Download Antibody Selection From Immunoglobulin Libraries Expressed in

Antibody Selection From Immunoglobulin
Libraries Expressed in Mammalian Cells
Ernest Smith, Ph.D. Senior Vice President, Research & CSO
[email protected] 585-271-2884 www.vaccinex.com
Vaccinex Corporate Summary
Headquarters: Rochester, NY
Employees: 51
History:
Founded in 1997
Core technology invented by founders
at the University of Rochester
Exclusive world-wide license in all fields of use
Intellectual Property: 70+ U.S. and foreign patents and patent applications.
Funding:
Closed a $50M Round in 2009
$33M in previous venture funding
$10M in research grants
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Focus:
Four antibodies in pre-clinical development
Two INDs expected to be filed in Q4 2010
Novel antibody discovery platform
Vaccinex Technology Summary: Discovering
Antigens and Therapeutic Antibodies
Vaccinia Virus Library Construction
Vaccinia Virus
____
Vaccinia virus infects most mammalian cells
____
Recombinant proteins expressed by vaccinia virus infected cells
undergo normal post-translational modifications and trafficking
____
Very versatile mammalian expression vector, used for >20 years
as a vector for basic scientific and vaccine research
____
The conventional recombination technology allows for the
introduction of one defined gene at a time into vaccinia virus
____
This is sufficient for expression of a single gene product but does
not enable functional cloning of unknown genes from a library
A New Technology was Needed to Enable More Efficient Creation
of Recombinant Vaccinia Virus
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Standard Homologous Recombination
Wt Vaccinia Virus
TK
plasmid
Infect
plasmid
Nucleus
Ligate
cDNA of interest
plasmid
Transfect
cDNA
cDNA
There is no selection against wild type virus
Selection (ex: BUdR)
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TK
cDNA
99.9 % Wild type Virus
0.1% recombinant virus
Strategy to Generate 100% Recombinant
Vaccinia Virus
V7.5/tk genomic DNA
N A
plasmid
TK
Cut with N and A
Ligate
cDNA of interest
Transfect arms + infect
defective helper virus
plasmid
plasmid
Nucleus
Transfect
cDNA
cDNA
Nature Medicine 7:967-972 (2001)
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Host cell containing ONLY recombinant
vaccinia virus
Vaccinex Technology Summary
Vaccinex has developed proprietary technology that allows for
the creation of large and diverse cDNA libraries in a vaccinia virusbased vector.
Millions of different vaccinia recombinants in each library.
Nature Medicine 7:967-972 (2001)
Technology for functional cloning in mammalian cells.
Efficient identification of immune target antigens
Selection of fully human monoclonal antibodies
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Antibody Libraries Expressed in
Mammalian Cells
We have developed a large library-based antibody discovery technology that can
efficiently express and select fully functional IgG antibodies in mammalian cells
Directly expresses complete, bivalent antibodies
Separate heavy and light chain libraries
107 Ig-H X 107 Ig-L =1014 combinations
rapid initial screening of 107 to 108 combinations
Affinity improvement by sequential substitution of heavy and light chains
Allows for efficient conversion of mouse MAbs to fully human
Expression of antibody libraries in secreted IgG format
Ig-H gamma I (no TM domain) + Ig-L = secreted MAb
Screen by ELISA (soluble antigen) or FMAT (cell surface antigen)
Expression of antibody libraries on the surface of mammalian cells
Ig-H gamma I (with TM domain) + Ig-L = surface display
Isolate specific antibodies by MACS/FACS
Isolate specific antibodies following induction of apoptosis
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Vaccinex Antibody Selection Platforms
1. Conversion of Mouse MAb to Human MAb
Secreted
SecretedAntibody
AntibodyPlatform
Platform
Membrane Antibody Platform: Selection by Cell Sorting
2. de novo Antibody Selection
3. Affinity Improvement of Human Antibodies
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Conversion of Mouse MAb to
Fully Human MAbs (Flow Chart)
Mouse Antibody
Clone V-genes, create vaccinia with chimeric Ig-H and Ig-K
Chimeric Ig-H
Use vaccinia with chimeric Ig-H to select replacement human Ig-K
Human Ig-K LIBRARY
Use selected human Ig-Ks to select replacement human Ig-H
Human Ig-H LIBRARY
1st Generation Human MAbs
Optimize (If necessary)
Lead MAb
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Conversion of Mouse Mab to
Human Antibody
Perform ELISA or FMAT Screen
Fixed Ag-specific chain:
complexity = 1
Irrelevant antigen
Plate out and amplify
mini complementary
libraries from complex
parent libraries
Co-infect Fixed chain
+ mini-libraries
in HeLa cells
Approximately 100 1000 antibodies/well
Antigen of interest
Complexity = 100 - 1000 pfu
Titer = 2x106/ml
100 replicate plates = 1x106 to 1x107
Immunoglobulin clones
Subclone specific chain from positive wells and rescreen
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Use Mouse Ig-H to Select
Human Ig-K: Round 1
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Use Human Ig-K to Select
Human Ig-H: Round 1
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Expression and Characterization
of Selected MAbs
The V genes in the clonal Ig-H chain and Ig-L chain vaccinia
recombinants are cloned from vaccinia virus into mammalian expression
plasmids.
