Nature Biotechnology 2000 18:1287

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Nature Biotechnology (2000) 181287
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Ribosome display
Absence of a stop codon is necessary to prevent
dissociation of the newly synthesized protein
from the ribosome.
RT-PCRproduct of affinity captured material
Affinity selection in rabbit reticulocyte
ribosome display is antigen-dependent and
requires the absence of a stop codon. In the
presenceof astop codon, the rprotien synthetic
complex dissociates.
From Hanes a et al., FEBS Letter, 450, 105-110,
1999)
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Starting material here HuCAL fully synthetic
human combinatorial antibody library based on
modular consensus frameworks and CDRs randomized
with trinucleotides. Framework consensus
sequences of 7 of heavy chain V-regions x 7
framework consensuses of light chain V-regions
49 combinations, randomized trinucleotides in
the variable regions.
Prokaryotic ribosome binding site
3 stem-loop
5 stem-loop
scFv
T7 promoter
3 spacer
DNA
mRNA

• DNA construct assembled by ligation and PCR no
  cloning in E. coli
• Here 2 x 109 molecules.
• Later conservative estimate of no. of molecules
  that can be screened
• 2.6 1011 per milliliter of reaction (Nature
  Methods 4269 (2007))

scFv single chain Fragment of variable chain
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Fully Synthetic Human Combinatorial Antibody
Libraries (HuCAL) Based on Modular Consensus
Frameworks and CDRs Randomized with
Trinucleotides Achim Knappik, Liming Ge,
Annemarie Honegger, Peter Pack Melanie Fischer,
Gunter Wellnhofer, Adolf Hoess, Joachim
Wolle Andreas Pluckthun and Bernhard Virnekas J.
Mol. Biol., 296 57-86 (2000)
CDR3 targeted here.
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• 7 different natural framework VH regions x 7
  different framework VL regions ? 49 combinations
• randomized CDR3s by inserting synthetic oligos
  beween restriction sites
• CDR3 site of VJ (light chain) and VDJ
  (heavy chain) joinings

Knappik et al, JMB, 296 57-86 (2000)
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Different amino acid combinations achieved after
insertion of sythetic oligonucleotides.
Planned Found
Figure 7. Comparison between design and
experimental composition of CDR3 libraries used.
For each position of the CDR3 region (numbering
according to Kabat et al., 1991 for HCDR3 the
position before H101 is numbered 100z, the length
variable region is numbered from H95 to H100s),
the amino acid composition in the planned
libraries (P, left columns) is compared with the
composition found from sequencing 257 clones of
the initial libraries (F, right columns). The
TRIM mixture indicates the mixtures of
trinucleotides used in the oligonucleotide
synthesis (see Table 3 of the Supplementary
Material). Occupied indicates the number of amino
acids encoded by the respective mixture and found
in the sequenced clones, respectively.
Knappik et al, JMB, 296 57-86 (2000)
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Repeat
Nature Biotechnology (2000) 181287
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via an E. coli S30 extract
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Anti-insulin?
insulin
B biotin
B
Avidin bead
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Along with specific insulin binding activity was
a larger amount of non-specific (or
different-specific) binding, as assayed by
binding of 35S-labeled translated protein in the
translational complex.
Note more background than foreground
non-specific binders, not competable by insulin
Insulin
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Plenty of framework mutations introduced by the
PCR (i.e., not in the HuCAL library)
Varied in the HuCAL library (CD3)
Length variations
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Only a 8 of the 49 possible framework
combinations were used. Three were used most
often. So there may be a preference for
particular frameworks for this antigen.
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Consensus WT HuCAL sequence(without the PCR
mutations) All selected clones exhibit tighter
binding. That is, the addiitonal mutations
introduced by PCR were selected.
Entirely cell-free no bottlenecks due to
relatively inefficient DNA transfer steps. One
is always dealing here with populations of
molecules.
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Screening for natural antibodies using yeast
Aga2 becomes associated with Aga1 in the
cell. Aga1 becomes covalently anchored to the
cell wall
Nature Biotechnology  21, 163 - 170 (2003)
Published online 21 January 2003
doi10.1038/nbt785 Flow-cytometric isolation of
human antibodies from a nonimmune Saccharomyces
cerevisiae surface display library Michael J.
Feldhaus1, Robert W. Siegel1, Lee K. Opresko1,
James R. Coleman1, Jane M. Weaver Feldhaus1, Yik
A. Yeung2, Jennifer R. Cochran2, Peter
Heinzelman2, David Colby2, Jeffrey Swers2,
Christilyn Graff2, H. Steven Wiley1  K. Dane
Wittrup2
C-myc

• Created a general scFv library from 58 people
  using PCR primers from various classes of Igs.
• Cloned in E. coli to maintain complexity of 109.
• Cloned into yeast for surface expression
  maintaining complexity
• Screen for antigen binding by 2-steps
• Magnetic bead capture (avidin micromagnetic beads
  20 nm, bind via biotinylated antigen) (too many
  cells for initial FACS )
  
  ?
• FACS
• Found Abs vs. many antigen tried.
• EGF, EGFR, p53 peptides, fluorescein, etc.
• Kd nM range, often.

