
Process Development,
Scale Up
Current Practice: Recombinant approaches to mAb
discovery & production requires 1.5 - 2 years from
discovery to drug in vials
Mouseimmunization
Mouse B-cellscreening
Recombinant DNA cloning, humanization,transfection, single cell cloning Cell line characterization & process development
GMP ManufacturingMonths
Cloned CDRs or H&L chains
of IgG’s
GMP production
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Aridis Pharmaceuticals 11
Fusion withFusion Partner
GMPproduction
NanocultureClonal Select
Selected B-cell
Bypassing Recombinant Approaches Using B-cell
Fusion & Immortalization
ImmortalizedHybridoma
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Engineering of
the Fusion
Partner Cell Line
Aridis Pharmaceuticals 12
Genetically engineered, non-secreting IgG mouse
heteromyeloma cell line developed with high cell fusion
efficiency
Genetic modifications introduced to activate master
transcription factors associated with increased transcription
and translation of H & L chain genes of monoclonal
antibodies. Exhibits
▪ Immortality
▪ CHO-level productivity
▪ Stability
Key FeatureContains stable integrated CRISPR machinery
▪ Cas enzyme, accessory proteins, and guided RNAs
necessary to activate endogenous master transcription
factors
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Aridis Pharmaceuticals 13
RSV-F antigen used for PoC study
▪ Availability of primary RSV-F+ B-cells from biobanks
▪ Well described structure of RSV-F protein
▪ Antigen specific tools, assays and reagents are available
B-cell Screening & Cell Fusion Proof-of-Concept Study:
RSV mAb Discovery
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Aridis Pharmaceuticals 14
Repertoire Screening & Cell Fusion Work Flow
Ag-Selective
B-cell expansion and
enrichment
B Cell Fusion with
Partner Cell LineNanoculture Screening Stable & high titer
mAb production
PBMC isolation from
convalescent patients
ISOLATE PBMCS B-CELL EXPANSION CLONAL SELECTION PRODUCTION
B cell B cellFusion
Partner
B Cell fused with
Partner Cell Line
IMMORTALIZED MAB
SECRETING PROGENY
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Aridis Pharmaceuticals 15
RSV-F Ag-specific Human B Cell Activation
0 5 10 15
0
2000
4000
6000
8000
DayE
nd
po
int
IgG
T
iter
(n
g/m
l)
90%4%
Post-Sorted Memory B Post-Sorted
RSVF+
Memory B
97%
Pre-sorted Memory B
Day 7 Ag-specific Human B-cell Activation
0 5 10 15
0
1
2
3
4
5
Day
% M
em
ory
B
ce
lls
RSV-F memory B-cell secretion
Day 7 RSVF B-cell
activationDay 7 PBMCs
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After Fusion
*
On-chip Single Cell Fusion: 10%-20% Fusion Efficiency [i.e. 10-20% of progenies that fused, survived, and are producing mAb]
Left: 16x16 array of fused hybridoma at Day 27 post-fusion; Right: distribution of hybridoma clones that are producing IgG. Fluorescence intensity is used as a metric of IgG secretion level
0
4000
8000
12000
16000
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
Mea
n F
luo
resc
ent
Inte
nsi
ty
Distribution of secreted IgG by fused hybridoma clones
Aridis Pharmaceuticals 16
Engineered Fusion Partner Exhibits High Fusion Efficiency
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Aridis Pharmaceuticals 17
Rapid identification of high-producing RSV-F specific hybridoma
A. Panel of nanowells of hybridoma producing Human anti-RSVF mAb. B. Distribution of fluorescent ring intensity of anti-RSVF production compared to CHO-Aer. ** Denotes production levels higher than CHO-Aer producing at 3.5 g/L.
A B
Anti-R
SV-F
Hyb
CHO
-AR10
5
0
5000
10000
15000
Mean
In
ten
sit
y U
nit
s
n = 62,500
** Top producers are rare (less than
0.05% of the population)
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Aridis Pharmaceuticals 18
High content screening allows for rapid identification of rare high-producing hybridoma clones
(A) Hybridomas generated from cell fusion compared to CHO-Sal were screened for mAb secretion on nanocultures. ~24,000
positives identified for MabIgX ® hybridomas/ ~ 33,000 positives identified for CHO-Sal.