Monoclonal antibody is expressed as full length IgG1/Kappa by
transfection of the plasmids into CHO cells. IgG is purified using Protein
A, and each selected MAb is tested for specificity, affinity and function.
As shown on the following slide, purified IgG was tested against an
antigen of interest as well as a panel of control antigens.
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Specificity of Antigen-Specific MAb
The V Genes From Selected MAbs are Transferred into Mammalian
Expression Vectors and expressed as soluble IgG
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Affinity of Antigen-Specific Antibodies
A large panel of human antigen-specific MAbs were selected
including the two leads MAb 2071 and MAb 2090:
MAb
Chimeric
MAb 2071
MAb 2090
Biacore
affinity (nM)
0.08
0.03
0.03
Selected Antibodies compete with the mouse antibody for binding to the
antigen of interest and demonstrate functional activity that is superior to
that of the chimeric antibody
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Vaccinex Antibody Selection Platforms
1. Conversion of Mouse MAb to Human MAb
Secreted Antibody Platform
Membrane Antibody Platform: Selection by Cell Sorting
2. de novo Antibody Selection
3. Affinity Improvement of Human Antibodies
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Vaccinex Antibody Platform Transformation
of Mouse MAb to Human MAb
Add labeled antigen
then anti-IgG
Magnetic
beads / FACS
Single antigen-specific
chimeric heavy chain in
vaccinia
Use specific
light chain to
identify human
heavy chain
Repeat infection
for additional
selection cycle(s)
Analyze and
select best light
chain clones
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Ig Expression
Human Ig light chain
library in vaccinia
Ag binding
Collect positive
cells
Extract virus
analyze positive
pool by flow
cytometry
Amplify
virus
Transformation of Mouse MAb
into Human MAbs
Chimeric Ig-H + Chimeric Ig-L
Fwd Scatter
Fwd Scatter
Wild Type
Ag Binding
Ag Binding
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Human Ig-H + Human Ig-L
Fwd Scatter
Fwd Scatter
Chimeric Ig-H +Human Ig-L
Ag Binding
Ag binding
Relative affinity of fully human
antibodies and chimeric MAb
The Ig-H and Ig-L chain genes are cloned from recombinant vaccinia virus into
mammalian expression plasmids. Antibody is produced as full length soluble
IgG1/Kappa by transfection of the plasmids into CHO cells.
IgG is purified using Protein A, and each selected MAb is tested for
specificity, affinity and function.
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MAb
Biacore
affinity (nM)
chimeric
0.05
Human Mab 416
0.27
Human Mab 926
0.15
Human Mab 1156
0.09
Human Mab 1338
0.07
Human Mab 1339
0.05
Human Mab 1259
0.09
All MAbs compete for binding to
antigen with the original mouse MAb
and demonstrate strong functional
activity
Advantages of
For the conversion of mouse MAbs into human MAbs, our library
technology allows for:
The selection of multiple candidate lead antibodies
derived from distinct VH and VL germ line genes
Multiple distinct Leads and backups
Conservation of epitope specificity
Affinity improvement from the original MAb
Built in selection for good expression levels in
mammalian cells
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Screening for Human MAbs
in Secreted IgG Format
Ig-H chain library:
complexity = 100 clones/well
Titer = 2x106/ml
1
Irrelevant antigen
Co-infect Ig-H + Ig-L minilibraries in HeLa cells
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Complexity = 1000
antibodies/well
Plate out and amplify
mini vv Ig libraries from
complex parent libraries
Perform ELISA
1
Ig-L chain library:
complexity = 10 clones/well
Titer = 2x106/ml
Isolate antigen-specific
Ig-L and Ig-H by limiting
dilution cloning and re-screening
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Antigen of interest
3
Affinity Improvement
Perform ELISA or FMAT Screen
Fixed Ag-specific chain:
complexity = 1
Irrelevant antigen
Plate out and amplify
mini complementary
libraries from complex
parent libraries
Co-infect Fixed chain
+ mini-libraries
In HeLa cells
Approximately 100 1000 antibodies/well
Antigen of interest
Complexity = 100 - 1000 pfu
Titer = 2x106/ml
Subclone specific chain from positive wells and rescreen
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Affinity Improvement of Human
MAbs by V-Gene Replacement
The V genes from positive clones are isolated and cloned into mammalian
expression plasmids. Antibody is produced as full length soluble
IgG1/Kappa by transfection of the plasmids into CHO cells.