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First purification to get numbers down to lt 108
for FACS magnetic (20 nm) microbeads
µ
Bead-labeled cells retreined on magnetic column
Biotin-
B
www.miltenyibiotec.com
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Isolation of engineered, full-length antibodies
from libraries expressed in Escherichia coli.
Mazor, Y., Van Blarcom, T., Mabry, R., Iverson,
B.L., and Georgiou, G. 2007. Nat Biotechnol 25
563-565.
Immunize mice ? spleen cells ? mRNA ? PCR Vh and
Vl cDNAs?clone 107 ? E. coli with ProteinA/mem
anchor fusion protein (ZZ) ? displayed outside
inner membrane ? lysozyme ? spheroplasts ?add
fluorescent antigen ? FACS
Fortunately, 100 pM avidity, 10x affinity.
Divalency?
PA B. anthacis protective antigen
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DNA shuffling (Stemmer - Maxygen)
(PCR)
recombination via PCR priming
PCR
Mutations can be purposely introduced prior to
shuffling (e.g., by chemical mutagenesis) Or by
error-prone PCR during the process Or by
faithful PCR during the process (lower
level) Or by using different members of a gene
family (paralogs or homologs)
Analogous functions in the same organism e.g.
families of proteases
Same function in a different organism e.g.,
interferons
Stemmer, W., Proc Natl Acad Sci U S A. 1994 Oct
2591(22)10747-51.                       
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Shuffling an engineered human gene with a mouse
gene
Boxed nts are not conserved. Low temp PCR was
required due to the poor homology (Klenow)
Stemmer, W., Proc Natl Acad Sci U S A. 1994 Oct
2591(22)10747-51.                       
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Advances in directed protein evolution by
recursive genetic recombination applications to
therapeutic proteins Aaron L Kurtzman, Sridhar
Govindarajan, Katherine Vahle, Jennifer T Jones,
Volker Heinrichs and Phillip A Patten
Current Opinion in Biotechnology 2001, 12361370
Maxygen Inc., 515 Galveston Drive, Redwood City,
CA 94063, USA e-mail aaron.kurtzman_at_maxygen.com
e-mail phil_patten_at_maxygen.com
Population genetics theory behind DNA shuffling
(not so clear) Different shuffling methods (not
explained) Table of 30 therapeutic proteins that
have been altered compared to their natural
state. But most were NOT altered by DNA
shuffling. But a good source of interesting
examples of proteins altered by DNA shuffling
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• Why alter a natural protein?
• For therapeutic proteins
• Tighter target binding (enabling lower doses,
  perhaps)
• Enable desirable side reactions
• Disable undesirable side reactions
• Imprive half-life in bloodstream
• Improve production characteristics (e.g.,
  secretion, stability)
• For industrial enzymes
• Altered (even new) substrate specificity

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Example 1 Human interferon alpha (IFNa ) 20
IFNa genes in a gene family Each human protein is
poor at inducing virus resistance in mouse
cells Shuffle the family, Only 2 rounds.
Mixing the non-conserved a.a. differences Select
for induction of viral resistance in mouse
cells Achieve 185X more activity in shuffled
protein. Best was even 3.5 X more active than
the best mouse IFN. And still equally effective
vs. human cells. So an example of a dramatic
change in biological specificity (binding?)
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• Example 2 Antibodies breaking the natural
  limit on affinity selection
• Natural affinity ceiling of 10-10 M (100 pM)
• Kd off time / on time
• Endocytosis rate 10 min to several hours
• So no selection for off rate longer than 3 h
  (104 sec)
• Diffusion-limited on-rate 10-6 M/s
• Selection limit of affinity of natural antibody
  evolution off rate / on rate
  (1/10-6)/(1/104) 10-10 Kd
• --------------------------------------------------
  -------------------------------------------------
• Directed evolution of antibody fragments with
  monovalent femtomolar antigen-binding affinity
• Eric T. Boder, Katarina S. Midelfort, and K. Dane
  Wittrup. Proc Natl Acad Sci U S A. 2000 97(20)
  1070110705.
• Used the FACS to selected single chain
  anti-fluorescein antibodies displayed on the
  surface of yeast cells. Competed with free
  fluorescein.
• DNA shuffled. 4 cycles. Selected for slow off
  times.
• Achieved 50 fM affinities. Thats femtomolar, 50
  x 10-15 M 5 x 10-14 M 0.05 pM (compare 100
  pM above)
• Slower off rate than biotin-avidin (gt5d).
• Others
• scMAb DNA shuffling ribosome display
  selection in DTT (reducing agent)

