Characterizing and engineering antibodies against
the epidermal growth factor receptor
by
Ginger Chao
B.S. Chemical Engineering
Rice University, 2002
Submitted to the Department of Chemical Engineering
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY IN CHEMICAL ENGINEERING
at the
Massachusetts Institute of Technology
February 2008
© 2007 Massachusetts Institute of Technology
All rights reserved
Signature of Author: _____________________________________________________________
Department of Chemical Engineering
October 15, 2007
Certified by: ___________________________________________________________________
K. Dane Wittrup
C.P. Dubbs Professor of Chemical Engineering and Bioengineering
Thesis Supervisor
Accepted by: ___________________________________________________________________
William M. Deen
Professor of Chemical Engineering
Chairman, Committee for Graduate Students
Characterizing and engineering antibodies against the epidermal
growth factor receptor
by Ginger Chao
Submitted to the Department of Chemical Engineering on October 15, 2007 in partial fulfillment
of the requirements for the degree of Doctor of Philosophy in Chemical Engineering
ABSTRACT
Epidermal growth factor receptor (EGFR) signaling leads to cellular proliferation and
migration, and thus EGFR dysregulation can significantly contribute to the survival of tumor
cells. Aberrant EGFR signaling due to receptor overexpression, mutation, or autocrine ligand has
been observed in a wide variety of malignancies, and antibody drugs which inhibit EGFR
signaling have been developed. However, the epitopes of most EGFR antibodies have not been
characterized, and the marginal efficacies of current antibodies underscore the need for improved
therapeutics.
In this thesis work, we have created a novel method of epitope mapping, which is the
determination of antigen residues responsible for mediating an antibody-antigen interaction. In
our technique, a library of random mutants of the EGFR antigen is displayed on the surface of
yeast, and the library is combinatorially selected for loss of binding to the antibody being
mapped. If a mutant shows loss of binding to an antibody, then that residue is a potential contact
residue. In addition, we found that many mutants caused a global misfolding of the antigen,
requiring the use of high-throughput sorting to remove the misfolded mutants. The development
of our epitope mapping method using random mutagenesis and yeast surface display enabled the
successful mapping of four different antibodies and three designed ankyrin repeat proteins
binding to EGFR.
In addition, we continued work on engineering novel antibodies against EGFR domains
II and IV. Antibodies against these domains are hypothesized to directly inhibit receptor
dimerization and subsequent activation, as opposed to traditional anti-EGFR antibodies which
block ligand binding. To accomplish this, peptide mimics of EGFR loops were used as antigens;
however, antibodies generated using both yeast surface display and rabbit monoclonal
technology were peptide-specific, but did not bind to EGFR protein. Finally, we developed a
mathematical model to describe equilibrium EGFR ligand binding and dimerization. Based on
observations that polyclonal antibodies against EGFR domain II or IV eliminate high affinity
EGF binding normally observed on the surface of cells, we hypothesized that preformed inactive
dimers were the high affinity component. Our model incorporating this hypothesis successfully
reproduced experimental data, resulting in characteristic concave-up Scatchard plots.
Thesis Supervisor: K. Dane Wittrup
Title: C.P. Dubbs Professor of Chemical Engineering and Bioengineering
2
Dedicated to my parents, Jim and Grace, and my brother Eric.
Thanks for your love and support.
3
Acknowledgements
I would like to thank my research advisor Dane Wittrup, who has been an ideal thesis advisor
these four years. He was always there with great advice and guidance while allowing me the
perfect amount of independence and autonomy. I am grateful for the way which he helped my
project evolve into several successful side projects and for his contributions to the development
of my abilities as a researcher. I also want to thank my thesis committee, Doug Lauffenburger
and Harvey Lodish, who were always willing to take time out of their extremely busy schedules
to offer great ideas and their unique perspectives.
Dane has created a great research environment, and I would like to thank everyone I’ve worked
with in the Wittrup lab. In particular, Jennifer Cochran originally trained me during my first
summer, getting me started with my project and providing invaluable assistance and ideas. Other
people who had been working on the EGFR project before I joined were extremely helpful:
Yong-Sung Kim, Mark Olsen, and Alejandro Wolf-Yadlin. I learned a lot from the people who
were in the lab before me: Katarina Midelfort, Jeffrey Swers, Bala Rao, Andy Yeung, Dave
Colby, Shaun Lippow, and Stefan Zajic. Of course, I also want to thank Andy Rakestraw for his
assistance in the lab and moral support outside the lab. Thanks to Steve Sazinsky, for his
insightful discussions and very detailed protocols; Ben Hackel, who has helped me learn about
library design and the intricacies of Microsoft Word; and Shanshan Howland, a great benchmate
and inventor of many ingenious lab tricks. Thank you to Dasa Lipovsek, who provided me with
excellent advice and got me a job! I also want to thank everyone else who has joined the lab over
the years: Wai Lau, Andrea Piatesi, Greg Thurber, Annie Gai, Mike Schmidt, Margie Ackerman,
Pankaj Karande, Eileen Higham, Kelly Davis, David Liu, and the new kids Jamie Spangler, Jordi
Mata-Fink, and Chris Pirie. Thanks everyone for letting me borrow reagents, answering
questions, and providing insight at group meetings!
I would like to thank my UROPs Heather Pressler and Nick Nguyen. Also thanks to everyone at
the flow cytometry core facility: Glen Paradis, Mike Doire, Mike Jennings, Yong Xie, and
Michelle Perry. I am grateful to the various people with whom I have had the opportunity to
collaborate: Arvind Sivasubramanian and Jeff Gray at Johns Hopkins, Jeff Way at EMD
Lexigen, and Daniel Steiner and Andreas Plueckthun at the University of Zurich.
Thank you Amy Lewis, Mark Styczynski, and Jared Johnson for being great friends and helping
me make it through my first semester at MIT. Thanks to my parents, Jim and Grace Chao, and
my brother Eric for being supportive through all these years.
My research was supported by a National Defense Science and Engineering Graduate
Fellowship, NIH grant CA96504, and a David Koch Graduate Fellowship from the MIT Center
for Cancer Research.
4
Table of Contents
Chapter 1: Introduction and background .......................................................................
1.1
EGFR signaling and trafficking ...................................................................
1.2
Biochemical and structural characterization of EGFR ................................
1.3
EGFR in cancer ............................................................................................
1.4
Current EGFR-targeted therapeutics ...........................................................
1.5
Overview of thesis work ..............................................................................
1.6
Works cited ..................................................................................................
7
7
11
15
18
25
27
Chapter 2: Epitope mapping of proteins binding to EGFR ..........................................
2.1
Background ..................................................................................................
2.2
Materials and methods .................................................................................
2.3
Results ..........................................................................................................
2.3.1 Creation of epitope mapping library ................................................
2.3.2 Epitope mapping of mAb 806 ..........................................................
2.3.3 Effects of EGFR mutations on binding of mAbs 806 and 175 ........
2.3.4 Epitope mapping of mAbs 225 and 13A9 .......................................
2.3.5 Epitope mapping of mAb EMD72000 .............................................
2.3.6 Epitope mapping of DARPins .........................................................
2.4
Discussion ....................................................................................................
2.5
Works cited ..................................................................................................
36
36
39
42
42
44
52
54
63
68
75
80
Chapter 3: Engineering antibodies against EGFR ......................................................... 84
3.1
Background .................................................................................................. 84
3.2
Materials and methods ................................................................................. 94
3.3
Results .......................................................................................................... 100
3.3.1 Characterization of antibody clones against EGFR peptides ........... 100
3.3.2 Initial round of selection for scFvs against peptides ........................ 103
3.3.3 Second round of affinity maturation ................................................ 107
3.3.4 Third round of affinity maturation ................................................... 110
3.3.5 Fab display libraries ......................................................................... 112
3.3.6 Rabbit monoclonal antibodies .......................................................... 115
3.4
Discussion .................................................................................................... 118
3.5
Works cited .................................................................................................. 120
Chapter 4: Mathematical modeling of high affinity EGF binding ................................ 123
4.1
Background .................................................................................................. 123
4.2
Results .......................................................................................................... 126
4.2.1 Model formulation and output .......................................................... 126
4.2.2 Model characterization ..................................................................... 134
4.3
Discussion ..................................................................................................... 137
4.4
Works cited ................................................................................................... 142
Appendix 1: Sequences ........................................................................................................ 145
Appendix 2: Protocol ............................................................................................................ 153
Curriculum vitae ................................................................................................................... 154
5
Abbreviations Used
ADCC
APC
CDR
DARPin
EGF
EGFR
Fab
FACS
FITC
IgG
Kd
koff
kon
mAb
MACS
pAb
PBS
PCR
PE
scFv
TGF-α
TKI
VH
VL
Antibody-dependent cellular cytotoxicity
Allophycocyanin
Complementarity determining region
Designed ankyrin repeat protein
Epidermal growth factor
Epidermal growth factor receptor
Fragment antigen binding portion of an antibody
Fluorescence-activated cell sorting
Fluorescein isothiocyanate
Immunoglobulin G
Apparent dissociation constant
Kinetic off-rate
Kinetic on-rate
Monoclonal antibody
Magnetic cell sorting
Polyclonal antibody
Phosphate-buffered saline
Polymerase chain reaction
Phycoerythrin
Single-chain variable fragment of an antibody
Transforming growth factor α
Tyrosine kinase inhibitor
Variable heavy chain of an antibody
Variable light chain of an antibody
6
Chapter 1: Introduction and background
1.1
EGFR signaling and trafficking
The epidermal growth factor receptor (EGFR/Her1/ErbB1) is a type I transmembrane
receptor tyrosine kinase and a member of the ErbB receptor family. This family includes three
other members: Her2 (ErbB2/Neu), Her3 (ErbB3), and Her4 (ErbB4). The receptors are
activated by a number of different ligands, such as epidermal growth factor (EGF) and
transforming growth factor alpha (TGF-α) which bind to EGFR, and epiregulin and betacellulin
which bind to both EGFR and Her4 (1). The exception to this is Her2, for which no natural
ligand has been discovered. The binding of ligand drives receptor dimerization, and the various
receptors in the EGFR family are capable of both homo- and heterodimerization with each other.
However, it does not appear that Her2 homodimerizes at physiological expression levels, and
homodimerization of Her3 receptors would not lead to receptor phosphorylation since Her3 is
kinase dead and thus must be trans-activated by heterodimerization with other ErbB receptors.
The receptor dimerization event allows for kinase activation and trans-phosphorylation of
residues in the intracellular domain. A number of different EGFR tyrosine residues can become
phosphorylated, depending on the stimulating ligand and dimerization partner. Following
stimulation of EGFR by EGF, the major sites of phosphorylation include tyrosines 1086, 1148,
and 1173 (2). Phosphotyrosine-binding proteins then associate with these residues in the ErbB
receptor tail and become activated, initiating various signaling cascades. Many of these
interactions have been characterized in detail, such as Grb2 binding to EGFR tyrosine 1068 and
SHC binding to EGFR tyrosines 1148 and 1173 (3,4). EGFR has been shown to activate a
number of signaling pathways, including the mitogen-activated protein kinase (MAPK),
7
phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC) pathways. In addition, EGFR
can be trans-activated by other proteins, such as G-protein-coupled receptors (GPCRs) (5). The
various signaling cascades associated with EGFR activation are summarized in Figure 1-1. It has
also been observed that activated EGFR may undergo nuclear translocalization and subsequently
regulate gene expression, although the contribution of this pathway compared to more traditional
signal transduction is unclear (6,7). The activation of EGFR thus leads to multiple cell responses,
including cellular growth, differentiation, and migration.
Figure 1-1: EGFR signaling network. (a) EGFR family ligands are shown with their binding specificities. For
simplicity, arrows are only drawn for EGF and neuregulin 4 (NRG4), although other ligand specificities are shown
in parentheses. (b) Signaling to the adaptor protein/kinase layer is only shown for two receptor dimers. (c) Possible
cellular outcomes based on EGFR signaling. Figure has been reproduced from (5) with permission from Elsevier.
In the absence of ligand, EGFR is constitutively internalized with a half life of ~30
minutes and quickly recycled back to the cell surface, leading to a total receptor distribution of
~80-90% on the cell surface at any given time (Figure 1-2) (8). The metabolic half-life of EGFR
8
in a tumor cell line is 20 hours (9); thus, a single receptor will cycle through the endocytic
pathway many times during its lifetime. Ligand activation increases the rate of EGFR
endocytosis 5-10 fold, but does not increase the internalization rates of other ErbB receptors
(10). In addition, activated EGFR dimers bound to EGF are preferentially retained in the
endosomes and not recycled (11), and these endosomes mature into multivesicular bodies and are
targeted to lysosomes (12). This leads to the specific degradation of activated receptors and
overall receptor downregulation. Earlier reports indicated that ligand-accelerated internalization
required EGFR kinase activity (13,14), but a recent study found that receptor dimerization was
sufficient to induce rapid internalization and that kinase activity was unnecessary (15). Although
receptor ubiquitination does not affect EGFR internalization rates (16,17), ubiquitination by
c-Cbl targets receptors to the lysosome and is dependent on kinase activity and an intact
C-terminal region (18).
While receptor internalization has often been viewed as a means of signaling attenuation,
it has been shown that activated EGFR continues to signal while in the endosome through certain
signaling pathways (19,20). Although Her2 has often been described as the preferred
heterodimerization partner for ErbB receptors (21), mathematical modeling and experiments
have shown that EGFR homodimerization and EGFR/Her2 heterodimerization occur with
comparable affinities (22), and EGFR/Her2 heterodimers appear to predominate because they
have a reduced rate of internalization and increased fraction of recycling (23). EGFR trafficking
has been the subject of much mathematical modeling, and the system serves as a paradigm for
other ligand-binding receptors (24). The constitutive trafficking patterns of EGFR can even
affect the global pharmacokinetics of antibody drugs which bind to the receptor due to antibody
9
internalization and subsequent degradation (25). Additional experiments on EGFR trafficking are
reviewed in (8) and (26).
Figure 1-2: Trafficking of EGFR. Activated EGFR homo- and heterodimers are internalized through clathrin-coated
pits and preferentially degraded. Times represent approximate mean time of the processes indicated. Figure has been
reproduced from (8) with permission from Elsevier.
10
1.2
Biochemical and structural characterization of EGFR
EGFR is a 170 kDa protein that is heavily N-glycosylated but not O-glycosylated (27),
with carbohydrate accounting for ~20% of the molecular mass. Mass spectrometry
characterization has determined that eight of the eleven canonical N-linked sites are glycosylated
in the A431 cell line, with two sites unglycosylated and one site glycosylated in a fraction of the
receptors (28). The EGFR extracellular region is divided into four domains, numbered I-IV
(Figure 1-3). Domains I and III (DI and DIII), also designated L1 and L2, are members of the
leucine rich repeat family and are similar to domains found in the insulin receptor family (29).
Prior to receptor crystallization, it was known that both DI and DIII contribute to ligand binding
(30). However, domain III appears to contribute most of the binding energy, since the affinity of
DIII alone for EGF is ~400 nM as measured solubly by isothermal titration calorimetry (31),
while soluble EGFR ectodomain binds immobilized EGF with an affinity of ~200 nM (32).
Domains II and IV (DII and IV), or the cysteine-rich (CR) domains, contain multiple small
disulfide-bonded modules similar to those in laminin (33).
Figure 1-3: Domains of EGFR. The extracellular region consists of domains I-IV, followed by a transmembrane
region (TM) and the intracellular portion. The regions inside the cell include the juxtamembrane portion (JM),
tyrosine kinase (TK), and C-terminal tail containing tyrosine residues which become phosphorylated upon receptor
activation.
11
The EGFR ectodomain has been crystallized in both monomer and dimer forms, and a
review of these structures can be found in (34). The monomer exists in a tethered, closed, or
autoinhibited state (35), with DII and DIV forming a tether contact (Figure 1-4, left). However,
in the dimer structures of EGFR bound to EGF (36) or TGF-α (37) ligand, the receptor is in an
untethered, open, or extended conformation, with ligand binding between DI and DIII (Figure
1-4, right). Although most cytokine receptors dimerize through ligand bridging the two receptor
monomers (38), all dimerization contacts in the EGFR dimer are fully receptor-mediated. The
dimer exists in a 2:2 receptor to ligand ratio, with multiple contacts through the DII dimerization
arm (residues 242-259). An additional DII loop, residues 271-283, also contributes important
dimer contacts (32). The dimerization interface may include DIV; however, in the dimer
structures, DIV is either unresolved (36) or was not present in the crystallized protein (37).
Peptides mimicking portions of DIV can inhibit EGFR heterodimerization with Her3 (39), and
DIV mutations can impair the ability of ligand to bind and induce EGFR phosphorylation (40).
However, these potential DIV contacts contribute <9% of the free energy of dimerization as
demonstrated by studies on soluble EGFR dimer formation (32). For illustrative purposes, DIV
has been added to the dimer structure in Figure 1-4 in the same relative orientation to DIII as
seen in the tethered monomer. A low-resolution molecular envelope structure of the full EGFR
dimer in solution has been obtained from small-angle X-ray scattering (SAXS), and it is
consistent with this dimer structure (41).
12
Figure 1-4: Crystal structures of monomeric and dimeric EGFR. Left, autoinhibited EGFR monomer (1NQL), with
domain I (DI), red; DII, orange; DIII, blue; DIV, green. A 130o rotation of DI and DII allows DI and DIII to come
together to bind EGF ligand. Right, dimeric EGFR bound to EGF ligand in yellow (1IVO). DIV is not resolved in
this structure, but has been added in the same relative orientation to DIII as seen in the monomer. This figure was
adapted from (34).