IgG is purified using Protein A, and each selected MAb is tested for
specificity, affinity and function
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Antibody
Biacore
affinity (nM)
Parental
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Replacement #1
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Replacement #2
6
Replacement #3
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Summary of Selected Projects
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Project
Approximate number of
Primary MAbs selected
A
10
De novo
B
60
mouse to human
X
10
mouse to human
I
20
De novo
P
20
De novo
IL6
60
mouse to human
C35
90
De novo and mouse to human
VX5
60
mouse to human
VX70
20
mouse to human
VX90
30
mouse to human
Selection type
Development of New Antibody
Selection Strategies
Chimeric Receptor Based
Selection Strategies
Because of their size, the throughput of MACS/FACS with mammalian cells
is still lower than what can be achieved with yeast or Phage
_______
Employ hybrid receptors whose signaling results in the induction of
apoptosis and loss of cell adherence (recombinant virus can be recovered
from floating apoptotic cells)
Candidate: Fas
Anti-Fas antibody induce apoptosis
Create Ig-Fas Chimeric molecules (ECD = Ig; ICD = Fas DD):
Infect Hela in monolayer
Add Ag
Harvest apoptotic cells (floaters) after overnight incubation
_______
Advantages:
Selection system
No FACS/Beads needed, so throughput is very large
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Selection of Human MAbs
using Hybrid Ig-Fas Receptor Libraries
Add Antigen
Heavy Chain Ig-Fas
Library in vaccinia
Ag binding
induces apoptosis:
cells detach
Human Ig light chain
library in vaccinia
Harvest Floating Cells
Repeat
infection to
enrich
Analyze clones
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Extract virus
analyze positive
pool by flow
cytometry
Amplify
virus
Test Cell Detachment with Ig-Fas
Recombinant Vaccinia Virus
Confluent monolayers of HeLa cells were infected at moi = 1
each of Ig-Fas and Ig-L
Antigen specific and control Ig-Fas used
_______
6 hours after infection, Antigen is added
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Cells allowed to detach for 24 hours
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Supernatant was harvested, the wells were washed two
times with PBS
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Remaining cells were stained with crystal Violet
Take Pictures
Solubilize with Acetic Acid and read OD570
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Ig-Fas Recombinant Vaccinia Virus
Mediates Antigen-Dependent Cell Detachment
Antigen Specific Ig-Fas
Cells remaining after
detachment
Control Ig-Fas
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Test Enrichment with Ig-Fas
Recombinant Vaccinia Virus
Confluent monolayers of HeLa cells were infected at moi = 1
with antigen specific Ig-Fas and co-infected with admixtures
of Ig-L virus
1.0% Antigen specific Ig-L + 99% Control Ig-L
0.1% Antigen specific Ig-L + 99.9% Control Ig-L
0.01% Antigen specific Ig-L + 99.99% Control Ig-L
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6 hours after infection, Antigen was added
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Cells allowed to detach for 24 hours
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Harvest Floating cells, extract and amplify virus
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Use this virus for a second round of enrichment.
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After the second round, take the virus and test for enrichment
by staining for Antigen specific binders by flow cytometry.
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Enrichment for Antigen specific Ig-L
1.0%
Starting
Sample
After
Enrichment
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0.1%
0.01%
Transformation of Mouse MAb to Human
MAb Using Ig-Fas Hybrid Libraries
Add Antigen
Ag binding
induces apoptosis:
cells detach
Single antigen
specific Ig-Fas
vaccinia
Human Ig light chain
library in vaccinia
Use specific
light chain to
identify human
heavy chain
Analyze and
select best light
chain clones
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Harvest Floating Cells
Sort for Ag
specific
binders after
2nd cycle
Extract virus
Amplify
repeat for 2nd
cycle of
enrichment
Transformation of Mouse
MAb into Human MAbs
Antigen Specific Control Ig-Fas
Ig Expression
Ig Expression
Control Ig-Fas
Ag Binding
Ag Binding
Ig Expression
Ag Binding
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Clone 11
Ig Expression
Clone 6
Ag binding
Competitive Advantages
Challenge
Vaccinex Technology Advantage
Selection of antibodies with conserved epitope specificity
Conversion of Non-human
and similar or even improved affinity and functional activity.
Antibodies to Fully Human
and Affinity Improvement of
Selection of multiple antibodies derived from distinct VH and
Existing Human Antibodies VL germ line genes with different biochemical properties.
Manufacturing
De novo Antibody
Selection
Re-engineering
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Intrinsic selection for high expression in mammalian cell lines,
easily adaptable to manufacturing.
Broader target range than mouse-based platforms
Very Large Throughput
Vaccinex expresses complete MAbs that do not require reengineering into IgG format.