J Foote and HN EisenKinetic and Affinity
Limits on Antibodies Produced During Immune
ResponsesPNAS 1995 92 1254-1256
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Example 3 Enzyme stabilization PAI-1, a
protease inhibitor (TPA inhibitor) Error prone
DNA shuffling ? 245X increase in stability Found
11 aa changes, presumably affecting protein
folding Back-cross to remove non-contributory
mutations DNA shuffle best clone with original
WT DNA. Maintain selective pressure. Analyze
progeny see 2 of the 11 lost, not needed,
replaced by WT sequence.
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• Example 4 Viral tropism murine leukemia virus,
  a retrovirus
• MLV, 6 strains, all ? poor infection of CHO
  cells.
• DNA shuffled envelope gene of 6 strains
• chimeric virus that can infect CHO cells
• And selected incidentally for non-inactivation
  under conditions of laboratory manipulation (100X
  centrifugation resistant)

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A more global version of DNA -- genome shuffling
A different (unnatural) kind of genetic mixing
using whole genomes
Zhang et al., Nature 415, 644 (2002) Can mix
Streptomyces (bacteria) genomes by protoplast
fusion. ? effective diploid bacteria The fused
cells will generate recombinant haploid
spores. Target tylosin production (an
antibiotic) Mutagenize a culture, collect 22,000
survivors. Screen them all for tylosin
synthesis, pick the top 11. Protoplast fuse all
11 with each other. Collect 1000
progeny. Screen 100 for tylosin, collect the
best 7. Protoplast fuse again. Collect 1000
again. Screen for tylosin again. Characterize
the best 2 Tylosin production is up 9-fold. So
productivity is up 9-fold, without a tremendous
amount of work (22000 screen the most)
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A more supervised version of DNA
shuffling Multivalent avimer proteins evolved by
exon shuffling of a family of human receptor
domains Nature Biotechnology 23 1556
(2005) Joshua Silverman, Qiang Liu, Alice
Bakker, Wayne To, Amy Duguay, Ben M Alba, Richard
Smith, Alberto Rivas, Peng Li, Hon Le, Erik
Whitehorn, Kevin W Moore, Candace Swimmer, Victor
Perlroth, Martin Vogt, Joost Kolkman  Willem Pim
C Stemmer Avidia, Inc., 2450 Bayshore Parkway,
Mountain View, California 94043
Strategy Create therapeutic proteins by
combining hundreds of known binding domains from
receptor proteins in new random combinations and
selecting for binding to a specific target by
phage display
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Organization of binding domains in typical
mammalian receptors
A-domains(35-40 AAs/domain) determine binding
speficicity of many receptors
Typical receptor structures
of 217 A-domains
(metaphorically?)
as a spacer between domains
2 domains cooperating
Bipartite domain
Dual specificity domain
Degenerate oligos synthesized to coding for 35-40
AAs of the A domains Only AAs naturally found at
each position were coded for. Conserved
structural AAs were kept constant. Complexity
1023 . Actually realized 1010 as phage display
particles Select one domain at a time, serially,
by panning
LRP LDL receptor related protein
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Isolation of a high affinity binding protein to
IL6 ( interleukin 6 ) by iterative selection
(IL6 is a target for cancer and
inflammation) IL6 immobilized on
plates. Recovered proteins from first cycle
cloned and tested for IL6 binding 20 top binders
pursued. Add the domian library to each of the
20 first round winning dominas. Again pick best
20 overall. After a third cycle pick the very
best binder C326
Monomer protein Screened for binding
Monomer displayed on phage coat
Build 20 dimers pools from 20 best monomers
M13 phage
IL6
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Add an IgG-binding domain at the end to prevent
rapid clearance (measured half-life of 89 hours
in monkeys)
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Binding measured by a competition assay
(AlphaScreen)
Reactive oxygen species can react only over a
short distance with and acceptor bead
Laser
Luminescence
Reactive Oxygen
IL6 receptor
IL6
Avidin bead biotinylated IL6 gp130-Fc
Protein A bead Competition IL6
(non-biotinylated) or C326 avimer
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More AlphaScreens effect of combining the 3
domains
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Physical assay Biacore surface plasmon
resonance to measure binding kinetics
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Biological assay Stimulation of proliferation of
TF-1 cells (erythroleukemia line) 16 h of 3H-TdR
incorporation to measure promotion of DNA
synthesis
Commercial anti-IL6 antibodies
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(No Transcript)
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Acute phase inflammatory response induced by IL6
is reversed by avimer C326 (in mice)
Specific for IL6-induced inflammation