From the crystal structures of the monomer and dimer, a model for EGFR activation has
been proposed (34). In this model, the monomer mostly exists in a tethered conformation, in
which the DII dimerization arm and other residues are obscured, thus preventing receptor
dimerization. The monomer equilibrates between the tethered conformation and an extended
conformation similar to that seen in the dimer. The binding of ligand between DI and DIII
stabilizes the extended conformation, which exposes the DII dimerization arm and other
residues, thus allowing the formation of the dimer. However, the breaking of the tether is
necessary but not sufficient for dimer formation, as demonstrated through mutations and
deletions of DIV (37,42,43). It also appears that certain types of receptor glycosylation can shift
the equilibrium toward the extended form (44,45). It has subsequently been found that the DIIDIV tether interaction is not necessarily responsible for maintaining the receptor in the
autoinhibited conformation; in fact, mutating every tether interaction in soluble EGFR still leads
13
to an autoinhibited conformation (41). In addition, it appears that ligand binding is necessary to
stabilize the conformation of the DII loop 271-283, which is an important dimerization contact
(32).
Ectodomain crystal structures are also available for the EGFR family members Her2 (46),
Her3 (47), and Her4 (48). The Her2 monomer, in contrast to EGFR, adopts an extended
conformation similar to that of EGFR in the dimer structure. Since Her2 has no activating ligand,
it is extended and poised to interact with other ErbB receptors. However, since ligand binding is
necessary to activate Her3 and Her4, these monomers adopt a tethered conformation.
EGFR has a single transmembrane domain, and the intracellular portion contains a short
juxtamembrane region, the tyrosine kinase, and a C-terminal tail containing tyrosine residues
which become phosphorylated upon receptor activation (Figure 1-3). The EGFR kinase domain
has been independently crystallized, revealing an intrinsically autoinhibited domain resembling
Src and cyclin-dependent kinases (CDKs) (49). In contrast to most kinases, phosphorylation of
the EGFR activation loop is not necessary for its activation (50). Instead, the EGFR kinase is
activated by an asymmetric dimer in which the C-terminal lobe of one kinase domain binds to
the second kinase domain in a manner analogous to cyclin in activated CDK/cyclin complexes.
Thus, ligand binding brings two receptor monomers together and allows for the dimerization and
subsequent activation of the kinase domains. In this model, the extent of EGFR kinase activation
depends on the effective local concentration of the receptor, since the probability of dimerization
will increase with increasing kinase domain density. This phenomenon has been demonstrated
experimentally using increasing concentrations of EGFR kinase domains tethered to the surface
of vesicles (49).
14
1.3
EGFR in cancer
EGFR was the first receptor to be directly linked to human cancer (51), and because
EGFR activation often leads to cellular growth, its signaling can provide tumor cells with
substantial survival advantages. In addition, EGFR signaling has been implicated in tumor cell
production of pro-angiogenic factors and cellular migration and invasion (52). EGFR
dysregulation has been observed in a wide variety of carcinomas, including head and neck,
breast, bladder, and non-small-cell lung cancer (5). Excessive EGFR signaling can arise from
receptor overexpression, autocrine signaling, or mutation. Normal cells usually express 4x104 to
1x105 EGF receptors per cell, but tumor cells can express as many as 2x106 (53). Receptor
overexpression commonly develops due to gene amplification, although it was recently reported
that the hypoxic microenvironment of tumors can also induce overexpression of EGFR by
increasing EGFR mRNA translation (54). In head and neck cancer, at least 80% of tumors
overexpress EGFR (55), and the observed percentages of tumors overexpressing EGFR in
various types of cancer are listed in Table 1-1. Furthermore, elevated levels of EGFR serve as a
prognostic indicator for poor survival rates (56).
Tumor type
Colon
Head and neck
Pancreatic
Non-small-cell lung
Breast
Renal
Ovarian
Glioma
Bladder
Percentage of tumors
overexpressing EGFR
22-75%
80-100%
30-50%
40-80%
14-91%
50-90%
35-70%
40-63%
31-48%
Table 1-1: EGFR overexpression in different tumor types. Table has been adapted from (53).
15
In addition, EGFR ligands can be overexpressed, resulting in high levels of autocrine
signaling when the receptor is present (57). Autocrine production of TGF-α or EGF is also
associated with reduced cancer survival (21). Finally, various activating EGFR mutations have
been observed in tumor samples. Gene rearrangements in glioblastoma often lead to truncation
mutants, the most common of which is EGFRvIII (EGFRΔ2-7), where amino acids 6-273
(encoded by exons 2-7) are deleted from the gene (58-60). This leads to a mutant which is
missing DI and most of DII, with EGFR amino acids 1-5 fused to the rest of the receptor through
a novel glycine residue resulting from the gene fusion. Although most frequently seen in
glioblastoma, EGFRvIII has also been observed in other types of tumors, including non-smallcell lung, breast, and ovarian cancers (61-63). EGFRvIII is not activated by ligand, but signals
constitutively at low levels (64) and thus confers enhanced tumorigenicity to cancer cells (65).
There is also evidence that EGFRvIII is downregulation defective, leading to prolonged
signaling (66). In addition, point mutants of the EGFR ectodomain have recently been observed
in glioblastoma, and select mutants showed high basal phosphorylation and conferred
tumorigenicity to NIH-3T3 cells (67).
In non-small-cell lung cancer (NSCLC), mutations in the EGFR kinase domain, which
are clustered around the ATP-binding pocket of the enzyme, have been observed (68). These
mutations appear to be restricted to a subset of NSCLC patients, particularly non-smokers,
women, and those of Asian ethnicity (69). Exon 19 in-frame deletions of some or all of residues
746-750 account for 45% of reported mutations, exon 21 substitutions (especially L858R)
account for 45%, and the remaining 10% involve exons 18 and 20, such as G719S and V765A
(68). Most of these mutations have been shown to hyperactivate the kinase and have oncogenic
16
activity. Although predominately seen in NSCLC, very rare kinase domain mutations have also
been observed in other types of tumors, including ovarian, colorectal, and head and neck (68).
Other EGFR family members have been shown to be dysregulated in cancer. Her2
overexpression due to gene amplification has been observed in various tumors, including breast,
ovarian (70), and bladder cancer (71). In particular, Her2 overexpression is an indicator of poor
prognosis in breast cancer (72). Her3 and Her4 signaling can also contribute to a malignant
phenotype (5).
17
1.4
Current EGFR-targeted therapeutics
Because of the importance of the EGFR family in cancer, the receptors are promising
targets for rational oncology therapies designed to inhibit receptor signaling. The two main
classes of such therapeutics are tyrosine kinase inhibitors (TKIs) and monoclonal antibodies.
TKIs are small molecule drugs which are designed to bind the ATP pocket of the kinase domain
and inhibit EGFR phosphorylation (73). Two EGFR-targeted TKIs have been approved by the
U.S. Food and Drug Administration (FDA) for the treatment of non-small-cell lung cancer:
gefitinib
(Iressa,
ZD1839;
AstraZeneca)
(74)
and
erlotinib
(Tarceva,
OSI-774;
OSI/Genentech/Roche) (75), which has been co-crystallized with the EGFR kinase domain (76).
Although these two TKIs are targeted to the EGFR tyrosine kinase, with IC50 values
(concentration required for 50% inhibition of activity) in the nanomolar range, they also will
inhibit the Her2 tyrosine kinase with IC50 values in the micromolar range (73). A small subset of
NSCLC patients dramatically respond to treatment with gefitinib (73) or erlotinib, and the
characterization of these patient tumors led to the discovery of activating EGFR kinase domain
mutations (described in Section 1.3). In fact, 77% of NSCLC patients who respond to gefitinib or
erlotinib harbor certain kinase domain mutations, as opposed to a 7% incidence of mutation in
non-responders. It has been hypothesized that these activating EGFR kinase domain mutations
lead to a state of oncogene addiction for tumor cells, thus explaining the extreme sensitivity of
mutation-bearing tumors to EGFR TKIs (68). Unexpectedly, in cells expressing the ectodomain
truncation mutant EGFRvIII, gefitinib induces signaling at low concentrations (77). Gefitinib
and erlotinib are reversible kinase inhibitors; however, irreversible TKIs which bind the kinase
domain covalently, such as HKI-357 (78), have also been developed. These have been shown to
18
be effective against reversible-TKI resistant cells expressing a secondary kinase mutation
T790M. Additional TKI drugs in development are reviewed in (79) and (68).
An alternative to TKI therapeutics is treatment with anti-EGFR monoclonal antibodies
(mAbs; for a general discussion on antibody structure, see Section 3.1). While TKIs can be
conveniently delivered orally and have a reduced chance of inducing allergic reactions,
monoclonal antibodies have superior stability in vivo and lower gastrointestinal toxicity (79).
Cetuximab (Erbitux, IMC-C225; ImClone Systems) was the first anti-EGFR monoclonal
antibody to be approved by the FDA. Originally approved for the treatment of EGFR-expressing
metastatic colorectal cancer, it has also recently been approved for head and neck cancer. The
antibody was initially isolated by immunization of mice with EGFR, and the murine monoclonal
antibody was designated mAb 225 (80). In vitro experiments revealed that this antibody inhibited
the binding of ligand to EGFR, resulting in decreased tyrosine phosphorylation and reduced cell
proliferation (80,81). The crystal structure of the cetuximab Fab bound to the EGFR ectodomain
reveals that the antibody directly competes with EGF ligand by binding to DIII on an epitope
which overlaps the EGF binding site (82). It was also found that mAb 225 can induce EGFR
internalization (83), which may reduce the number of receptors available to bind ligand. The
bivalency of the mAb 225 molecule was shown to be important for its ability to internalize and
downregulate EGFR and inhibit subsequent growth (84). In addition, cetuximab has been shown
to exhibit anti-angiogenic properties, leading to even more effective treatment of tumor
xenografts in vivo (85). Cetuximab may also induce antibody-dependent cellular toxicity
(ADCC) in vivo (86), and the antibody’s various mechanisms of action are further discussed in
(87). To decrease its potential immunogenicity, the mouse mAb 225 was chimerized with human
IgG1 by adding the mouse antibody variable regions to the human constant IgG regions. This
19
chimeric antibody, designated C225, was shown to have a higher affinity for EGFR and was
effective against human tumor xenografts (88). Additionally, cetuximab is able to inhibit the
growth of lung cancer xenografts expressing the L858R/T790M EGFR kinase mutants, which
are resistant to reversible TKI treatment (89). In clinical trials, cetuximab was well-tolerated with
mild toxicities, which included acneiform rash, fatigue, and hypersensitivity (90). The effects to
normal tissue appear to minimal, since many healthy cells expressing EGFR are not rapidly
turned over, with the intestinal epithelium as an exception (53). In a Phase III clinical trial with
329 irinotecan-refractory metastatic colorectal cancer patients, 10.8% responded to cetuximab
therapy alone while 22.9% responded to cetuximab plus irinotecan (90), leading to the FDA
approval of this drug. Interestingly, cetuximab treatment was able to reverse tumor cell
resistance to the chemotherapeutic irinotecan.
Other antibodies have been developed which similarly inhibit ligand binding to EGFR as
cetuximab does. Panitumumab (Vectibix, ABX-EGF; Amgen/Abgenix) has also been FDAapproved for the treatment of metastatic colorectal cancer and blocks the binding of ligands to
EGFR (91). Panitumumab has one potential advantage over cetuximab; it is a fully human
antibody developed from the XenoMouse technology, in which human antibody genes were
cloned into mice (91). However, panitumumab is a class IgG2 antibody and thus incapable of
activating ADCC, although it is unclear how important this is for tumor inhibition (92). Data
from clinical trials shows that panitumumab produces an 8% overall response rate in metastatic
colorectal cancer, with progression-free survival extended from 60 days to 96 days (92). Another
cetuximab-like antibody is matuzumab (EMD72000; EMD Pharmaceuticals/Merck KgaA),
which is currently in various phase II clinical trials. The antibody was originally isolated from
mice (mAb 425/EMD55900) and inhibits the binding of ligands to EGFR (93). The murine
20
version of the antibody was humanized by grafting the murine complementarity-determining
regions (CDRs) onto a human IgG1 framework (94). A phase I study of matuzumab treatment of
patients with advanced pancreatic adenocarcinoma showed partial responses and stable disease
in eight of twelve patients (95). However, in a recent phase II trial, matuzumab showed no
evidence of significant clinical activity in ovarian tumors (96). In addition, a bispecific version of
the humanized mAb 425, named MDX-447 (Medarex/Merck KgaA), has been developed, with
the other arm of the antibody binding to the high affinity Fc receptor (FcγRI) (97). MDX-447 is
currently undergoing further clinical trials in combination with activated monocytes in an effort
to direct these immune cells to tumor sites. Finally, nimotuzumab (hR-3, TheraCIM; YM
Biosciences/Center of Molecular Immunology, Cuba) is a humanized antibody which also blocks
ligand binding to EGFR (98). Phase I/II trials of glioblastoma and anaplastic astrocytoma
patients showed a 38% objective response rate (99), and the antibody is currently undergoing
further trials in the U.S.
Antibodies which target the truncation mutant EGFRvIII have also been developed. Since
EGFRvIII contains a novel glycine residue at the junction between two EGFR fragments, this
junctional peptide is a potential target for antibody binding and represents an epitope specific to
tumor cells. One antibody targeting this peptide is MR1-1, which is an affinity-matured single
chain variable fragment (scFv) specific for EGFRvIII (100). However, antibodies against the
junctional peptide have no intrinsic anti-EGFRvIII activity, and thus must be conjugated to
cytotoxic agents such as immunotoxins in order to kill tumor cells (101). Another antibody, mAb
806, was isolated from immunization of mice with EGFRvIII, and it was shown to bind and
inhibit the growth of cells expressing the mutant protein. However, it was discovered that mAb
806 also bound to a small portion of wild-type EGFR (~5-10%) on cells overexpressing the
21
receptor and inhibited cell growth, but it did not bind to or inhibit cells expressing normal EGFR
levels (102-104). Therefore, mAb 806 has the unique ability to recognize EGFR expressed by
tumor cells but not on normal tissues. Characterization of mAb 806 revealed that it binds a
peptide epitope shared by both EGFRvIII and EGFR (105). However, this epitope is only
exposed in certain situations: in EGFRvIII due to the deletion of DI and DII, when wild-type
EGFR transitions from the autoinhibited to open conformation, or by high-mannose forms of the
receptor in cells overexpressing EGFR (44). The mAb 806 epitope is discussed in further detail
in Chapter 2. mAb 806 has also been chimerized to reduce murine sequences, generating
antibody ch806 (106). In mouse models of cancer, 806 was effective in inducing regression of
lung tumors expressing either EGFR harboring an activating kinase domain mutation or
EGFRvIII (107). However, 806 is ineffective against kinase-dead EGFRvIII, suggesting that it
functions by blocking receptor trans-phosphorylation (108). Phase I clinical trial data showed
that ch806 was well-tolerated by patients, with highly specific targeting to tumors versus normal
tissues (109), and further trials are currently ongoing.
The only FDA-approved antibody which targets Her2, trastuzumab (Herceptin, 4D5;
Genentech), was isolated by mouse immunization (110) and humanized (111). Trastuzumab
inhibited the proliferation of breast tumor-derived cell lines (110) and also supported ADCC
killing against the SK-Br-3 tumor line (111). Trastuzumab was shown to be effective against
human gastric carcinoma xenografts (112), as well as breast cancer BT-474 xenografts (113). In
phase III trials, the addition of trastuzumab treatment to chemotherapy was associated with a
longer time to disease progression (7.4 vs. 4.6 months) and a higher objective response rate (50%
vs. 32%) (114), leading to the FDA approval of this drug for the treatment of Her2-positive
metastatic breast cancer. The main toxicities were cardiac adverse events, since Her2 signaling
22
appears to be important for adult heart function (115). Recent trials have shown that adjuvant
trastuzumab therapy reduces the 3-year risk of recurrence by half in early breast cancer patients
(116). Despite the success of trastuzumab, its mechanism of action is not immediately apparent.
A crystal structure of trastuzumab bound to Her2 reveals that it binds the C-terminal portion of
domain IV (117); however, it does not block Her2 heterodimerization with Her3 (118). Although
initial reports suggested that trastuzumab could inhibit tumors through downregulation of the
Her2 receptor (119,120), a more recent study demonstrated that the antibody is internalized and
recycled back to the cell surface without mediating receptor degradation (121). The Her2
ectodomain undergoes cleavage, and the resulting membrane-bound kinase fragment shows
increased tyrosine kinase activity (122). Trastuzumab blocks this Her2 shedding (123),
presumably by restricting access to the protease cleavage site, thus preventing the formation of
the activated kinase fragment (119). Finally, trastuzumab can induce ADCC killing of tumor
cells (124), but this cannot be its sole mechanism since ADCC cannot explain the antibody’s
efficacy in vitro. Additional experiments with trastuzumab are reviewed in (125). Another antiHer2 antibody named pertuzumab (Omnitarg, 2C4; Genentech) binds to domain II of Her2
(demonstrated by co-crystallization (126)), and thus it inhibits Her2/Her3 heterodimerization and
breast and prostate xenograft tumor growth (118). The murine version, mAb 2C4, was
humanized (127), and initial results from a phase II trial treating prostate cancer showed no
objective responses (128). However, a clinical trial evaluating pertuzumab treatment plus
trastuzumab is currently ongoing, motivated by preclinical studies where the two antibodies
synergistically inhibited the survival of breast cancer cells (129).
Although therapeutics directed at the EGFR receptor family have been somewhat
effective in inhibiting the growth of tumor cells, optimal cancer treatments will likely include
23
some form of combination therapy. Since multiple genetic alterations contributing to a malignant
phenotype normally arise during the course of tumor development (130), it may be difficult to
inhibit these tumors by blocking only one receptor. Synergistic inhibition effects of combining
anti-EGFR antibodies with chemotherapeutic agents in vitro were initially reported by (131).
Treatments of trastuzumab plus chemotherapy (114) or cetuximab plus irinotecan (90) both
improved patient outcome in clinical trials. Additional trials examining combinations of antiEGFR antibodies with other chemotherapeutics have been and are currently being investigated
(3,53,132). However, not all combinations are synergistic or even additive, as demonstrated by
failed clinical trials combining gefitinib or erlotinib with chemotherapy (133). Using two
different classes of anti-EGFR inhibitors may be effective, since tumor xenograft experiments
have shown synergy between mAb 806 and the TKI AG1478 (134). In addition, the combination
of two anti-EGFR antibodies directed against different epitopes has been shown to stimulate
receptor downregulation and inhibit signaling (135,136). Other successful combinations include
simultaneously inhibiting EGFRvIII and c-Met (137), and rationales for treatment using ErbB
plus mTOR inhibitors or ErbB plus estrogen inhibitors are discussed in (132).