(B) Mean fluorescence intensity of the (B) 50 highest producing clones and (C) Correlation of mean intensity units to
cumulative volumetric titer of cell lines with experimentally-determined productivity (*** p<0.0001 by Student’s t-test, n=50.
(D-E) Representative panel showing single cell IgG production of (D) Hybridoma and (E) CHO-Sal (1 g/L volumetric titer)
A
CHO
-AR30
1
Mab
IgX H
ybrid
oma
0
5000
10000
15000
Mean Inte
nsi
ty U
nits
p<0.0001
Top 50 Lead Candidates
✱✱✱
B C D
E
Fused cells
CHO-AR301
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Stable Hybridoma Productivity Comparable to CHO over 60-Generations
▪ Endpoint titer of 3 hybridoma clones producing human
IgG demonstrates consistent productivity at each time
point
▪ Titer at generations 6, 24, and 60 shown
▪ CHO-Sal producing at 1 g/L was used as a comparator for
all stability studies
Aridis Pharmaceuticals 19
F2-1-
1
F6-33
-18b
207-
4-12
CHO
-AR30
1
0
1
2
3
4
En
dp
oin
t I
gG
T
ite
r (
g/m
l)
G6 G24 G60
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Aridis Pharmaceuticals 20
Cell Fusion to Create Hybridoma: Advantages & Disadvantages
Fusion withFusion Partner
Advantages:▪ Well established process
Disadvantages:▪ Up to ~1 year longer than the hybridoma
approach
Advantages:▪ Convenient B-cell starting point▪ Bypasses recombinant steps▪ Little to no process development needed▪ Fastest to clinical manufacturing
Disadvantages:▪ Involves hybridoma*
*Can derive CHO while in Phase 1-2 clinical studies
Current
Aridis
Engineered Fusion Partner
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Aridis Pharmaceuticals 21
Outline
▪ Single cell cloning & mAb discovery
▪ B-cell immortalization using cell fusion
▪ Enhanced mAb production of established cell lines
• CRISPR-enabled activation of transcription regulatory genes
▪ mAb production CHO cell line technology
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Aridis Pharmaceuticals 22
Key Master Transcription Factor Genes of Interests
▪ Transcription factor genes selected
for CRISPR mediated addition &
activation
• Drm2
• Fr5
• Bxp2
▪ Promotor activation is universal and
can be applied to any host cell
irrespective of B cell lineage
FR5 Drm2 Bxp2 BPRs
Ig TRANSLATION
ANTIBODY PRODUCTION
Ig TRANSCRIPTION
Ig mRNA TRANSPORT
LiE2
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CRISPR
endonuclease
D10A
H84A
activator
Binding
Transcription inhibition
Transcription activation
Binding
assembly
DSB
sgRNA
TSS TSS
Native NHEJ
Random deletions
Precise editing
Precise deletion
/ replacement
HDR
cEdit-X TM
Reconstituted NHEJ
CRISPRi
RNAP Block
CRISPRaCRISPRa
Cas9/ gRNA Complex RNA-guided binding Function
Cas9
dCas9
dCas9
TSS TSS
RNAP
RNAP
RNAP
mRNA
Recruiting the RNAP
TSS TSS
RNAP
Repair system Final results
Aridis Pharmaceuticals 23
*Transcriptional state site (TSS), homology-directed repair (HDR), non-homologous end joining (NHEJ)
CRISPR-Cas Targeted Gene Activation
Cas
Cas
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Aridis Pharmaceuticals 24
1. In traditional activation, open reading frames (ORF) of genes
need to cloned under a ubiquitous promoter. The size of ORF
is a rate-limiting step as only 1-2 genes can be activated
2. Short gRNA (20 nt) for transcriptional activation allows for
multiple concomitant activation of genes
▪ Potent activation of genes from 20-100 fold
3. Stable expression of dCas-VP64 recognizes canonical
protospacer motif (PAM) on gRNA targeting regions within
200bp of the transcriptional start site (TSS) of the plasma cell
master regulators: Drm2, Fr5 and Bxp2.