24
1.5
Overview of thesis work
In this thesis work, we have sought to characterize and engineer antibodies against the
epidermal growth factor receptor. Multiple anti-EGFR antibodies have been developed, but their
binding epitopes had not been determined due to a lack of convenient epitope mapping methods.
To alleviate this, we developed a novel method of epitope mapping using random mutagenesis
and yeast surface display. In our approach, we disrupted the antibody-antigen interaction through
point mutations. However, since we did not know the epitopes of the antibodies, we made a
library of random mutations, and then combinatorially screened this library using yeast surface
display. EGFR mutants which displayed a loss of binding to the antibody being mapped were
thus potential epitope residues. It was also necessary to screen for retention of binding to a
conformation-specific antibody to remove misfolded EGFR mutants. The development of our
novel method enabled the epitope mapping of a total of 4 antibodies: mAbs 806, 225, 13A9, and
EMD72000. The determination of the antibody epitopes enabled further understanding of their
mechanisms of action. Since mAbs 806, 225, and EMD72000 have high therapeutic relevance,
this information may have clinical impact as well. In addition, the epitope mapping method was
extended to mapping the binding of three designed ankyrin repeat proteins (DARPins) against
EGFR.
Instead of using the common inhibition strategy of using antibodies to block ligand
binding, we opted to engineer antibodies to directly inhibit receptor dimerization. Such
antibodies could have advantages in tumors driven by autocrine signaling or the EGFRvIII
mutant. We selected epitopes in EGFR domain II and domain IV to target with antibodies, and
synthesized peptide mimics of these loops to use as antigen. Starting from a nonimmune human
scFv library in yeast, candidate binders to the peptide epitopes were isolated and characterized.
25
The majority of binders were heavy chain only constructs, many of which were likely mediating
binding through the VH/VL interface instead of the antibody CDRs. In addition, rabbit
monoclonal antibodies against a domain IV peptide were developed, but appeared to only bind
the peptide and not the EGFR ectodomain.
Finally, previous experiments had shown that polyclonal antibodies (pAbs) against either
EGFR domain II or domain IV could eliminate the high affinity facet of EGF binding normally
observed on the surface of cells. Based on these results, we hypothesized that the pAbs inhibited
receptor dimerization, and thus inactive preformed EGFR dimers could represent the high
affinity binding component. We developed a mathematical model incorporating these hypotheses
to determine whether such a model could be consistent with experimental observations. The
equilibrium ligand binding and receptor dimerization model was fit to the experimental data
using three parameters: the high affinity binding constant, the dimerization constant, and the
maximum binding signal. The model was able to successfully reproduce the experimental data,
and it could produce characteristic concave-up Scatchard plots for a wide range of values.
Overall, we have increased understanding of the mechanisms of action of a number of antiEGFR antibodies.
26
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35
Chapter 2: Epitope mapping of proteins binding to EGFR
2.1
Background
Epitope mapping is the process of determining which antigen residues are responsible for
mediating antibody-antigen interactions. This information can be important in understanding the
mechanism of action of the antibody and can aid in the design of novel antibodies against the
same antigen. One method of determining the epitope of an antibody is through co-crystallization
of the antibody in complex with its antigen. However, this can be very time-consuming and
technically difficult, especially for a complex antigen such as EGFR. Only within the past few
years was the EGFR monomer itself crystallized (1), and the only antibody-EGFR complex
structure was published recently (2). While co-crystallization is the “gold standard” of
characterizing the epitope of an antibody, other methods exist to determine which residues of the
antigen are important for antibody binding.
One class of epitope mapping methods utilizes peptide fragments of the antigen.
Antibody reactivity to each of these fragments is tested, and the epitope can be localized to
certain stretches of amino acids. SPOT synthesis involves the creation of synthetic peptides,
which are spotted on cellulose membranes and assayed for antibody binding (3). Alternately,
peptides can be expressed on the surface of bacteriophage (4), Escherichia coli (5), or yeast (6),
with subsequent antibody binding analysis. Mass spectrometry has also be used to identify
digested peptides of the antigen which retain binding to an antibody (7). Phage display and
SPOT techniques have been utilized to determine the epitopes of various antibodies against ErbB
receptor family members (8-10). However, peptide-based methods are limited in that they can
only identify continuous, non-conformational epitopes. If the binding of the antibody depends on
36
the proper tertiary fold of the antigen, other methods must be used to determine its epitope.
Peptides have been used to mimic epitopes discontinuous in sequence (11,12), although it is not
guaranteed that for any given epitope, a peptide “mimotope” exists.
To identify a discontinuous epitope, H/2H-exchange mass spectrometry has been used to
localize an epitope to discontinuous proteolytic fragments (13). Antibody epitopes can also be
resolved to protein fragments or domains displayed on the surface of phage (14) or yeast (15).
Another useful tool in dissecting protein-protein interactions is site-directed mutagenesis, in
which residues of interest are mutated and subsequent changes in binding are measured. This
allows for characterization of antibody binding in the context of the entire protein antigen.
Initially, only conservative mutations to amino acid homologs (16) or alanine (17) were made to
minimize disruption to the protein secondary and tertiary structure. This method requires soluble
protein expression and characterization of each mutant to ensure proper folding, which can be
time consuming. Also, some knowledge of the general location of the epitope is necessary to
indicate which site-directed mutants should be constructed. Alternately, shotgun alanine or
homolog scanning mutagenesis is a high-throughput method using phage display libraries and
can incorporate saturation mutagenesis so that no knowledge of the epitope is necessary. Shotgun
scanning has been used to map paratopes and protein-protein interactions (18-20). However, the
prokaryotic expression system used by this method may not be amenable to epitope mapping of
complex eukaryotic proteins such as EGFR ectodomain, which contains 25 disulfide bonds and
10 N-linked glycosylation sites (21). Finally, other methods have utilized random mutations to
all amino acids in order to disrupt antibody-antigen interactions. Random mutagenesis of the
antigen followed by secretion by yeast in 96-well plates enabled epitope mapping in a semi-highthroughput manner (22). Libraries of randomly mutagenized antigen have also been displayed on
37
the surface of filamentous phage (23) and mammalian cells (24), and selection of nonbinding
mutants was performed in a combinatorial manner in these studies.
To overcome some of the shortcomings of existing techniques, we developed a novel
method of epitope mapping using random mutagenesis and yeast surface display. In this method,
a library of mutated antigen was displayed on the surface of yeast and screened using
fluorescence-activated cell sorting (FACS). We applied this method to antibodies and designed
ankyrin repeat proteins (DARPins) against EGFR, and we successfully determined the epitopes
of several proteins, many of which bind in a conformation-specific manner.
38
2.2
Materials and methods
Construction and expression of epitope mapping libraries
The epitope mapping libraries were constructed using the Stratagene GeneMorph random
mutagenesis kit to give a low mutagenesis rate. The template used for library construction was a
pCT302 yeast-display plasmid backbone containing either the EGFR fragment 273-621 with a
C283A mutation to prevent disulfide mispairing (15) (Appendix 1.2) or the 404SG EGFR
ectodomain mutant (25) (Appendix 1.3). The PCR products were gel purified and extracted using
a Qiagen Qiaquick gel extraction kit. The library was transformed into Saccharomyces cerevisiae
strain EBY100 (26) by electroporation and homologous recombination using a Bio-Rad
(Richmond, CA) Gene Pulser Transfection Apparatus (27). The final library contained a roughly
Poisson distribution of amino acid changes to the EGFR fragment as demonstrated by plasmid
recovery using a Zymoprep kit (Zymo Research) and sequencing of 100 library clones (MIT
Biopolymers Laboratory). Yeast were grown in SDCAA media overnight at 30 oC and were
induced to express protein on the cell surface by induction at a starting OD600 of 0.5 at 30 oC in
SGCAA media (27) with a 1/25 volume of SDCAA.
Labeling and sorting of yeast
The anti-human EGFR mouse monoclonal antibodies 806 and 175 were generously
provided by the Ludwig Institute for Cancer Research, mAb 13A9 was provided by Genentech
(San Francisco, California), and mAb EMD72000 was provided by EMD-Lexigen
Pharmaceuticals. mAb 225 was purchased from LabVision (Fremont, CA), anti-c-myc chicken
IgY fraction was purchased from Invitrogen (Carlsbad, CA), and mAbs 528 (28), 199.12,
EGFR1 (29), and ICR10 (30) were purchased from Abcam (Cambridge, UK).
39
Yeast cells were labeled as described (27). Briefly, yeast cells (at least 10X library size)
were washed with PBSA buffer (phosphate buffered saline containing 1 mg/ml bovine serum
albumin). The cells were incubated with 1:250 anti-c-myc chicken IgY and the appropriate
concentration of mAb for 30 min at 25 oC. The cells were then washed with PBSA and incubated
with 1:50 phycoerythrin (PE)-labeled goat anti-mouse IgG (Sigma) or 1:100 goat anti-human-PE
IgG (Rockland) and 1:100 goat anti-chicken-Alexa488 IgG (Invitrogen) for 20 min at 4 oC. The
labeled cells were washed with 1 ml PBSA, and cell libraries were sorted using either a MoFlo
(DakoCytomation) or Aria (BD Biosciences) FACS machine at the MIT flow cytometry core
facility. In addition, a positive control of antibody binding to wild-type antigen was labeled to
assist in drawing the selection window.
Identification and testing of single clones
Plasmids from the sorted library populations were recovered using a Zymoprep kit and
sequenced at the MIT Biopolymers Laboratory. Site-directed mutants were made using
QuickChange mutagenesis (Stratagene). Single clones were transformed into yeast using EZ
Yeast Transformation (Zymo Research) and grown and induced as above. For each clone, 106
cells were labeled as before in a final volume of 50 μL. Fluorescence data was obtained using a
Coulter Epics XL flow cytometer (Beckman-Coulter) and was analyzed using either
DakoCytomation Summit or FloJo (Tree Star, Inc.) software.
Titration of EGFR fragments against mAb806
Cells were grown, induced, and labeled as above. Mean fluorescence data of c-myc
positive yeast were obtained using a Coulter Epics XL flow cytometer and were normalized by
40
maximal and minimal mean fluorescence intensities. The binding interaction was assumed to be
a single site binding model with no ligand depletion. Titration data was fit to the equation:
f mAb =
[mAb]
[mAb] + K d
(1)
where fmAb is the fractional binding of mAb 806 to yeast-surface displayed EGFR 273-621,
[mAb] is the concentration of mAb 806, and Kd is the apparent dissociation constant. A global fit
of three data sets was performed using Microsoft Excel, and 68% confidence intervals were
calculated according to (31).
Protein images and surface area calculations
All EGFR protein images were generated using PyMOL software (DeLano Scientific
LLC, at http://www.pymol.org). The solvent accessible surface area of each residue of EGFR
(using structure 1NQL) was calculated using Getarea 1.1 (Sealy Center for Structural Biology,
University of Texas Medical Branch, at http://www.scsb.utmb.edu/cgi-bin/get_a_form.tcl). A
water probe size of 1.0 was used to allow for correct identification of EGF contact residues as
being on the surface. Residues with a value of 20 or above were considered surface residues.
DARPin epitope mapping
Designed ankyrin repeat protein (DARPin) clones E1, E67, E68, E69, and E72 containing
C-terminal His tags were provided by Dr. Andreas Plueckthun (University of Zurich). DARPin
binding to 404SG on the surface of yeast was detected using 1:100 biotinylated mouse Penta-His
antibody (Qiagen) followed by 1:50 streptavidin-PE (Invitrogen). All other cell growth, sorting,
and clone analysis was performed as above.
41
2.3
Results
2.3.1
Creation of epitope mapping library
In order to determine the epitopes of antibodies binding to EGFR, we created a novel
method of epitope mapping using random mutagenesis and yeast surface display. Previously,
Cochran et al. reported domain-level epitope mapping through yeast surface-displayed fragments
of EGFR (15). Large polypeptide fragments of EGFR were expressed on the surface of yeast
cells and were used to localize antibody binding to particular regions of the receptor for both
continuous and discontinuous epitopes. Yeast surface display is a tool originally developed for
protein engineering (32), in which the gene for a protein of interest is fused the yeast native Aga2
protein. The yeast cells are then induced to display this protein fusion on the cell wall, where it is
accessible for binding to other soluble proteins (Figure 2-1).
Figure 2-1: Schematic of yeast surface display. The yeast native proteins Aga1 and Aga2 are displayed on the yeast
cell wall, allowing the fusion protein Aga2p-EGFR to be displayed. Fluorescent antibody against either the HA or
c-myc epitope tag allows for the detection of EGFR on the yeast surface, while antibody binding to the EGFR can be
detected through a differently colored fluorescent probe.
42
In creating our novel fine epitope mapping method, we sought to further resolve antibody
epitopes to individual residues involved in antibody-antigen contacts. To do so, we specifically
disrupted the interaction between the antibody being mapped and the EGFR antigen through
point mutations. If a single mutant displayed of a loss of binding to the antibody, then that
particular residue was potentially a contact residue. However, mutating each individual residue
of the EGFR ectodomain (621 amino acids) would be prohibitively time-consuming. To make
the process high-throughput, we employed random mutagenesis of a fragment of the EGFR
ectodomain (residues 273-621), and transformed this library into yeast. Then, using yeast surface
display, we could induce each yeast cell to display a different EGFR mutant on its surface. Highthroughput screening of this library for EGFR mutants with a loss of antibody binding was
accomplished using fluorescence-activated cell sorting (FACS). Once the nonbinding population
was enriched, the individual clones were sequenced to identify potential epitope residues. The
process of epitope mapping using yeast display is summarized in Figure 2-2.
A low mutation-rate library was constructed by error-prone PCR of the EGFR fragment
273-621. The initial library size was 5x105, and sequencing of 100 clones from the unscreened
library indicated that 72% were wild-type EGFR, 17% contained single amino acid mutations,
and 11% contained multiple mutations or a frameshift. This corresponds to an effective library
size of 8.5x104, which is an order of magnitude higher than the largest theoretical diversity of
single amino acid mutants of this 349-residue protein fragment (6.6x103), and nearly two orders
of magnitude higher than the 1.0x103 diversity of amino acids accessible by a single nucleotide
mutation. Yeast were transformed with the DNA library, and the cells were induced the express
the EGFR mutants on their surfaces. Using this epitope mapping method, we sought to determine
the epitopes of several therapeutically relevant antibodies against EGFR.
43
Figure 2-2: Summary of epitope mapping method. A mutant library of the antigen is transformed into yeast, and the
yeast cells are induced to express the mutant antigens on their surfaces. The antibody to be mapped is added to the
yeast mixture, and cells with reduced antibody binding are isolated using FACS.
2.3.2 Epitope mapping of mAb 806
mAb 806 against EGFR served as a positive control since its epitope had previously been
shown to reside in the disulfide-bonded loop between EGFR residues 287-302 (33). In order to
map the mAb 806 epitope, the library of EGFR mutants displayed on the surface of yeast was
labeled with chicken anti-c-myc IgY to detect full-length EGFR 273-621 expression. The cells
were also labeled with a high concentration of mAb 806 to allow for differentiation between
cells possessing wild-type binding and those with a decrease in binding affinity. The library was
sorted using successive rounds of FACS to isolate those cells displaying mutants with a decrease
in affinity to mAb 806 (Figure 2-3).
44
Figure 2-3: Flow cytometry data for mAb 806 binding to epitope mapping library. EGFR expression as detected
through the c-myc tag is shown on the abscissa, and mAb 806 binding is shown on the ordinate. The yeast EGFR
mutant library was labeled with 10 nM mAb 806 for sort 1 and 75 nM mAb 806 for sort 2. The yeast were also
labeled with chicken anti-c-myc IgY, followed by goat anti-chicken-Alexa488 IgG and goat anti-mouse-PE IgG.
The cell collection gates are indicated by the dotted lines, and the double negative population results from yeast
plasmid loss.
Three sorts were performed for mAb 806, with round 1 labeled at 10 nM mAb 806, and rounds 2
and 3 labeled at 75 nM mAb 806. Clone isolation and DNA sequencing were performed after
sorts 2 and 3. Out of 100 sequenced clones, ~20% contained multiple amino acid mutations and
were omitted from subsequent analysis to facilitate interpretation of the data and to eliminate
spurious neutral mutations. The single mutants isolated from the library for loss of binding to
mAb 806 are listed in Table 2-1.
45
EGFR mutant
mAb 806
binding
+
+
−
+
−
+
−
+
+
−
C287G,R,S,W,Y
E293D,G
E293K
D297Y
G298D,S
R300C
R300P
K301E
C302F,R,Y
C302G,S
Table 2-1: EGFR mutants isolated for loss of binding to mAb 806. Binding scores were determined by testing for
binding at 75 nM mAb 806 to mutants displayed on the surface of yeast. + indicates a partial loss of binding and −
indicates a large loss of binding.
After the mutations were sequenced, the plasmids containing the EGFR mutants were retransformed into yeast, and each mutant was retested to ensure that all mutations were correctly
identified as having reduced binding to mAb 806. The mutations were also scored according to
the degree of binding loss at a concentration of 75 nM mAb 806 (Table 2-1). Example flow
cytometry data for various EGFR mutants binding to mAb 806 is shown in Figure 2-4.
46
Figure 2-4: Flow cytometry data for representative EGFR mutants and their binding scores to 75 nM mAb 806.
Yeast displaying the mutants were also labeled with chicken anti-c-myc IgY, followed by goat anti-chickenAlexa488 IgG and goat anti-mouse-PE IgG. ++ indicates wild-type binding, + indicates a partial loss of binding, and
− indicates a large loss of binding (equal to negative control at this concentration).
To quantitatively correlate mAb 806 labeling scores with particular affinity constants,
binding titrations on the yeast surface were performed to determine the apparent dissociation
constant (Kd) of mAb 806 for wild-type EGFR 273-621 (++), mutant C287R (+), and mutant
E293K (−). The titration curves are shown in Figure 2-5. The data were fit to a single site
binding model with no depletion. Although the bivalent mAbs could potentially result in avidity
effects, these apparent dissociation constants could still be used for comparison between EGFR
mutants. The apparent dissociation constant of mAb 806 for yeast surface-displayed wild-type
EGFR 273–621 is 2.13 nM (68% confidence interval of 1.83–2.50 nM), which is consistent with
47
the affinity of mAb 806 binding to mammalian cells expressing EGFR vIII (34). The C287R
substitution increases the Kd to 127 nM (68% confidence interval of 103–160 nM), which gives a
ΔΔG value of +2.4 kcal/mol when compared to wild-type and, in alanine scanning terms, is a
significant loss of binding (35). The E293K substitution leads to a Kd value of at least 30 μM,
corresponding to a ΔΔG of at least +5.7 kcal/mol. This large loss of binding is consistent with the
charge-reversing nature of the amino acid substitution. The above ΔΔG values demonstrate the
relative energetic importance of these mutations, and the results from these three representative
clones were used to roughly estimate the energetic importance of the other untitrated mutants
based on their binding scores.
fmAb, fractional binding
1
0.5
0
0.001
0.01
0.1
1
10
100
1000 10000
[mAb 806], nM
Figure 2-5: Titration of mAb 806 against yeast surface-displayed EGFR 273-621 and mutants. Red, wild-type (++);
green, C287R (+); blue, E293K (−). A global fit to a single site binding model was performed with three
independent sets of data. Square, triangles, and diamonds represent separate data sets.
To verify the results from the fine epitope mapping method, we performed alanine
scanning through site-directed mutagenesis on the residues comprising the EGFR disulfidebonded loop 287-302 (Table 2-2). All amino acid sites with a loss of mAb 806 binding by
48
substitution by alanine corresponded to mutants identified using our yeast-displayed library
method (287, 293, 298, and 302). Conversely, residues 297, 300, and 301 were not identified as
important for mAb 806 binding by alanine scanning, yet form an energetically important
component of the epitope as determined by our method. Clearly, using mutations to residues
other than alanine allows for greater discrimination of the epitope of an antibody.
EGFR mutant
C287A
G288A
A289K
D290A
S291A
Y292A
E293A
M294A
E295A
E296A
D297A
G298A
V299A,K
V299D
R300A
K301A
C302A
mAb 806
binding
+
++
++
++
++
++
+
++
++
++
++
+
++
−
++
++
−
Table 2-2: EGFR mutants created by site-directed mutagenesis. Binding was tested at a concentration of 75 nM
mAb 806 against mutants displayed on the surface of yeast. ++ indicates wild-type binding, + indicates a partial loss
of binding, and − indicates a large loss of binding.
All of the mutations leading to a decrease in mAb 806 binding were localized to the
disulfide loop between cysteine residues 287 and 302, consistent with published results (33).
However, the residue-level resolution afforded by our method provides additional insight into the
mAb 806 binding epitope. The residues identified as energetically important for mAb 806
binding are shown in terms of the EGFR structure are shown in Figure 2-6. These residues are
49
clustered on one face of the disulfide loop between cysteines 287 and 302, indicating that this
region is the mAb 806 binding epitope. Interestingly, Val299 is located in the middle of these
residues, but was not identified in the library or alanine scan. Thus, the V299K and V299D sitedirected mutants were created and tested, and although the epitope can accommodate a lysine
residue without effect, an aspartic acid substitution ablates detectable binding (Table 2-2). This
further indicates that mAb 806 is likely to contact this face of loop 287–302.
Although mAb 806 binds to heat and SDS-denatured EGFR (33), implying a linear
binding epitope, the library analysis performed here suggests that the epitope is not entirely
continuous in sequence. Examination of the structure of the loop shows that the sidechain of
Glu293 projects from one side of the loop to the putative contact face. In addition, the mAb 806
epitope is constrained by a disulfide bond between cysteines 287 and 302, and two salt bridges
(Glu293-Arg300 and Asp297-Lys301) (Figure 2-6c). All six residues involved in these
interactions were isolated from the library for loss of binding to mAb 806, highlighting the
importance of the constrained nature of the epitope for high affinity binding. It is unclear
whether these residues directly make contact with the antibody, or if they are only important for
constraining the peptide and reducing its entropy. The cysteine at position 287 tolerated a wide
variety of substitutions that led to an intermediate loss of binding (Table 2-1), indicating that its
energetic contribution to the epitope may arise more from constraining the loop rather than
directly contacting the antibody. In contrast, the cysteine at position 302 may be a contact
residue since it only tolerated substitutions to larger residues with aromatic character.
50
Figure 2-6: mAb 806 binding epitope on EGFR. (a) and (b) Front and back views of residues 287-302 in chain a of
the EGF-bound dimerized EGFR structure (1IVO), which was used because Glu293 is not resolved in the monomer
structure (1NQL). Residues shown in color are those which were identified as important for mAb 806 binding using
our yeast library method. Red, residues that also caused loss of binding upon alanine substitution; orange, residues
that did not; gray, residues that were not identified as important for binding by both library and alanine scanning
methods. (c) The epitope is constrained by a disulfide bond and two salt bridges. Negatively charged residues, red;
positively charged residues, blue; cysteine residues, yellow. Image includes residues 287-302 on both EGFR
molecules in dimer structure (1IVO). (c) mAb 806 epitope in context of full tethered EGFR monomer (1NQL),
colored as in (a), with the rest of EGFR in blue.
51
The mAb 806 epitope in context of the tethered EGFR monomer structure (1) is shown in Figure
2-6d. From viewing the structure, it appears that in this conformation, an antibody would be
partially blocked from accessing the epitope. This is consistent with the observation that mAb
806 does not bind soluble EGFR, but does bind to an untethered EGFR mutant perturbed away
from the autoinhibited conformation (33).
2.3.3
Effects of EGFR mutations on binding of mAbs 806 and 175
Because of the convenience of performing titrations on the surface of yeast, the binding
of mAb 806 to additional EGFR mutants was characterized in further studies. This work was
conducted in collaboration with Arvind Sivasubramanian and Jeff Gray at Johns Hopkins
University. We combined computational protein-protein docking with computational and
experimental mutagenesis to predict the structure of the complex formed by mAb 806 and
EGFR. Initially, Arvind docked mAb 806 to its peptide epitope of EGFR residues 287-302.
Potential mAb 806-EGFR structures, or decoys, were generated, and computational mutagenesis
was used to filter them according to their agreement with the previously generated experimental
mutagenesis data in Tables 2-1 and 2-2. Further computational mutagenesis suggested additional
mutations, which we tested to differentiate between three potential docked models. The sitedirected mutants were created and transformed into yeast, and mAb 806 binding to these mutants
was scored as before in Section 2.3.2. The results are listed in Table 2-3, and they allowed
Arvind to arrive at a final structure with the highest agreement between computational and
experimental mutagenesis data, which is described in (36).
52
EGFR mutant
E293Y
E293F
E293W
M294Y
M294F
M294W
E295Y
E295F
E295W
E296W
D297W
V299Y
R300Y
R300F
R300W
K301W
mAb 806
binding
−
−
−
++
++
++
++
++
++
++
+
++
++
++
++
++
Table 2-3: mAb 806 binding to EGFR mutants used to discriminate between computational models. Binding was
tested at a concentration of 75 nM mAb 806 against mutants displayed on the surface of yeast. ++ indicates wildtype binding, + indicates a partial loss of binding, and − indicates a large loss of binding.
Another antibody, mAb 175, was also characterized using binding to EGFR mutants
displayed on the surface of yeast. mAb 175 was isolated at the same time as mAb 806, and
displays similar reactivity to the peptide of EGFR residues 287-302 (T.G. Johns et al.,
manuscript in preparation). Therefore, the mutants originally tested for binding to mAb 806 were
tested for mAb 175 reactivity. The binding scores for mAb 175 compared to those for mAb 806
are shown in Table 2-4. The results show only minor binding differences between mAbs 175 and
806, with mAb 175 showing slightly reduced binding to mutants D297A, V299A, and V299K.
This is consistent with the crystal structures of mAbs 175 and 806 each bound to the EGFR
peptide 287-302, which show identical contact residues for the two antibodies (T.G. Johns et al.,
manuscript in preparation).
53
EGFR mutant
mAb 175
binding
+
++
++
++
++
++
+
−
++
++
++
+
+
+
−
+
−
++
+
−
++
+
−
+
C287A,G,R,S,W,Y
G288A
A289K
D290A
S291A
Y292A
E293A,D,G
E293K
M294A
E295A
E296A
D297A
D297Y
G298A
G298D,S
V299A,K
V299D
R300A
R300C
R300P
K301A
K301E
C302A,G,S
C302F,R,Y
mAb 806
binding
+
++
++
++
++
++
+
−
++
++
++
++
+
+
−
++
−
++
+
−
++
+
−
+
Table 2-4: Binding of mAb 175 to EGFR mutants compared to mAb 806. Binding was tested at a concentration of
75 nM mAb 175 or mAb 806 against mutants displayed on the surface of yeast. ++ indicates wild-type binding, +
indicates a partial loss of binding, and − indicates a large loss of binding.
2.3.4 Epitope mapping of mAbs 225 and 13A9
The binding of mAbs 806 and 175 to EGFR did not depend on the correct tertiary fold of
the protein; therefore, their epitopes were not strictly conformational. However, epitope mapping
of mAbs 225 and 13A9 presented a greater challenge because these two antibodies have
conformational epitopes (15). mAb 225 is the murine precursor to the FDA-approved antibody
drug cetuximab (Section 1.4). Both antibodies bind to EGFR domain III, compete with each
other (data not shown), and compete with TGF-α ligand binding (37,38). However, mAb 225
54
competes with EGF ligand binding, while mAb 13A9 does not (38,39). This is unexpected,
because it appears from the crystal structures of EGF and TGF-α bound to EGFR that the two
ligands bind in the same location (40,41). We sought to determine the epitopes of these
antibodies to better understand their behavior and mechanisms of action.
For epitope mapping of mAb 225, the EGFR mutant library described in Section 2.3.1
was screened for loss of binding to 50 nM mAb 225 for sort 1 and 100 nM for sorts 2-4, with
clone sequencing after sorts 3 and 4. Out of 185 clones sequenced, ~40% contained multiple
amino acid mutations and were omitted from subsequent analysis. In total, 84 unique single
amino acid substitutions were isolated from the library and were re-transformed and retested in
yeast to confirm loss of mAb 225 binding. These mutations are shown in Figure 2-7a. The
majority of the mutations are localized or adjacent to domain III, consistent with the observation
that mAb 225 blocks ligand binding to this domain. Since mAb 225 recognizes a conformational
epitope (15), a properly folded domain III is required for antibody binding. Clearly, all of the
residues shown in Figure 2-7a cannot comprise the mAb 225 epitope; therefore, it seems that
most of these mutations are instead causing a perturbation of the domain III conformation,
leading to the observed loss of mAb 225 binding. As shown by this analysis, it appears that
domain III is very sensitive to conformational perturbation through mutation. Although it was
expected that the yeast secretory quality control apparatus would not allow misfolded mutants to
be displayed on the cell surface (42,43), it appears that many of these conformationally altered
EGFR mutants are able to circumvent quality control. In fact, the intertwined disulfides Cys482Cys491 and Cys486-Cys499 of EGFR domain IV are required for the proper folding of domain
III (44), and, accordingly, all of these mutations were isolated from the library for loss of binding
to mAb 225.
55
Figure 2-7: (a) All single amino acid mutation sites isolated from the library that exhibited a loss of binding to mAb
225, yellow. EGFR fragment 273-621 (1NQL) is shown in surface representation in grey. (b) and (c) Front and back
view of surface only mutation sites that were not mutated to/from proline, glycine, or cysteine or from a
glycosylation site, yellow. (d) Residues identified after antibody cross-testing; mAb 225 residues, yellow; mAb
13A9 residues, red. (Note: Lys465 and Glu472 side chains are not resolved in this structure.)
56
In an attempt to extract useful contact residue information from these mutational data, we
developed two criteria to eliminate those mutations which seemed particularly disruptive to
protein folding. First, we only considered mutations of surface residues (based on solventaccessible surface area), since mutations to the protein core could be expected to disrupt protein
conformation. The mutations with loss of binding to mAb 225 and disregarded from analysis in
this step are listed in Table 2-5, top. However, the remaining mutations were still dispersed
across the surface of EGFR domain III in a fashion that would not allow simultaneous contact
with a single antibody-binding surface. Mutations to or from a proline, glycine, or cysteine
residue could potentially perturb the EGFR domain III secondary structure, and such mutations
were found to be abundantly represented amongst the pool of non-binding mutants. Therefore,
we applied a second criterion to disregard mutations of a potentially disruptive nature, and
residues that were mutated to or from a proline, glycine, or cysteine or from a glycosylation site
were eliminated from the mAb 225 epitope analysis (Table 2-5, middle). Since elimination of all
of these mutations is overly restrictive, the sites eventually identified by this procedure can
formally only be a subset of the energetically significant contact residues. While the use of these
two criteria reduced the number of potential mAb 225 contact residues, the remaining mutations
still were not localized to a plausible contact surface (Figure 2-7b,c and Table 2-5, bottom).
57
Table 2-5: EGFR mutants with loss of binding to mAb 225
Surface inaccessible:
C313Y
F352Y
A385V
V437A
G315R
H356Y
W386C
I439N
S326Y
T360N
A395S
C475Y
N331Y
L363Q
F396S,V,Y
C486S
F335S
L368M
G404D,S
G490D
I341S
L371M
S413F
C491R,Y
G343D
T378I
A415T
W492C
L345H
F380L
L429F,H
G493D
I347N
L381F,S
I432K
C499Y
A351E
I383N
G435R
T546I
Mutated to/from Pro, Gly, Cys, glycosylation site:
V312G
P361H
R427C
C482Y
D344G
P362T
S428P
S487P
P349L,T
P365L
G441E,V
N544I
G354S
R403C
G471R
Remaining mutants:
V312A
V350M
K465E
I467M
S324L
T358I
I466F
S487F
A329T
T373A,I
mAb 13A9, another antibody that binds to a conformational epitope, was also screened
against the EGFR mutant library. The library was labeled at 50 nM mAb 13A9 for sort 1 and 100
nM for sorts 2-5, with individual clone analysis after sort 5. Out of 170 clones sequenced, ~40%
contained multiple amino acid mutations and were omitted from subsequent analysis. A total of
73 unique single amino acid mutations were identified as leading to a loss of binding to mAb
13A9. As seen with mAb 225, the majority of mutations were located in or adjacent to domain
III but were not localized to an epitope. The same criteria used to eliminate potentially misfolded
mAb 225 mutants were applied to the mAb 13A9 mutants (Table 2-6). Notably, many of the
eliminated mutants were isolated for loss of binding to both mAb 13A9 and mAb 225. This
further supports the hypothesis that these mutants are conformationally altered such that they can
58
no longer bind conformation-specific antibodies and justifies their elimination from the epitope
mapping analysis.
Table 2-6: EGFR mutants with loss of binding to mAb 13A9
Surface inaccessible:
G315E,R
T360A
G404D
C475Y
F321V
L363Q,R
L414I,P
C486Y
S326P
V374A
S423F
C491Y
I327K
G379W
L429F
C499G
F335I
L381S
I432M
T546I
C338Y
A385V
V437M
C567R
T339I
P387L,S
N442D
P572L
I341N
L393H,P
C446S
K585N
L345H
F396C,V
F457L
A351E,S,V
L399I
N469I
Mutated to/from Pro, Gly, Cys, glycosylation site:
G317D
P365L,S
G441R
C482Y
G354A,C
R403C
G471A,S
N544I
P362H
Remaining mutants:
K322I
D344V
K443N
E472D,K
A329V
T358I
S468I
S487F
E388K
H359Y
The recurrence of the same mutations in the mAb 225 and mAb 13A9 sorts led to the
concept of antibody cross-tests, in which the conformation-specific antibodies were used as
probes for the proper folding of EGFR domain III. We hypothesized that loss of binding to only
one but not the other antibody indicated a specific, energetically important contact residue for the
antibody that showed loss of binding to the mutant. Representative antibody cross-tests are
shown in Figure 2-8. For example, the EGFR mutant I467M does not bind to mAb 225 but
shows wild-type binding for mAb 13A9. Therefore, the loss of binding to mAb 225 is most
likely due to the mutation of a specific contact residue for the antibody, and not due to protein
misfolding. Although unlikely, the possibility exists that a mutation might cause a
59
conformational change in domain III that permits binding of one antibody but not the other,
therefore generating a false positive. For the EGFR mutant A329V, loss of some degree of
binding to both mAb 225 and mAb 13A9 indicates that it is either altered in domain III
conformation or the residue is in an overlapping portion of the two antibody epitopes. Such
mutants with loss of binding to both antibodies were eliminated from the analysis, although this
will also eliminate any residues that form important energetic contacts with both antibodies.
Using the antibody cross-tests, we were able to identify two epitope residues each as
energetically important for the binding of mAbs 225 and 13A9. EGFR mutants K465E and
I467M showed loss of binding to mAb 225, but retained binding to mAb 13A9; therefore,
Lys465 and Ile467 were implicated as energetically significant contact residues for mAb 225.
Likewise, S468I and E472K showed loss of binding to mAb 13A9, but retained binding to mAb
225; therefore, Ser468 and Glu472 were identified as the mAb 13A9 epitope. The epitopes in
context of EGF and TGF-α ligand binding are shown in Figure 2-9.
60
Figure 2-8: Flow cytometry data for antibody cross-tests of mAbs 225 and 13A9 binding to yeast surface-displayed
EGFR 273-621 mutants at 50 nM mAb. EGFR display fluorescence is shown on the abscissa, and mAb binding is
shown on the ordinate. Negative controls and wild-type data are shown along with mutants isolated from the library.
61
Figure 2-9: mAb 225 and mAb 13A9 epitopes in context of ligand binding. Blue, mAb 225 epitope; red, mAb 13A9
epitope; green, ligands. (a) Domain III of EGF-bound EGFR dimer (chain a of 1IVO). (b) Domain III of TGF-αbound EGFR dimer (chain a of 1MOX).