4. Activation of the three transcription factors: Drm2, Fr5 and
Bxp2 trigger a cascade of events to enhance antibody
production:
Multiplex CRISPR Guided Activation (CRISPRa)
MPH
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Current Challenges
▪ gRNA that allow for potent transcriptional activation
needs to be empirically be determined
▪ Not all gRNAs will activate gene transcription
▪ Multiple transfection and transduction are required for
a single gene
▪ Current CRISPRa technologies at best can only activate
two gRNAs at a time
Multiplex TF Activation
by CRISPRa
Aridis Multiplex CRISPRa
▪ Utilizes a unique and modular gRNA scaffold design
for multiple processing of gRNAs in a single vector
▪ Synergistic activation of multiple genes in the sample
molecular pathway superior to a single gRNA activation
Aridis Pharmaceuticals 25
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Potent synergistic activation of transcription factors in Fusion
Partner by multiplex guided RNAs
Aridis Pharmaceuticals 26
Synergistic activation of master
transcription factor genes
• Predicted synergistic activation
of targeted transcription
factors with single gRNA
• Multiplex approach showed
higher transcriptional
activation transcription factors
compared to single gRNA
activation
Drm2 Fr5 Bxp2
Drm
2
Bxp
2
Drm
2
Fr5
Bxp
2
Drm
2
Fr5
Bxp
2
Drm
2
Fr5
Bxp
2
Drm
2
Fr5
Bxp
2
Fold
Ch
ange
in m
RN
A E
xpre
ssio
n
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Aridis Pharmaceuticals 27
Hypothesis: CHO production cell lines have similar master
transcription regulatory elements, but are not comparably activated or
utilized as in B-cells
Question: Can B-cell MTREs Enhance Productivity of Existing mAb
Production CHO Cell Lines?
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Aridis Pharmaceuticals 28
CRISPR-Activation of Endogenous Master TF Regulatory
Elements Increases Productivity a CHO Production Cell Line
A-B. Significant increase in single cell IgG production measured using nanoculture arrays. B. Expression of Drm2 & Bxp2 in CRISPRa CHO C. Representative panel. Right panels show an inset of single well containing 100,000 nanowells. Data represents average titer over 100,000 wells. *** p<0.0001 by Student’s t-test, n = 100,000.
CHO-AR105
CHO-AR105+CRISPRa
CHO-AR105
CHO-AR105
CHO
-AR10
5
CHO
-AR10
5 + C
RIS
PRa
0
5000
10000
15000
Mean
In
ten
sit
y U
nit
s
***
Prdm
1a
Xbp1
0
2
4
6
8
10
Fo
ld C
han
ge E
xp
ressio
n CHO-AR105
CHO-AR105+CRISPRa
(Titer: 3.5 g/L) Drm2 Bxp2CHO-Aer CHO-Aer
Activated
CHO-Aer
CHO-AerActivated
CHO-Aer
CHO-Aer Activated
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*Pools of APEX CHO-Sal after 3 weeks selection in antibiotics were seeded on nanowells and top 50 clones were isolated using micromanipulator. E2 clone was further subcloned and
expanded for 3 weeks in CHO CD medium. Following expansion, CHO-Sal and APEX CHO-Sal (E2) clone was plated on nanowells for IgG Diffusion Assay. Figure on left shows top 100 lead
clones ring intensity distribution after 1 day for original CHO-Sal (volumetric titer: 1 g/L) and stable APEX CHO-Sal (E2 clone) with stable MTRE CHO
-AR30
1 (A
1)
CHO
-AR30
1 (E
2)+C
RIS
PRa
0
5000
10000
15000
20000
Top 100
Rin
g Inte
nsity U
nits
(Average of top 100 clones)*
Case Study #2: CRISPR-Activation of Endogenous Master
Transcription Regulatory Elements of CHO-Sal Cell Line
Aridis Pharmaceuticals 29
(Titer: 1 g/L)
CHO-Sal CHO-Sal
CRISPRa
(n=3 separate trials)
96-well T-flask
Rel
ativ
e U
nit
s
Rel
ativ
e U
nit
s
Shake flask
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
CHO-AR301 CHO-A AR301
Rela
tive U
nit
s
CHO-Sal CHO-Sal
CRISPRaCHO-Sal CHO-Sal
CRISPRa
CHO-Sal CHO-Sal
CRISPRa
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Aridis Pharmaceuticals 30
Outline
▪ Single cell cloning & mAb discovery
▪ B-cell immortalization using cell fusion
▪ Enhanced mAb production of established cell lines
• CRISPR-enabled activation of transcription regulatory genes
▪ mAb production CHO cell line technology