The experimental work leading to the identification of the epitopes of mAbs 806, 225, and 13A9
(described in Sections 2.3.1-2.3.2 and 2.3.4) was published in (45). After the publication of this
paper, the crystal structure of the Fab fragment of mAb 225 bound to EGFR was solved (2). The
residues Lys465 and Ile467, which were identified by our library method, were found to be in the
62
center of the mAb 225/EGFR complex interface; thus, the results of the epitope mapping method
have been independently verified by co-crystallization.
2.3.5 Epitope mapping of mAb EMD72000
In a partnership with EMD-Lexigen Pharmaceuticals, the epitope of mAb EMD72000
was determined. mAb EMD72000 is a humanized antibody, and like mAb 225, inhibits the
binding of EGF to EGFR (46). However, initial experiments using EGFR fragments on the
surface of yeast revealed that mAb EMD72000 did not bind EGFR 273-621. Although mAbs
225 and 13A9 were able to detectably bind EGFR 273-621, at least a portion of the protein
displayed on the yeast surface was misfolded and thus unreactive to the antibodies, as evidenced
by the lower fluorescence signal at saturating binding conditions compared to mAb 806 binding
(Figure 2-8 vs. Figure 2-4). mAb EMD72000 binding may be extremely sensitive to
conformational perturbation or yeast hyperglycosylation (O-linked or hypermannosylation of Nlinked sites). Although the full EGFR ectodomain is not correctly expressed by yeast, previous
work has identified an EGFR ectodomain mutant, 404SG, which is properly folded on the
surface of yeast (25). 404SG contains two point mutations in domain I (A62T and L69H) and
two mutations in domain III (F380S and S418G). This receptor is able to bind the conformationspecific antibodies 199.12, ICR10, EGFR1, 225, and 13A9 (25). mAb EMD72000 also bound
the 404SG mutant displayed on the surface of yeast and did not compete for binding with mAb
225 or mAb 806 (Figure 2-10). However, the binding of mAb 225 to EGFR appears to slightly
decrease the affinity of mAb EMD72000 for the receptor. One possibility is that mAb 225
perturbs the conformation of EGFR domain III upon binding, thus decreasing the binding of
mAb EMD72000 to the receptor.
63
Figure 2-10: mAb EMD72000 binding to yeast surface-displayed 404SG EGFR ectodomain mutant. (a) Binding
histograms to yeast labeled with either 40 nM mAb 225 or 0.5 μM mAb EMD72000. (b) Binding histograms to
yeast labeled with mAb 225 and mAb EMD72000 simultaneously. (c) Binding histograms to yeast labeled with
either 75 nM mAb 806 or 0.5 μM mAb EMD72000. (d) Binding histograms to yeast labeled with mAb 806 and
mAb EMD72000 simultaneously.
The epitope of mAb EMD72000 was determined as before using random mutagenesis
and yeast surface display (Section 2.3.1). However, a new low mutation rate epitope mapping
library was created using 404SG as the DNA template. The library had a diversity of 6.5x105,
and sequencing of 10 random clones from the library yielded 5 wild-type clones, 1 single mutant,
and 4 multiple mutants and/or frameshifts. The library was sorted to enrich for mutants with loss
of binding to mAb EMD72000 with four rounds of FACS at 0.5 µM EMD72000 (Figure 2-11a).
Then, instead of antibody cross-tests as were performed in Section 2.3.4, high-throughput FACS
selection was then used to remove the misfolded 404SG mutants from the analysis. The library
was sorted for retention of binding to mAb 225 with 2 rounds of positive FACS (Figure 2-11b).
Since mAb 225 binding is dependent on the correct conformation of EGFR domain III, this
64
ensured that only properly folded EGFR mutants were subsequently sequenced and analyzed.
These selections also abrogated the need to apply filtering criteria to remove potentially
misfolded mutations from the epitope analysis, as was necessary for mAbs 225 and 13A9.
Figure 2-11: FACS selections for mAb EMD72000 epitope mapping. 404SG expression as detected through the cmyc tag is shown on the abscissa, and antibody binding is shown on the ordinate. Selection windows are shown in
red. (a) 404SG mutant library labeled with 0.5 μM mAb EMD72000, sorted for a loss of binding to the antibody.
The yeast were also labeled with chicken anti-c-myc IgY, followed by goat anti-chicken-Alexa488 IgG and goat
anti-human-PE IgG. (b) Enriched mutants from (a) labeled with 40 nM mAb 225, sorted for retention of binding to
mAb 225. The yeast were also labeled with chicken anti-c-myc IgY, followed by goat anti-chicken-Alexa488 IgG
and goat anti-mouse-PE IgG.
After the selections using FACS were completed, the plasmids from the yeast were
rescued, and a total of 100 clones were sequenced. All unique sequences were then retransformed into yeast to ensure their loss of binding to mAb EMD72000 and retention of
binding to mAb 225, and these mutants are listed in Table 2-7. For this selection, a total of seven
residues were identified as being energetically important for mAb EMD72000 binding, instead
of only two residues as before with mAbs 225 and 13A9. One reason was that the elimination of
misfolded mutants was high-throughput through the use of FACS, thus enabling the sequencing
and identification of many more properly folded mutations than before. Also, since the probe for
proper EGFR mutant folding, mAb 225, did not compete with mAb EMD72000, more epitope
residues could be identified. The mutants showed different degrees of loss of binding to mAb
65
EMD72000 (Figure 2-12), and these residues thus represent the mAb EMD72000 epitope. The
epitope is compared to other antibody epitopes on EGFR domain III in Figure 2-13 and the
EGFR dimer in Figure 2-14.
N
K
F
S
G
Q
K
452
454
457
460
461
462
463
I
E*, Q, N
I*
A, F, P, T, Y
D
K*, L
E, I, N, T
Table 2-7: Mutants with loss of binding to mAb EMD72000 and retention of binding to mAb 225. Mutants marked
with an asterisk were only found in the library as clones with additional irrelevant mutations, which were mutated
back to wild-type sequence, and the single mutant was then re-tested to confirm its behavior.
Figure 2-12: Examples of single-clone analysis of 404SG and mutants displayed on the surface of yeast. 404SG
expression as detected through the c-myc tag is shown on the abscissa, and antibody binding is shown on the
ordinate. Yeast were labeled with 0.5 μM mAb EMD72000 or 40 nM mAb 225 and chicken anti-c-myc IgY,
followed by goat anti-chicken-Alexa488 IgG and goat anti-human-PE or goat anti-mouse-PE IgG.
66
Figure 2-13: Space-filled representation of EGFR domain III bound to EGF ligand (1IVO). Green, EGF; red, mAb
EMD72000 epitope; Yellow, mAb 13A9 epitope; Blue, mAb 225 epitope residues identified by this work; cyan,
additional mAb 225 epitope residues from the mAb 225/EGFR crystal structure (2).
Figure 2-14: Space-filled representation of EGFR dimer bound to EGF (1IVO). EGFR residues 1-272 (the residues
absent from EGFRvIII) are shown in beige and brown, while the remaining EGFR residues are shown in grey and
olive. EMD72000 epitope, red; mAb 806 epitope, violet; all other coloring as in Figure 2-13.
67
2.3.6 Epitope mapping of DARPins
Designed ankyrin repeat proteins (DARPins) are a novel class of proteins which, like
antibodies, can be engineered for binding specificity to other proteins (47). DARPins that bind to
a number of proteins have been isolated, including binders against Her2 (48), maltose binding
protein (49), and caspase-2 (50). Originally, the molecules were designed from structure and
sequence alignments of natural ankyrin repeats to generate a module with fixed framework
residues and randomized residues for interaction with other proteins. For the full molecule,
various numbers of this module are assembled (2 to 6) between N- and C-terminal capping
repeats, and thus the randomized positions on several adjacent repeats comprise the binding
interface. DARPins are extremely stable, with midpoints of thermal denaturation in the range of
65 to 85 oC (47) and thus are easily expressed and purified from E. coli. Five DARPin clones
(E1, E67, E68, E69 and E72) binding to EGFR were isolated by Daniel Steiner of the Andreas
Plueckthun laboratory at the University of Zurich. Initial characterization of the DARPin clones
performed by Daniel is detailed in Table 2-8. It was determined that E1, E67, and E68 competed
with mAb 225 for binding to EGFR, while E69 did not. In addition, E67 and E68 competed for
binding with E1.
Repeats
E1
E67
E68
E69
E72
3
3
3
4
3
Apparent
MW
28.0
26.8
58.8/26.8
33.4
26.8
koff
mAb 225
E1
kon
competition competition [104 M-1s-1] [10-3 s-1]
yes
yes
43.00
0.21
yes
yes
2.95
0.22
yes
yes
314.00
1.89
no
no
7.25
1.05
-
Kd
[nM]
0.48
7.30
0.60
14.52
>100
Table 2-8: Summary of characterization of DARPin clones performed by Daniel Steiner. MW, molecular weight;
kon, on-rate; koff, off-rate; Kd, apparent dissociation constant. Because of a low binding signal for clone E72,
competition data was inconclusive.
68
First, domain-level mapping of the DARPins was accomplished using EGFR fragments
on the surface of yeast as before (15). E1 was found to bind yeast-surface displayed 404SG but
did not bind EGFR 273-621 or 294-543, most likely for the same reasons that mAb EMD72000
binding to these fragments was not observed (Section 2.3.5). The same binding pattern was seen
for E67 and E68. Based on the previous experimental results, it was inferred that these three
clones bound to EGFR domain III, and thus binding to the other EGFR fragments not containing
this domain was not tested. E69 showed binding to 404SG and EGFR 1-294, very slight binding
to EGFR 1-176, and no binding to EGFR 1-124. Therefore, it appears that E69 is binding either
domain I or domain II, very possibly to residues spanning from just before amino acid 176 to
amino acid 294. However, we cannot exclude the possibility of misfolding of the fragments
which do not show E69 binding; therefore, formally, the epitope is located between amino acids
1-294. E72 only showed binding to 404SG and not to any of the other EGFR fragments tested.
These binding results are summarized in Table 2-9.
EGFR
fragment
E1 E69 E72
E67
E68
1-124
Ν
−
−
1-176
~
Ν
−
1-294
+
Ν
−
273-621
−
−
−
294-543
−
−
−
475-621
Ν
−
−
1-621 (404SG)
+
+
+
Table 2-9: Binding of DARPins to yeast surface-displayed EGFR fragments. +, binding; −, no binding; ~, very
slight binding; N, not tested.
Binding of the DARPins to yeast surface-displayed 404SG is shown in Figure 2-15.
Although each DARPin was labeled at a concentration to give >90% receptor occupancy
69
according to its affinity, appreciable differences in binding signal were observed for the different
DARPins. Since 404SG has two mutations in domain III, these mutations may be affecting the
binding of the DARPin clones to the yeast-displayed protein, with binding of clone E67
especially affected.
Figure 2-15: Labeling of yeast surface-displayed 404SG with DARPin clones. 404SG expression as detected
through the c-myc tag is shown on the abscissa, and DARPin binding is shown on the ordinate. The yeast were
labeled with each of the DARPins and detected using biotinylated Penta-His antibody followed by streptavidin-PE.
Yeast were also labeled with chicken anti-c-myc IgY and goat anti-chicken-Alexa488 IgG to detect display level of
404SG. Cells were labeled with (a) 50 nM E1 (b) 150 nM E67 (c) 50 nM E68 (d) 150 nM E69 (e) 500 nM E72.
Competition binding experiments were conducted for DARPins and conformationspecific anti-EGFR antibodies binding to yeast-displayed 404SG. The DARPin and antibody
were added simultaneously to the yeast mixture, and the change in DARPin binding was
measured using FACS (Figure 2-16). E1 competed with mAb 225 and mAb EMD72000 for
binding to 404SG, but not with mAb 528. Likewise, E67, E68, and E72 all displayed similar
behavior to E1. E69 competed with ICR10 and 199.12 binding, but not with EGFR1.
70
Figure 2-16: DARPin competition with anti-EGFR mAbs binding to 404SG on the surface of yeast. Cells were
labeled with DARPins at the same concentrations as Figure 2-15. mAbs were added at a concentration of 50 nM
simultaneously with the DARPins. DARPin binding fluorescence as detected using biotinylated Penta-His antibody
followed by streptavidin-PE is shown on the x-axis.
DARPins E1, E68, and E69 were selected for further fine epitope mapping. The 404SG
mutant library used for mAb EMD72000 epitope mapping (Section 2.3.5) was also utilized for
the DARPin mapping. First, misfolded EGFR mutants were removed from the library through
one FACS selection for retention of binding to either 50 nM mAb 528 or mAb EGFR1. Then, the
mAb 528-positive library was sorted for loss of binding to E1 (sort 1 at 50 nM and sorts 2-3 at
100 nM) or E68 (sort 1 at 50 nM and sorts 2-3 at 100 nM). The mAb EGFR1-positive library
71
was sorted for loss of binding to E69, with sort 1 at 150 nM and sorts 2-3 at 300 nM. The
enriched populations from the sorts were sequenced, and individual mutants were transformed
into yeast and tested for proper folding and loss of binding to the DARPins. From 98 clones
sequenced for DARPin E69, the epitope was localized to ten residues in EGFR domain I (listed
in Table 2-10). The E69 epitope on the EGFR ectodomain is shown in Figure 2-17.
Y
D
A
N
K
T
P
H
L
G
101
102
103
104
105
106
130
159
160
161
H
E
P
I
E, I, N
P
H, L, S
Y
P
D
Table 2-10: Mutants with loss of binding to DARPin E69 and retention of binding to mAb EGFR1.
Figure 2-17: Epitope of E69. Epitope residues are shown in red, with the rest of the EGFR ectodomain (from 1IVO,
chain a) shown in grey. EGF ligand is shown in stick representation in green.
72
For E1 and E68, 48 clones were sequenced from each enriched population, yielding 14
residues for both epitopes. As seen in Table 2-11, many residues were shared between the two
DARPins. The mutants Q408H, H409Y, Q411K, K465I, and G471D, which led to loss of E1
binding, were tested against E68 and retained binding to this DARPin. The mutant Q384H
showed loss of binding to E68, but retained binding to E1 (data not shown). However, some
mutants isolated for loss of binding to one DARPin (H409Y, F412V, A415E, I438K,
K463E/I/N/T, K465E, I467M/T, N469D) were not assayed for loss of binding to the other. Thus,
these mutants may not be unique to either the E1 or E68 epitopes, but perhaps were not sampled
in the clones sequenced. Amino acid 418 is an epitope residue for both E1 and E68, and it is
mutated from serine to glycine in the 404SG EGFR ectodomain mutant, likely causing the lower
than expected binding of these DARPins to 404SG (as seen in Figure 2-15).
Residue
L 381
Q 384
Q 408
H 409
Q 411
F 412
A 415
V 417
G 418
I
438
G 441
K 463
K 465
I
467
S 468
N 469
G 471
E1
V
E68
V
H
H
D, P, Q, Y
K, R
T, V
A
D
K
E, R
E, I, N
M
G, R
H
D
D, P, Q
R
V
E, T, V
A
D
E, R
E, I, N, T
N
T
G, R
H, D
Table 2-11: Mutants with loss of binding to DARPins E1 and E68 and retention of binding to mAb 528. 404SG
wild-type residues are shown in the left hand column, and mutations isolated for loss of binding to each DARPin are
shown in the right columns.
73
The E1 and E68 epitopes are shown in Figure 2-18. Both DARPins bind to EGFR domain
III and are expected to directly compete with ligand binding to the receptor. In addition, the
DARPin epitopes share many residues with the mAb 225 epitope, consistent with these two
DARPins competing with mAb 225 binding (as seen in Table 2-8 and Figure 2-16).
Figure 2-18: (a) E1 and (b) E68 epitope residues (red) shown on EGFR domain III (chain a of 1IVO) bound to EGF
ligand (green), compared to (c) mAb 225 epitope residues as identified by the crystal structure (2).
74
2.4
Discussion
This work describes a novel method of fine epitope mapping using screening of randomly
mutagenized antigen displayed on the yeast cell surface. The method is capable of identifying
conformational epitopes of antibodies binding to complex eukaryotic proteins without prior
knowledge of potential contact residues, which are several advantages over peptide epitope
mapping and alanine scanning. The yeast surface display system enables convenient protein
expression without the need to solubly express and purify each individual mutant. Clone
characterization and titration are also efficiently performed on the surface of yeast. Initially, we
identified the epitope of mAb 806, whose binding is not dependent on antigen tertiary structure.
Because of this, all single mutations initially isolated from the library for loss of binding to mAb
806 were localized to a single plausible antibody contact surface. In contrast, in order to identify
the epitopes of mAb 225 and mAb 13A9, it was necessary to apply filtering criteria and antibody
cross-tests to remove misfolded mutants from the analysis. Later, while mapping mAb
EMD72000, we improved our epitope mapping method through the use of high-throughput
selection of mutants which retained binding to a different conformation-specific antibody, thus
ensuring that all sequenced clones were properly folded.
This method can be generalized to map any protein-protein interaction. Yeast must be
able to surface display one binding partner, and the other binding partner must be made solubly.
Finally, another conformation-specific binder to the yeast-displayed protein is necessary to
eliminate misfolded mutants. Preferably, this protein should not compete with the interaction
being mapped, although useful information can still be gained if they do compete (as in the case
of mAbs 225 and 13A9). For a multi-domain protein such as EGFR, the probe for proper
conformation should preferably bind the same domain as the interaction being mapped.
75
Additionally, if yeast are unable to properly surface display a protein of interest, a small number
of mutations can often be found through directed evolution that enable the correct folding of the
protein, as in the case of EGFR and the 404SG mutant (25). Our method of random mutagenesis
and yeast surface display has since been used to map the paratope of a single-chain antibody (51)
and to determine the epitope of antibodies against botulinum toxin (52) and the West Nile virus
envelope protein (53).
A potential shortcoming of the fine epitope mapping method is that it is impossible to
isolate all possible mutants which lead to a loss of antibody binding. For example, C302A is not
an amino acid change accessible by a single nucleotide mutation; therefore, with a low mutation
rate library, it is extremely unlikely that this mutation will be seen. In addition, although the
epitope mapping library size was theoretically large enough to accommodate all amino acid
changes accessible by a single nucleotide mutation, the mutant V299D was not isolated from the
library. However, when this site-directed mutant was constructed, it led to a large loss of binding
to mAb 806. Since the generation of the library is random, by chance, the initial library may not
have contained this mutant. Alternately, the mutant may have been lost during growth or sorting
of the library prior to single clone analysis, or it may have been necessary to sequence more
clones to identify this mutant. To avoid such events, a larger library could be made to increase
the number of mutations, or a larger number of cells could be grown, sorted, and sequenced.
Despite the potential for loss of relevant mutants, the significantly reduced effort required in this
approach outweighs the alternatives of site-directed mutagenesis or saturation mutagenesis of the
entire gene. In any case, the fine epitope mapping method can be used to localize antibody
binding to certain residues, followed by the creation of site-directed mutants to confirm and/or
expand the epitope.
76
In this method, substitutions other than those to alanine can be sampled. As demonstrated
here, some energetically important residues are not significantly impacted by mutation to alanine
(e.g. Asp297, Arg300, and Lys301 for mAb 806), and consequently would lead to false negatives
in an alanine scan. In some instances, the mutations in the library led to reversals of amino acid
charge. Such mutations at some distance from the antibody contact interface might conceivably
contribute to a loss of binding through electrostatic steering effects. However, this formal
possibility did not appear to pertain in the cases of the mAb 806, mAb 225, and mAb 13A9
epitopes, given the clustering of mutants in contiguous locations.
Using this epitope mapping method, the binding site for mAb 806 was localized to one
face of the constrained disulfide loop of EGFR 287-302, with Asp297-Cys302, Glu293, and
possibly Cys287 acting as contact residues. The mAb 806 epitope is an example of a cystine
noose, which is a disulfide-constrained, surface-exposed loop important in binding specificity
(54). Cystine nooses have also been identified as major antigenic epitopes on various proteins,
including protein G of bovine respiratory syncytial virus (55) and measles virus hemagglutinin
protein (56). This suggests that a disulfide-constrained loop is a favorable antigenic structure;
since it is already constrained, there is a smaller entropic cost upon antibody binding. Thus, a
number of other disulfide loops on EGFR are potential epitope targets for antibody binding. mAb
806 has been shown to exhibit increased binding to EGFR on cells lacking the domain II
dimerization arm (33). Therefore, it has been hypothesized that mAb 806 binds to a transitional
form of the receptor as it changes from a closed to extended monomer (33). It is thought that
upon mAb 806 binding, the EGFR monomer can no longer dimerize and activate the receptor,
accounting for its anti-tumor activity. The mAb 806 epitope presented here and as a result of
protein docking simulations (36) is consistent with this hypothesis. The epitope is only partially
77
accessible in the tethered monomer structure, with residues Glu293 and Cys302 obscured (Figure
2-6d). These residues could become exposed upon a conformational transition and allow binding
of mAb 806. The mAb 806 epitope of one EGFR monomer is adjacent to the other monomer in
the EGFR dimer structures, and antibody binding to this epitope could sterically prevent EGFR
dimerization. Likewise, the mAb 175 epitope was found to be highly similar to that of mAb 806,
and the two antibodies are expected to possess the same characteristics.
Two energetically important residues for each of the mAb 225 and mAb 13A9 epitopes
were also identified. Because of a lower fluorescence binding signal to noise ratio for these
conformation-specific antibodies, only mutants with a complete loss of binding could be
differentiated from wild-type. The location of these energetically important contact residues
might provide insight into the mechanisms of these antibodies (Figure 2-9). The mAb 225
epitope is positioned adjacent to the binding sites of both EGF and TGF-α, and thus mAb 225
most likely blocks binding of both ligands sterically. The epitope of mAb 13A9 appears to be
located further away from the ligand binding site; although Ser468 is adjacent to the EGF
binding site, Glu472 is farther towards EGFR domain IV. Therefore, it is possible to envision
both mAb 13A9 and EGF simultaneously binding to EGFR, consistent with the experimental
observation that mAb 13A9 does not compete with EGF binding (38). As viewed in the crystal
structures of EGF- and TGF-α-bound EGFR, it does not appear possible to sterically block
TGF-α binding without also sterically preventing EGF binding. However, mAb 13A9 prevents
TGF-α binding while allowing EGF binding (38). Based on the location of these residues and the
ligand binding site on EGFR, mAb 13A9 may prevent TGF-α binding through mediating
conformational changes to domain III that are sufficient to prevent TGF-α, but not EGF binding.
We have already shown that domain III is highly sensitive to conformational perturbation
78
through mutation. Conceivably, therapeutic antibodies could bind to a number of epitopes on
EGFR domain III to mediate such a conformational change without the need to sterically occlude
ligand binding. However, it also possible that these crystal structures do not give a complete
picture of ligand binding sterics, and mAb 13A9 may have a different mechanism of action.
Seven residues in EGFR domain III were identified as the epitope of mAb EMD72000.
Based on its location on the crystal structure, it does not appear that mAb EMD72000 directly
inhibits EGF binding (Figure 2-13). However, mAb EMD72000 may inhibit EGF binding by
preventing EGFR from adopting the fully extended conformation necessary to sandwich the
ligand between domains I and III (Figure 2-14). This has also been suggested as an additional
mechanism of mAb 225 (2). Alternately, mAb EMD72000 may perturb the conformation of
domain III and prevent ligand binding in the same manner as we hypothesize mAb 13A9 does.
Finally, the yeast surface display epitope mapping method was extended to the mapping of
DARPin molecules binding to EGFR. Clone E69 bound to EGFR domain I and is not expected
to inhibit ligand binding. However, clones E1 and E68 bound to domain III and are expected to
inhibit ligand binding in a manner similar to mAb 225. The E1 and E68 DARPin epitopes highly
overlap with the mAb 225 epitope, and all of these molecules also share contact residues with
ligands which bind to EGFR. This portion of domain III represents a hot spot for protein
recognition, with three very different types of proteins binding to the same location on EGFR.
In conclusion, we have developed a novel method of fine epitope mapping using random
mutagenesis and yeast surface display. The technique was used to identify the binding epitopes
of a total of four anti-EGFR antibodies and three DARPin molecules. The locations of these
epitopes may aid in the understanding of EGFR antibody mechanisms of action and suggests
new targets for EGFR inhibition.
79
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West Nile virus, Nat Med 11, 522-530.
Lapthorn, A. J., Janes, R. W., Isaacs, N. W., and Wallace, B. A. (1995) Cystine nooses
and protein specificity, Nat Struct Biol 2, 266-268.
Langedijk, J. P., Meloen, R. H., Taylor, G., Furze, J. M., and van Oirschot, J. T. (1997)
Antigenic structure of the central conserved region of protein G of bovine respiratory
syncytial virus, J Virol 71, 4055-4061.
Putz, M. M., Hoebeke, J., Ammerlaan, W., Schneider, S., and Muller, C. P. (2003)
Functional fine-mapping and molecular modeling of a conserved loop epitope of the
measles virus hemagglutinin protein, Eur J Biochem 270, 1515-1527.
83
Chapter 3: Engineering antibodies against EGFR
3.1
Background
Directed evolution is a common procedure for engineering antibodies against an antigen
of interest. In directed evolution, mutations of the antibody are made at the DNA level, and these
mutants are then expressed as proteins. The mutants with improved binding to an antigen of
interest are isolated, and these improved clones can be further mutated for increased antigen
binding. The entire process is repeated multiple times until the desired antibody properties are
attained (Figure 3-1).
Figure 3-1: Process of directed evolution.
In order to engineer antibodies through directed evolution, a display system must be used
to link the genotype of an antibody to its phenotype. Tools to accomplish this include phage
84
display, mRNA display, and yeast surface display. Since it is difficult for these systems to
display or express full antibody immunoglobulin (IgG) molecules, smaller fragments of the IgG
are often used instead (Figure 3-2). The IgG molecule contains two identical antigen-binding
sites at the end of each variable arm, and a Fab consists of one set of variable domains and CH1
and CL domains. A single chain variable fragment (scFv) molecule contains the minimal
sequences necessary for antigen binding, with the variable heavy and light chains connected by a
flexible polypeptide linker, usually (Gly4Ser)3. The complementarity-determining regions
(CDRs) are the antibody residues responsible for mediating antigen binding and consist of six
loops in the variable domain (Figure 3-2c).
Figure 3-2: Immunoglobulin (IgG) protein and its fragments. (a) Schematic of an IgG. Ag, antigen; scFv, single
chain variable fragment; VH, variable heavy region; VL, variable light region; CH, constant heavy region; CL,
constant light region. (b) Crystal structure of an immunoglobulin molecule (1HZH). Heavy chain, blue; light chain,
yellow. (c) scFv of the antibody in (b), with CDRs shown in yellow and blue for the light and heavy chains,
respectively.
85
Phage display is the most widely used method of antibody isolation and affinity
maturation. scFvs or Fabs are displayed on the surface of either fd or M13 bacteriophage and
selected for binding to an antigen of interest (1). Alternately, mRNA or ribosome display is an in
vitro method where the DNA encoding an scFv is stably linked to the protein through a
puromycin linker or ribosome, respectively (2). In yeast surface display (3), the scFv is displayed
on the yeast cell wall as a fusion to the native yeast Aga2 protein (Figure 3-3a). Expression of
the protein on the yeast surface can be detected through the use of the C-terminal c-myc epitope
tag, and binding to a biotinylated antigen of interest is detected through a different colored
secondary reagent. Expression of the Aga2p-scFv is under the control of a galactose-inducible
promoter on the yeast display plasmid, which is maintained in yeast episomally with a nutritional
marker. Each yeast cell typically displays 104 to 105 copies of the scFv.
86
Figure 3-3: (a) Schematic of yeast surface display. The scFv (cyan) is displayed as an Aga2p (pink) fusion on the
yeast surface. Expression can be detected through fluorescent antibodies binding to the epitope tags (tan), and
binding of the scFv to a biotinylated antigen (orange) can be detected using fluorescent avidin (violet). HA,
hemagglutinin; VL, variable light chain; VH, variable heavy chain. (b) Yeast cells labeled as in (a) for surface
expression (abscissa) and antigen binding (ordinate). The double negative population of yeast cells is due to plasmid
loss. As yeast surface display increases, so does the antigen binding signal, giving rise to a diagonal binding curve.
The fluorescence data for two different clones are overlaid, with a mutant with two-fold higher affinity in red and
wild-type in blue. Because of the expression normalization in yeast surface display, a diagonal sort window can be
drawn to capture even slightly improved mutants efficiently. (c) The populations in (b) are difficult to differentiate
based on the antigen-binding histogram alone. PE, phycoerythrin. Figures were reproduced from (4).
In addition, Fab fragments have been displayed on the surface of yeast (5,6). In the
system described in (6), the heavy chain is expressed as an Aga2p fusion, with the light chain
expressed from a separate plasmid (Figure 3-4). The two antibody chains associate in the yeast
endoplasmic reticulum (ER) and are displayed as a full Fab construct on the yeast surface.
Separate epitope tags on each chain can be used to ensure the presence of both heavy and light
regions.
87
Figure 3-4: Schematic of Fab display on the surface of yeast (6). The heavy chain variable and constant regions
(cyan) are expressed as an Aga2p fusion and can be detected using an Alexa488-labeled antibody binding to the cmyc epitope tag (violet). The light chain (yellow) is captured by the heavy chain in the yeast ER and can be detected
through a PE-labeled antibody binding the V5 epitope tag. Binding to an antigen such as biotinylated peptide
(orange) can be detected using avidin (magenta) labeled with a third fluorophore such as allophycocyanin (APC).
Yeast surface display offers several advantages for the directed evolution of proteins. It
enables quantitative screens through the use of fluorescence-activated cell sorting (FACS),
allowing the equilibrium activity and statistics of the sample to be observed directly during the
screening process. Furthermore, the antigen-binding signal is normalized for expression,
eliminating artifacts due to host expression bias and allowing for fine discrimination between
mutants (Figure 3-3b,c). Antibodies can be engineered for improved stability, as expression is
measured directly and has been shown to correlate with the stability of the displayed protein (7).
Using a two-color labeling scheme as in Figure 3-3a, stability and affinity engineering can be
accomplished simultaneously. Once maturation is complete, antibody affinity can be
conveniently titrated while displayed on the surface of the yeast, obviating the need for
expression and purification of each clone. In almost every case for dozens of different antibodies,
the binding properties on the yeast cell surface are in quantitative agreement with those measured
88
in solution or by biosensor methods (8). Finally, the displayed proteins are folded in the ER of
the eukaryotic yeast cells, taking advantage of ER chaperones and quality-control machinery.
A theoretical limitation of yeast surface display is the potentially smaller functional
library size (~107-109) than that of other selection methods (phage display, ~106-1011;
mRNA/ribosome display, ~1011-1013). However, it is difficult to determine the true functional
diversity of any display library, and bias-free propagation of yeast libraries has been confirmed
over an amplification of 1010-fold (9). Furthermore, all of these methods greatly undersample the
theoretical sequence space of scFv CDR variation (~1080). To realize the advantages of kinetic
screening and expression normalization, yeast surface display also requires more complex
equipment than other display methods. Various technologies for screening recombinant antibody
libraries and their relative strengths and weaknesses have been reviewed in (10). However, in
parallel selections from the same DNA library, it has been shown that yeast display can yield
three times as many unique scFv clones as phage display does (11).
Yeast surface display has been used to engineer antibodies against various antigen
targets, including T cell receptors (12), huntingtin protein (13), carcinoembryonic antigen (14),
and botulinum neurotoxin (15). In addition, an antibody to fluorescein has been engineered to
femtomolar affinity, the highest affinity yet reported (16). A nonimmune human library of 109
scFv clones displayed on the surface of yeast has been created (9) and can serve as an initial
library for the isolation of antibodies against an antigen of interest.
Although there are currently two anti-EGFR antibody drugs (cetuximab and
panitumumab) approved by the FDA for the treatment of cancer and many more in clinical trials
(see Section 1.4), these antibodies are only marginally effective. Cetuximab treatment leads to
objective responses in only 10.8% of patients as monotherapy and 22.9% in combination with
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irinotecan (17). Panitumumab produces an 8% overall response rate in metastatic colorectal
cancer, with progression-free survival extended from 60 days to 96 days (18). In a recent phase II
trial, another anti-EGFR antibody, matuzumab, showed no evidence of significant clinical
activity in patients with ovarian cancer (19). The low response rates are particularly noteworthy
since the clinical trials only involved patients who tested positive for EGFR expression in tumor
biopsies. Clearly, there is room for improved novel antibodies against EGFR.
In this antibody engineering work, we have opted to use an alternate inhibition strategy
from existing EGFR therapeutics. Traditional anti-EGFR antibodies such as cetuximab,
panitumumab, and matuzumab bind to EGFR domain III and inhibit ligand binding, thus
preventing receptor activation and downstream signaling (Figure 3-5a). However, we sought to
engineer antibodies against EGFR domains II and IV to directly inhibit receptor dimerization
(Figure 3-5b). Such antibodies could have advantages over ligand-competitive antibodies in
certain types of tumors. In particular, cancer cells driven by EGFR autocrine signaling (20) have
high local effective concentrations of ligand, and it may be difficult for an antibody diffusing
into a tumor from the plasma to compete with ligand binding. In addition, ligand-competitive
antibodies such as cetuximab are ineffective against EGFRvIII (21), a truncation mutant which
signals constitutively in the absence of ligand (22). Finally, novel engineered antibodies could be
used synergistically with existing EGFR antibody therapeutics.
Figure 3-5: EGFR inhibition strategies for antibodies. (a) Ligand-competitive antibodies such as cetuximab. (b)
Signaling inhibition by blocking receptor dimerization. Blue, receptor; green, ligand; yellow, antibody.
90
Based on the EGFR dimer crystal structures, antibodies binding to either the domain II
dimerization arm or portions of domain IV should inhibit receptor dimerization (see Figure 1-4).
Although domain IV is not resolved in the EGFR dimer crystal structures, it may contain
receptor dimerization contacts for reasons discussed in Section 1.2. Even if domain IV does not
mediate receptor dimerization, it is predicted that the two domains are sufficiently close in the
dimerized form such that antibody binding to domain IV should sterically inhibit dimerization.
In order to target specific regions of EGFR domains II and IV with antibodies, a former
postdoctoral associate in the laboratory, Mark Olsen, designed peptide mimics of loops in the
receptor to use as antigen. Five peptides, one in domain II comprising the dimerization arm and
four in domain IV, were selected and designed (Figure 3-6). In this thesis, the peptides will be
referred to according to the first three amino acids in the corresponding EGFR sequence (i.e.
LYN, CRN, CAH, MGE, and LVW). All five peptides were synthesized with an N-terminal
biotin molecule for detection, and a glycine amino acid to separate the peptide epitope from the
biotin molecule. Because of the small size of the MGE peptide, a larger spacer molecule,
6-aminohexanoic acid, was used instead of glycine. The peptides were cyclized through a
disulfide bond to mimic the structures of the loops in EGFR.
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Figure 3-6: EGFR peptide epitopes targeted for antibody engineering. Peptides are shown in space-filled
representation on the tethered EGFR monomer structure (1NQL). Peptides synthesized for use as antigen are shown
on the right, with the corresponding EGFR residues shown below the peptide sequence. Aha, 6-aminohexanoic acid.
The peptide epitopes were chosen for their potential to inhibit EGFR dimerization. The
LYN peptide consists of the tip of the domain II dimerization arm (23,24), and proteins binding
to this epitope would be analogous to pertuzumab, an antibody which binds to domain II of Her2
(25). The CRN peptide was chosen as a control peptide in domain IV. The other three domain IV
peptides (CAH, MGE, and LVW) are analogous to the binding epitope of Herceptin on Her2
(26). The CAH peptide corresponds to part of the Herceptin epitope on Her2 residues 570-573,
the LVW peptide corresponds to another part of the epitope on Her2 residues 593-603, and MGE
corresponds to residues immediately N-terminal of the LVW peptide. In addition, the LVW
peptide has previously been synthesized, and its binding to EGFR has been shown to inhibit
receptor heterodimerization with Her3 (27). However, antibodies against this peptide have not
been published. A monoclonal antibody (2D2) against the peptide
92
556IQCAHYIDGPHC567,
which includes the CAH peptide, has been generated for diagnostic purposes (28), but the effect
on EGFR dimerization of binding to this epitope has not been tested.