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Aridis Pharmaceuticals 31
Current State of the Art in mAb Manufacturing
Random integrationinto genome
mAb
Single cell screening & Clonal selection
mAb gene expression vector
Cell clone-specific manufacturing process development
GMPProduction
Months~$0.5m ~$1m $2m-$4m
5-8 months 8-10 months 4-6 months
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Aridis Pharmaceuticals 32
Product Concept:
▪ Engineered CHO cell lines with CRISPR-mediated transcription activation
harboring a full length mAb H/L genes that is designed to be swapped out
for the CDR or H/L of interest
• Integration sites and H/L gene copy numbers are known and unchanged
▪ CDR or H/L can be switched from the cell line without significant
modification of established upstream & downstream manufacturing
processes
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Aridis Pharmaceuticals 33
B.R.E.A.T.HTM B-cell Regulatory Elements Assisted Transcription Host
√ MTRE activatedBy CRISPR
√ Manufacturing Process to be Developed
APEX’s BREATHTM CHO Master Cell Line:
Development of a Manufacturing Process Template
Master Cell LineHarboring IgG1
√ Scalability to GMP Manufacturing to be
demonstrated
Ready for IgG1
Gene Swap
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Aridis Pharmaceuticals 34
Site-specificCRISPR guided
integration
B.R.E.A.T.HTM B-cell Regulatory Elements Assisted Transcription Host
Selected B-cell
Cloned CDRs or H&L of IgG’s
B.R.E.A.T.HTM
CHONanocultureClonal Select
GMPproduction
APEXTM CHO Cell Line: CDR or H/L Chain Swappable
CHO Host
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APEXTM Technology Solutions
Random integrationinto genome
mAb
Single cell screening & Clonal selection
mAb gene expression vector
Cell clone-specific manufacturing process development
GMPProduction
Months
APEX Nanoarrays
~12-15 months time savingAPEX: Discovery, Development, and Manuf.
~$0.8m - $1.2m cost saving
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CRISPR
endonuclease
D10A
H84A
activator
Binding
Transcription inhibition
Transcription activation
Binding
assembly
DSB
sgRNA
TSS TSS
Native NHEJ
Random deletions
Precise editing
Precise deletion
/ replacement
HDR
cEdit-X TM
Reconstituted NHEJ
CRISPRi
RNAP Block
CRISPRaCRISPRa
Cas9/ gRNA Complex RNA-guided binding Function
Cas9
dCas9
dCas9
TSS TSS
RNAP
RNAP
RNAP
mRNA
Recruiting the RNAP
TSS TSS
RNAP
Repair system Final results
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*Transcriptional state site (TSS), homology-directed repair (HDR), non-homologous end joining (NHEJ)
CRISPR-Cas Targeted Gene Replacement
Cas
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Genetically engineered transfection-ready production CHO
cell line harboring H &L chains of a human IgG1 and B-cell
master transcription factor regulatory elements
Proprietary CRISPR-Mediated Activation
Genetic modifications introduced to activate transcription
factors to promote productivity and stability
▪ High productivity
▪ Stability
Engineered for Productivity
▪ Engineered as a host cell line to accommodate cloning of
human H & L chain CDRs or full-length H & L chains by
homologous recombination
▪ Proprietary vector system allows for dual promoter, high-
copy amplification of plasmid DNA
Engineering of CHO with B-cell Master Transcription Factors
(B.R.E.A.T.HTM CHO)
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Target Specific Insertion of Heavy and Light Chain CDR3
Genes
Aridis’ approach technology utilizes
CRISPR/Cas to achieve site specific
integration of antibody heavy and light
chain genes for maximum productivity
Site-specificCRISPR guided
CDR3 gene swap
SalmAb
GFPmAb
BREATHTM CHOMaster Cell Line
producing Sal
Green = GFP switched cellsRed = Non-switched, Sal secreting
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APEXTM Technology Suite
B-cell Repertoire Screening:
APEXTM NanoArrays
BREATHTM CHO Master cell line
designed for CDR swapping
mAb Productivity Enhancement:
APEXTM CRISPR-assisted TF
activation
B-cell immortalization using cell
fusion