To isolate antibodies binding to these peptides, Mark Olsen performed the first round of
screening against the nonimmune human scFv library expressed on the surface of yeast (9). For
each of the peptides, he first performed one magnetic bead isolation (MACS) at 1 μM peptide,
followed by three selections using fluorescence-activated cell sorting (FACS). These screens
yielded candidate clones binding to all of the peptides except the CAH peptide.
In this thesis work, antibody engineering against the above EGFR peptides was
continued. It was discovered that all the clones isolated previous to this thesis work were heavy
chain only binders. Full scFv molecules were engineered by adding random light chains to the
selected heavy chains. In addition, rounds of affinity maturation through random mutagenesis
using both peptide and soluble EGFR ectodomain as antigen were performed. Fab display on the
surface of yeast and rabbit monoclonal antibody technology were utilized to further engineer
binding to peptides LYN and MGE. However, affinity maturation of Fabs to the MGE peptide
led again to binders consisting of only the heavy chain, and the rabbit monoclonal antibodies
binding to the MGE peptide did not bind the full EGFR ectodomain.
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3.2
Materials and methods
Peptide and 404SG antigen
The peptides listed in Figure 3-6 were synthesized by Cell Essentials, Inc. (Boston, MA).
The molecular weights of the peptides were confirmed by mass spectrometry to 95% purity:
LYN, 1973; CRN, 1516; CAH, 1452; MGE, 1208; and LVW, 1565.
For the secretion of soluble 404SG, the S. cerevisiae strain YVH10 was used, which is
modified from BJα5464 to overexpress protein disulfide isomerase (29). The DNA for 404SG, a
mutant of the EGFR ectodomain (30), was cloned using NheI and BamHI into the secretion
plasmid pBala, which contains an N-terminal flag epitope tag (DYKDDDDK) and C-terminal
His tag (HHHHHH). YVH10 was transformed with this DNA in addition to an empty pRS314
vector (which enables tryptophan synthesis) using the EZ yeast transformation kit
(ZymoResearch). Transformed cells were plated on SDCAA plates (yeast media preparation is
described in (4)), and individual colonies were grown in 5 ml SDCAA media at 30 oC overnight.
The seed culture was transferred to 1 L SDCAA media in a 2 L Tunair flask (Shelton Scientific)
and grown at 30 oC for 20-22 h. The cells were harvested and pelleted in 500 ml centrifuge tubes
for 15 min at 5000 rpm and 4 oC. The yeast were resuspended in 1 L SGCAA containing 0.5 g
lysozyme as a carrier protein and were induced in a 2 L glass shake flask with no baffles at 20 oC
for 3 d. The supernatant was separated from the yeast as above, and the pH was adjusted to 7.4.
404SG protein was initially purified according to the manufacturer’s instructions using a batch
protocol with TALON Superflow resin (BD Biosciences), which binds to the His6 tag.
Alternately, the yeast supernatant was concentrated to ~50 mL volume using an Amicon stirred
cell concentrator (Millipore). The protein was then digested with EndoH (New England
Biosciences) to remove excess glycosylation and was purified using mAb 528 agarose resin
94
(Santa Cruz Biotechnology). The protein was eluted with 0.1 M glycine HCl, pH 3.5 and was
neutralized with 1 M Tris, pH 8. The protein concentration was determined using an extinction
coefficient of 55,900 M-1cm-1 at 280 nm absorbance.
Yeast labeling for FACS and clone characterization
Yeast were labeled as in (4). Additional yeast surface display protocols are available in
(31) and (32). Briefly, EBY100 yeast transformed with the pCT2 yeast display plasmid were
grown in SDCAA media at 30 oC and induced in SGCAA at 20 oC. For FACS analysis and
sorting, 1x106 yeast (for individual clones) or 10X the library diversity were labeled for display
using either mouse anti-HA 12CA5 IgG (Abcam; 1:100) or chicken anti-c-myc IgY (Invitrogen;
1:250), followed by goat anti-mouse Alexa488 or goat anti-chicken Alexa488 (Invitrogen;
1:100). Yeast were labeled with biotinylated peptides, followed by either streptavidin-PE (1:50),
streptavidin-APC (1:100), or neutravidin-PE (1:50; all from Invitrogen). For 404SG labeling,
404SG was pre-incubated with up to 0.5 μM biotinylated M2 anti-flag antibody (Sigma) for at
least 1 h on ice, followed by streptavidin-PE. All incubations were performed in PBS buffer with
0.1% BSA, with primary incubations for 30 min at room temperature and secondary incubations
for 20 min on ice.
For characterization of individual clones, DNA was harvested from yeast cells using a
Zymoprep kit (ZymoResearch), transformed into XL-I Blue E. coli (Stratagene), and sequenced
by the MIT Biopolymers Laboratory. BLAST searching against germline genes was performed
using IgBlast at http://www.ncbi.nlm.nih.gov/projects/igblast. Fluorescence data was collected
using a Coulter Epics XL flow cytometer (Beckman-Coulter) and was analyzed using FloJo
software (Tree Star, Inc.).
95
Construction of scFv libraries
For the first round of affinity maturation pairing selected heavy chains with light chains,
plasmid DNA from the original scFv library construction (9) was amplified by transformation
and growth in E. coli strain XL-I Blue (Stratagene). Plating of transformed cells showed that the
maximum diversity of this amplified DNA was 8.5x105, which is slightly lower than the original
library’s reported VL diversity (1.2x106). However, in order to conserve the original library
DNA, it was determined that this was sufficient to proceed. The library DNA was digested with
NheI and BamHI to remove the original VH sequence. Then, the DNA containing the originally
selected VH clones (LYN-1, etc.) were digested with KpnI and NotI to generate the insert
fragment. These two DNA fragments were transformed into EBY100 yeast as described to
generate libraries of full scFvs (4). It was determined that this method yielded more full-length
scFvs and thus was preferable to using the digested VH clone as backbone and a PCR product
from the library insert. Libraries were labeled as above and sorted using either a MoFlo or Aria
FACS machine at the MIT flow cytometry core facility. For the generation of subsequent
random mutagenesis libraries, the nucleotide analog mutagenesis protocol in (32) was used.
Yeast were transformed and libraries were sorted as above.
Fab display
Fab display vectors pPNL200 and pPNL300 (adapted from (6)) were generously
provided by Mike Feldhaus. For individual clone analysis, both plasmids were transformed into
the yeast strain JAR200 (Ura-, Trp-) and grown and induced as strain EBY100 above. To clone
scFv genes into the Fab display vectors, 5’ NheI and 3’ MluI restriction sites were added to the
variable heavy region using PCR, and for the light chain, 5’ DraIII and 3’ BsiWI sites were
96
added. Populations of yeast were cloned into the Fab display vectors by designing primers based
on sequenced clones and PCR using the zymoprep DNA as template. The primers used for
nucleotide analog mutagenesis of antibodies in the Fab vectors are listed in Table 3-1. For library
backbone generation, pPNL200 was digested with NheI and MluI and pPNL300 with DraIII and
BsiWI. Heavy and light chain libraries were constructed separately, with the heavy library
transformed into JAR200 and grown in SDCAA+20 mg/L uracil (added from 0.2% stock) and
the light library transformed into YVH10 and grown in SDCAA+20 mg/L tryptophan (added
from 1% stock). The Fab library was created using yeast mating by resuspending 109 freshly
grown cells from each of the libraries in 10 mL YPD. The yeast were pipetted onto 4 YPD plates
(15 cm diameter) in a pool (not spread) and incubated at 30 oC overnight. The mated cells were
scraped off and resuspended in SDCAA, and dilutions were plated to determine mating
efficiency. This protocol was adapted from (6).
Table 3-1: Sequences of primers used in Fab display
VH:
VH:
VL:
VL:
VH,
200for 5’-CTAAAGAAGAAGGGGTATCTCTCGAAAAAAGAGAGGCTGAAGCAACGCGT-3’
200rev 5’-CTCTTGGAGGAGGGTGCCAGGGGGAAGACCGATGGGCCCTTGGTGCTAGC-3’
300for 5’-TAAAGAAGAAGGGGTATCTCTCGAAAAAAGAGAGGCTGAAGCACGATGTG-3’
300rev 5’-TGCTCATCAGATGGCGGGAAGATGAAGACAGATGGTGCAGCCACCGTACG-3’
VL sequencing: 5’-CTATTGCCAGCATTGCTGC-3’
Growth and labeling of mammalian cells
A431NS cells (A431 cells passaged to become less adherent) were obtained from
American Type Culture Collection (ATCC) and grown in DMEM (ATCC) supplemented with
10% fetal bovine serum (FBS). NR6 cells (33) were obtained from Doug Lauffenburger (MIT)
and grown in MEM-α supplemented with L-glutamine, sodium pyruvate, non-essential amino
acids, and 350 mg/L G418. Mammalian cell monolayer panning was conducted as described in
97
Appendix 2. For FACS labeling, A431 cells were lifted from the plate using versene (Invitrogen)
at 37 oC for 30 min. 2.5x105 cells were labeled in 200 μL/tube in PBS + 1% BSA on ice. Goat
anti-rabbit-PE (Invitrogen) was used to visualize rabbit antibody binding. 7-aminoactinomycin D (7-AAD) for labeling dead cells was purchased from Calbiochem.
Production and growth of rabbit hybridomas
Rabbit hybridoma production was performed by Epitomics, Inc. (Burlingame, CA). LYN
and MGE peptides (lacking biotin) were conjugated to KLH and injected into rabbits. Rabbit
hybridoma clones were grown in RPMI 1640 supplemented with 0.05 mM 2-mercaptoethanol,
Epitomics RabMab supplement A, 10% FBS, and 1X HAT (Hypoxanthine, Aminopterin, and
Thymidine). Hybridoma cells were grown in 10 cm culture dishes and expanded to T-175 flasks
(60 mL media). For rabbit monoclonal antibody production, cells were adapted to serum-free
conditions by diluting growth media 1:4 in serum-free media (BD cell mAB medium (BD
Biosciences) supplemented with 1% glutamax (Gibco)). After 1-3 days of adapting, full serumfree media was used and changed every 3 days until antibody harvesting was complete (usually 3
media changes).
Purification and sequencing of rabbit monoclonal antibodies
Rabbit hybridoma supernatants were concentrated in an Amicon stirred cell concentrator
(Millipore) and diluted 1:1 in Ig binding buffer (Pierce). Antibodies were purified using Protein
A resin (Pierce) and eluted using Ig elution buffer (Pierce). Elutions were buffer-exchanged with
PBS using an Amicon ultra-15 centrifugal filter unit (Millipore). Typical yields were ~2 mg from
1 L of culture media. For rabbit monoclonal antibody sequencing, total hybridoma mRNA was
98
harvested using an RNeasy kit (Qiagen). The mRNA was reverse-transcribed using an
Omniscript RT kit (Qiagen) and primers IgGHrev (primes to heavy constant region) and
IgGKrev (primes to kappa constant region). The reverse transcription product was amplified
using PCR with primers IgGHfor (primes to heavy leader sequence)/IgGHrev and
IgGKfor/IgGKrev for kappa light chains. Primers are listed in Table 3-2, with additional unused
primers for rabbit IgGL (lambda light chain) sequences. For PCR of clone 31, the reverse primer
IgVHJrev was used to prime to the variable J region since this clone was not an IgG. PCR
products were sequenced using the reverse primers, and cloned into an scFv format using overlap
extension PCR.
Table 3-2: Primers used for sequencing rabbit monoclonal antibodies
IgGHfor
IgGHrev
IgGKfor
IgGKrev
IgGLfor1
IgGLfor2
IgGLrev
IgVHJrev
5’-ATGGAGACTGGGCTGCGCTGGCTT-3’
5’-GGTCACCGTGGAGCTGGGTG-3’
5’-GGGCCCCCACTCAGCTGCTGGG-3’
5’-CATCCACCTBCCAGGTGACGGTG-3’
5’-GGAGACTGGGCTGCGCTGGC-3’
5’-GGCCTGCACCCCGCTCCTCC-3’
5’-GGGTAGAAGTCATTGATCAGAC-3’
5’-GGTGACCAGGGTGCCYKGGCCCCA-3’
B = C/G/T, Y = C/T, K = G/T
99
3.3
Results
3.3.1
Characterization of antibody clones against EGFR peptides
The antibody clones enriched for binding to peptides CRN, CAH, MGE, and LVW after
one round of selection by Mark Olsen were sequenced. Unexpectedly, all of the enriched clones
were not full-length scFvs, but only consisted of the heavy chain (VH) and not the light chain
(VL). Such clones may have arisen from errors in the PCR construction of the library or in yeast
homologous recombination, leading to a frameshift and eventual premature termination of
translation. The closest germline genes to the selected clones as determined by a BLAST search
engine IgBlast are listed in Table 3-3, and the amino acid sequences of the selected clones are
shown in Figure 3-7. Overall, each of the two clones isolated for binding to a particular peptide
were highly similar in sequence, with the exception of CRN-1 and CRN-2. The VH clones
contain most of the (Gly4Ser)3 linker region and additional amino acids after the linker,
terminating in a stop codon. These additional amino acids are not light chain residues, as they are
frame-shifted from the original sequence. Each clone listed in Figure 3-7 was tested for binding
to its cognate peptide (Figure 3-8). Given the intermediate labeling of the clones at 1 μM
peptide, it appeared that the affinity of these VH domains for the peptides was on the order of 10
μM or greater. It was confirmed that the VH clones did not bind any secondary reagents. In
addition, each VH was tested for binding to all of the other peptides. No appreciable crossbinding was observed at 1 μM peptide except for clones LYN-1 and LYN-2 binding to CAH
peptide (Figure 3-8(c-d)), although these clones do show a higher affinity for the LYN peptide.
100
VH clone
LYN-1, LYN-2
CRN-1
CRN-2
MGE-1, MGE-2
LVW-1, LVW-2
Germline
V3-30
V6-1
V4-34
V4-39
V2-5
Table 3-3: Heavy chain germline genes of VH clones selected for binding to peptides.
FR1
CDR1
LYN-1 QVQLVQSEGGVVQPGRSLRLSCAAP GFTFSSYAMH
LYN-2 ------------------------S ----------
FR2
CDR2
WVRQAPGKGLEWVA VISYDGSNKY
-------------- ----------
CRN-1 QVQLQQSGPGLVKPSQTLSLTCDIS GDSVSSDSAAWN WIRLSPSRGLEWLG RTYYWSKWYTD
CRN-2 ------W----L---E------GV- -G-L-GYYWS
---Q--GKE---I- EINQGGSTN
MGE-1 QVQLQESGPGLVKPSETLSLTCTFS GGSIRSSSDYWG WVRQPPGKGLEWIG SISSSGSTY
MGE-2 -----------------------V- ----SN-DY--- -I--------R--- --NYY-T-LVW-1 QITLKESGPTVVKPTQTLTLTCTFS GFSLSTSGVGVG WIRQPPGKALEWLA LISWDDDKR
LVW-2 ------------------------- ------------ -------------- ---------
FR3
CDR3
LYN-1 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DEGYYGSGCIDY
LYN-2 --------------------------------------- -----------CRN-1 YAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYFCAR ERYCSSTSCYLDAFDI
CRN-2 –NP-LR--V--SV----K-L--KV----AS-----Y--- -TFRG-NCFDS
MGE-1 YNPSLKSRVTISVDTSRNQFSLRLSSVTAADTAIYYCAR YNHYWYFDL
MGE-2 ---------AM-----K-----K----------V----- RGANSWFFDL
LVW-1 YSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYFCAH LNNFVDAIELRTGWCFDV
LVW-2 --------------------------------------- -I----------------
Linker
LYN-1 WGQGTLVTVSSGILGSGGGGSGGGGSGGGGLQEF*
LYN-2 ----------------------------------*
CRN-1 WGQGTMVTVSSGILGSGGGGSGGGGSGGGGLQEF*
CRN-2 -----L--------------I-------------*
MGE-1 WGRGTLVTVSSGILGSGGGGSGGGGSGGGGSKRHSRSLQHSSQRLQETKSTSPAKPAKTLMMI*
MGE-2 ------------------------------LQEF*
LVW-1 WGRGTLVTVSSGILGSGGGGSGGGGSGGGVLKLC*
LVW-2 ---------------------------------R*
Figure 3-7: Amino acid sequences of VH clones selected for binding to peptides. FR, framework region; CDR,
complementarity determining region; -, conserved residue; *, stop codon.
101
Figure 3-8: Characterization of individual VH clones isolated for binding to peptides. Yeast cells displaying the VH
clones were labeled with anti-HA antibody 12CA5 (abscissa) and 1 μM peptide (ordinate), followed by goat antimouse-Alexa488 IgG and streptavidin-PE. (a-b) Clones LYN-1 and LYN-2 binding to LYN peptide. (c-d) LYN-1
and LYN-2 binding to CAH peptide. (e-f) CRN-1 and CRN-2 binding to CRN peptide. (g-h) MGE-1 and MGE-2
binding to MGE peptide. (i-j) LVW-1 and LVW-2 binding to LVW peptide.
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3.3.2
Initial round of selection for scFvs against peptides
Single-domain antibodies have proven useful for some applications, such as intracellular
expression (13) or increased stability (34). However, for our purposes, single-domain antibodies
would provide little advantage over full scFvs; therefore, we sought to pair our selected VH
clones with VL domains. This was accomplished by creating libraries joining the VH DNA with
light chain DNA from the original library (9). The resulting scFv DNA was transformed into
yeast using electroporation, yielding libraries designated LYN-A (~107 diversity), CRN-A
(9x105), MGE-A (~107), and LVW-A (~107). In addition, the original nonimmune human scFv
library on the surface of yeast (9) was screened for binders against the CAH peptide using one
round of MACS, since no clones were previously isolated against this peptide.
Selections of the above five populations of yeast were performed using FACS to screen
for binding to the peptides. For the LYN peptide, 4 FACS selections were performed at 1 μM
peptide, and clones from the enriched population were sequenced. However, only one clone
(LYN-A4-8, sequence in Appendix 1.4) was a full scFv, while 9/10 clones only had heavy chains
with no C-terminal c-myc tag. Since the anti-c-myc antibody used was a chicken IgY polyclonal,
it appeared that some molecules in this population were able to weakly bind a motif in the VH
clones. The enriched cells after 3 FACS selections (LYN-A3) were re-sorted with an improved
sorting window drawn to avoid these VH clones. Nine clones from the enriched population
(LYN-A5) were sequenced, revealing 7 unique clones (Appendix 1.4). However, tests of these
clones for binding to 1 μM LYN peptide did not show appreciable binding. For the CRN peptide,
3 FACS selections were performed at 1 μM peptide, and the enriched cell population (CRN-A3)
was sequenced. Out of 10 clones sequenced, all were unique (listed in Appendix 1.5) and showed
103
binding to 1 μM CRN peptide (Figure 3-9). It was further confirmed that these clones indeed
bound the peptide and did not bind the secondary streptavidin reagent.
Figure 3-9: Binding of CRN-A3 clones selected for the CRN peptide. Yeast were labeled with chicken anti-c-myc
IgY (abscissa) and 1 μM CRN peptide (ordinate), followed by goat anti-chicken-Alexa488 IgG and streptavidin-PE.
Clones CRN-A3-1 through CRN-A3-10 are shown in (a)-(j), respectively.
For the CAH peptide, the MACS-enriched population was subjected to 3 FACS
selections at 1 μM CAH peptide. At this point, it appeared that the sorted population contained
some binders to streptavidin instead of the CAH peptide. However, two more FACS selections
using alternating secondary reagents allowed the elimination of the streptavidin binders, resulting
in the CAH-A6 population. Out of 10 clones sequenced, 8 were unique and tested for binding,
and 4 of these clones bound to CAH peptide (Figure 3-10). It was confirmed that these clones did
not bind the streptavidin secondary reagent, and sequences for these clones are listed in
104
Appendix 1.6. For the MGE peptide, 4 FACS selections were performed at 1 μM peptide, and
the enriched population (MGE-A4) was sequenced. Out of 10 clones sequenced, 1 clone was a
VH only, with 8 unique scFv sequences. However, only 4 of these clones (sequences in Appendix
1.7) showed appreciable binding to 1 μM MGE peptide (Figure 3-11), and it was confirmed that
these clones did not bind streptavidin-PE. Finally, for the LVW peptide, 3 FACS selections were
performed at 1 μM peptide. 10 clones from the enriched population (LVW-A3) were sequenced,
revealing 1 VH only clone and 8 unique scFv sequences (Appendix 1.8). Of the 7 scFv clones
tested, all appeared to bind peptide to different degrees (Figure 3-12) and did not bind
streptavidin.
Figure 3-10: Binding of selected CAH-A6 clones. Yeast cells were labeled with 1 μM CAH peptide, followed by
streptavidin-PE. Binding histograms for clones CAH-A6-2 (labeling similar to CAH-A6-1) and CAH-A6-5 (labeling
similar to CAH-A6-4) were omitted for clarity.
Figure 3-11: Binding of MGE-A4 clones selected for the MGE peptide. Yeast were labeled with chicken anti-c-myc
IgY (abscissa) and 1 μM MGE peptide (ordinate), followed by goat anti-chicken-Alexa488 IgG and streptavidin-PE.
(a) MGE-A4-2 (b) MGE-A4-3 (c) MGE-A4-4 (d) MGE-A4-7.
105
Figure 3-12: Binding of LVW-A3 clones selected for LVW peptide. Yeast were labeled with chicken anti-c-myc
IgY (abscissa) and 1 μM LVW peptide (ordinate), followed by goat anti-chicken-Alexa488 IgG and streptavidin-PE.
(a) LVW-A3-2 (b) LVW-A3-3 (c) LVW-A3-4 (d) LVW-A3-5 (e) LVW-A3-7 (f) LVW-A3-8 (g) LVW-A3-9.
As shown above, full scFv clones binding to the various peptides were successfully
isolated in the first round of selections. However, based on binding data at 1 μM peptide, it
appeared that these clones were of similar or worse affinity than the VH only clones. Since the
antigen consisted of only a small peptide, there may not have been enough surface area for the
light chain to also make contacts to the antigen. The closest germline genes for the light chains of
the selected clones listed in Appendix 1.4-1.8 are shown in Table 3-4. Selections for binding to
the five different peptides yielded only 7 unique germline genes out of 34 unique sequences.
These genes are from the most abundantly represented germline families in the original
unselected nonimmune library (9). The observation that many VL germline genes are shared
between the scFv clones binding to different peptides lends further support to the hypothesis that
the light chain is not significantly contributing to antigen peptide binding. In this round, it
appears highly possible that we have selected for light chains that are merely compatible with the
original VH only clones.
106
LYN
CRN
CAH
MGE
LVW
κ (Kappa) germline
V1-39 V3-20 V5-2
X
X
X
X
X
X
X
X
λ (Lambda) germline
V1-44 V2-14 V2-23 V6-57
X
X
X
X
X
X
X
X
Table 3-4: Light chain germline genes represented by selected clones against peptides are indicated with an X.
3.3.3 Second round of affinity maturation
To improve the affinities of the clones from the first round of scFv selection, new
antibody libraries were constructed. Random mutagenesis of the entire scFv gene was
accomplished through PCR with nucleotide analogs, using DNA from the entire enriched
population of yeast as a template. Therefore, this DNA population most likely contained
additional clones which had been enriched, but had not been sequenced or characterized. The
DNA was transformed into yeast to create libraries with the following diversities: LYN-B,
6.4x105; CRN-B, 4.4x106; CAH-B, 1.7x107; MGE-B, 1.6x106; and LVW-B, 1.3x106.
Sequencing of random clones from the libraries showed a wide range of mutations (discernible
by comparison of the heavy chains to the original VH clones), from 0-20 amino acid changes. At
this point, the antigen was changed from the peptides to the full EGFR ectodomain for library
screening. Since the light chain did not appear to be contacting the peptide antigen based on the
previous round’s analysis, it was hypothesized that moving to a large protein antigen would
provide more potential surface area for the light chain to bind, thus improving the affinity of the
scFv. The specific antigen used was 404SG, an EGFR ectodomain mutant which has been
engineered to be expressed solubly by yeast. 404SG contains four point mutations, none of
which are in domains II or IV (30). Excess glycosylation was removed from the 404SG antigen
using EndoH digestion prior to use.
107
The LYN-B library was enriched for binding to 404SG using 4 FACS selections. The
CRN-B library was similarly selected using 4 FACS sorts. Clones from the enriched population
(CRN-B4) were sequenced (listed in Appendix 1.9-1.11). The selection appeared to enrich for
some binding artifacts, including light chain only clones and peptides. The CAH-B library was
enriched for binding to 404SG through 4 FACS selections, yielding only one scFv clone due to
library contamination with yeast-displayed EGF (CAH-B4-1 sequence in Appendix 1.12). The
MGE-B library was sorted using 6 FACS screens against soluble 404SG antigen, and the
enriched MGE-B6 clones were sequenced (Appendix 1.13). Finally, the LVW-B library was
selected using 4 FACS sorts, and the enriched yeast population (LVW-B4) was sequenced. In
addition to scFvs, this library also yielded 2 clones consisting of a light chain only (Appendix
1.14).
Clones CAH-B4-1 and MGE-B6-2 from the second round of selections were tested for
their 404SG antigen-binding properties and compared to negative (D1.3, a lysozyme-binding
scFv) and positive (EGF) controls displayed on the surface of yeast (Figure 3-13). Initially,
binding could only be seen at very high 404SG concentrations, with cells labeled at 20 and 140
μM. The selected scFv clones also showed an unusual pattern of binding; instead of the normally
observed diagonal, the binding curves were more vertical. Such binding is normally indicative of
avidity effects, which may occur at high levels of yeast scFv expression and multimeric antigen.
This is a possibility since a bivalent molecule, biotinylated anti-flag IgG, was pre-incubated with
the 404SG to allow binding to the N-terminal flag tag. However, it is still unusual that there is a
population of cells which show no antigen labeling even though they express a high number of
scFv molecules.
108
Figure 3-13: Clone binding to soluble 404SG. Yeast were labeled with chicken anti-c-myc IgY (abscissa) and
404SG antigen which had been pre-incubated with biotinylated M2 anti-flag IgG (ordinate), followed by Alexa488
goat anti-chicken IgG and streptavidin-PE. (a-d) D1.3 negative control, EGF positive control, CAH-B4-1, and
MGE-B6-2, respectively, displayed on the surface of yeast and labeled with 20 μM 404SG. (e-h) Yeast displaying
proteins as in (a-d), labeled with 140 μM 404SG. (i) MGE-B6-2 labeled with 200 nM 404SG (mAb 528 affinitypurified). (j) MGE-B6-2 labeled as in (i) plus 3.6 μM soluble EGF.
For the labeling data in Figure 3-13 a-h, the 404SG antigen was affinity purified using the
C-terminal His6 tag and digested with EndoH to remove excess glycosylation. However, the high
concentration necessary to label even the positive control suggested that a portion of the 404SG
may have been misfolded. Therefore, the EndoH-digested protein was affinity purified using a
conformation-specific EGFR antibody, mAb 528. 404SG purified in this manner could label
cells at a much lower concentration of 200 nM 404SG (Figure 3-13i). Pre-incubation of the
404SG with soluble EGF ligand only slightly increased antigen binding (Figure 3-13j). In
addition, clones CAH-B4-1 and MGE-B6-2 were tested for binding to EGFR on the surface of
109
A431 cancer cells (expressing ~2x106 receptors/cell) using monolayer cell panning (35). In this
technique, yeast displaying antibodies are captured by adherent mammalian cells expressing an
antigen of interest (protocol in Appendix 2). No appreciable binding of these clones was seen in
this assay (data not shown), but their affinities may have been below the sensitivity of the
method. scFvs selected against 404SG were tested for binding to their cognate peptides, showing
that some clones still retained affinity for the peptides while other no longer did (Figure 3-14).
Figure 3-14: Binding of clones to peptides. Yeast were labeled with chicken anti-c-myc IgY (abscissa) and 1 μM
peptide (ordinate), followed by goat anti-chicken-Alexa488 IgG and streptavidin-PE. (a) Clone CAH-B4-1 labeled
with CAH peptide. (b-k) Clones MGE-B6-1 to MGE-B6-10 labeled with MGE peptide.
3.3.4
Third round of affinity maturation
In this round, the affinity maturation against 404SG antigen was narrowed down to
libraries based on two peptides, LYN and MGE. Random mutagenesis libraries were again
110
constructed using nucleotide analog mutagenesis of the DNA from the population enriched in the
previous round. The libraries were transformed into yeast with diversities of 5.4x106 for LYN-C
and 1.8x106 for MGE-C. For the LYN-C library, 2 FACS selections were performed, with the
first selection against 0.5 μM 404SG and the second at 200 nM 404SG. The enriched population
(LYN-C2) was sequenced, and representative clones are listed in Appendix 1.15. Individual
clones were also tested for their binding to the 404SG antigen (Figure 3-15). The vertical pattern
of binding seen in the previous round was again observed. The addition of soluble EGF did not
improve 404SG binding, and clones MGE-C2-1, 3, 5, 6, and 7 did not show appreciable binding
to 1 μM LYN peptide (data not shown).
Figure 3-15: Binding of 404SG to LYN-C2 clones. Yeast displaying the scFvs were labeled with chicken anti-cmyc IgY (abscissa) and 200 nM 404SG antigen which had been pre-incubated with biotinylated M2 anti-flag IgG
(ordinate), followed by goat anti-chicken-Alexa488 IgG and streptavidin-PE. (a) LYN-C2-1 (b) LYN-C2-3 (c)
LYN-C2-5 (d) LYN-C2-6 (e) LYN-C2-7 (f) LYN-C2-8 (g) LYN-C2-10 (h) LYN-C2-11.
Library MGE-C was sorted against 500 nM 404SG using 4 FACS selections with no
enrichment for antigen binders. In addition, it was noted that the quality of the 404SG protein
varied between different preparations. Thus, we returned to using peptide as an antigen source,
111
and the CRN peptide was used for new selections. A library based on CRN-A from the first
round was made, and this library (CRN-2B) had a diversity of 1.2x107. 6 FACS selections were
performed, starting at 500 nM CRN peptide decreasing to 10 nM. Clones from this enriched
population (CRN-2B6) were sequenced and are listed in Appendix 1.16. Based on the labeling of
these clones, they appeared to have affinities on the order of 100 nM. To increase this affinity,
the CRN-C library (6x107 diversity) was made based on random mutagenesis of the selected
clones from the previous round. After 5 FACS selections at 10 nM peptide, the enriched CRNC5 population was sequenced. The two best clones, CRN-C5-1 and CRN-C-11 (sequences in
Appendix 1.16) were titrated on the surface of yeast and found to have ~50 nM affinity for the
CRN peptide (data not shown). However, clone CRN-C5-1 showed no appreciable binding to
400 nM 404SG or 1 μM Alexa488-labeled EGFR ectodomain produced by insect cells
(generously supplied by Jennifer R. Cochran). Likewise, clone CRN-C5-11 did not bind 200 nM
404SG or 400 nM insect cell EGFR.
3.3.5
Fab display libraries
At this point, all antibody selections were moved from scFv display to Fab display on the
surface of yeast. Eventually, we envisioned reformatting our selected scFv clones into full IgG
molecules for cancer therapy. However, it has been observed that antibody affinity may change
during reformatting (36). Therefore, Fab display enabled testing of the effects of adding the
antibody constant regions to the variable regions, while still being able to work conveniently
with yeast. The scFv clones LYN-C2-6, LYN-C2-13, CRN-C5-1, and CRN-C5-4 were
reformatted to Fabs displayed on the surface of yeast. The yeast displaying these clones were
tested for the presence of both heavy and light chains (Figure 3-16a to d). The LYN-C2 clones
112
appeared to reformat well, with good association of the heavy and light chains, and the Fab
clones retained binding to the antigen (Figure 3-16e,f). However, the CRN-C5 clones did not
reformat well, with little light chain captured by the heavy chain and displayed on the surface.
Consequently, these clones also showed poor antigen binding, demonstrating that both heavy and
light chains were necessary for peptide binding.
Figure 3-16: Properties of Fab-reformatted clones. For (a-d), yeast displaying Fabs were labeled with chicken antic-myc and mouse anti-V5 to detect the VL (ordinate), followed by goat anti-chicken-Alexa488 and goat anti-mousePE IgG. (a) Clone LYN-C2-6 (b) LYN-C2-13 (c) CRN-C5-1 (d) CRN-C5-4. For (e-h), the clones in (a-d) were
labeled with chicken anti-c-myc to detect the VH (abscissa) and antigen (ordinate). For (e-f), yeast were labeled with
200 nM 404SG antigen which had been pre-incubated with biotinylated M2 anti-flag IgG, followed by streptavidinPE. For (g-h), yeast were labeled with 1 μM CRN peptide followed by streptavidin-PE. Cells in the c-myc positive,
V5 negative quadrant may represent yeast which contain the heavy chain plasmid but have lost the light chain
plasmid.
scFv clones from the MGE-A4 and MGE-B6 populations were also reformatted into Fab
display vectors and showed good association between the heavy and light chains on the surface
of yeast. Therefore, a new library was made using nucleotide analog mutagenesis of the variable
regions. The heavy and light libraries were transformed separately into two different strains of
yeast, and the Fab library was created using yeast mating of the heavy and light libraries.
Because of the uncertainty of the quality of the 404SG antigen, the Fab library was sorted against
113
MGE peptide using 6 FACS selections starting at 1 μM peptide decreasing to 250 nM. 3-color
sorts (as shown in Figure 3-4) were performed to select clones that showed both heavy and light
chain expression, as well as antigen binding. The enriched population (MGE-Fab-M6) was
sequenced (listed in Appendix 1.17). Of the 5 sequenced light chains, 4 contained only a
fragment of the variable light chain consisting of the first 12 amino acids of a variable region
fused directly to the constant region (Figure 3-17). Sequencing of the original unselected library
demonstrated that this was a rare event in the starting library, and thus these clones had been
selected for better antigen binding. It appeared these clones preferred to be heavy chain only
binders since they were originally selected as such. Most likely, the MGE peptide was not
binding to the antibody CDRs, but instead to somewhere near the VH/VL interface. Another
single-domain antibody, a light chain clone, was previously selected using yeast surface display
(13) and has been shown to bind antigen at the VH/VL interface (Arne Skerra, personal
communication). During selections of light chains compatible with the original heavy chains, we
may have simply selected for light chains which did not associate well with the heavy chains (but
were still connected through the linker), thus allowing the peptide access to the binding site.
Clones MGE-Fab-M6-1, 2, and 3 were tested by mammalian cell panning for binding to EGFR
expressed on NR6 cells (~105 receptors/cell), and no appreciable binding was observed.
<----VL----><----------------------CL--------------------->
DIQVTRARMETKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
Figure 3-17: Light chain sequence of clone MGE-Fab-M6-1. The variable light region (VL) is truncated to 12
amino acids, and the beginning portion of the constant region (CL) is shown.
114
3.3.6
Rabbit monoclonal antibodies
Since the leads isolated from the nonimmune human library in yeast did not appear
promising, we sought to obtain antibodies from rabbits against the LYN and MGE peptides.
Rabbits typically produce higher affinity antibodies than mice due to the use of both gene
conversion and somatic hypermutation for antibody diversification (37). In addition, previous
work had demonstrated that rabbits injected with these peptides were capable of generating
antibodies against EGFR (38). Thus, rabbits were immunized with the peptides, and the binding
of the rabbit antisera to A431 cancer cells was tested. The rabbits did not appear to produce a
response against the LYN peptide (data not shown), but one rabbit showed a response against the
MGE peptide (Figure 3-18). The binding of the rabbit antiserum to the A431 cells could also be
competed off by excess MGE peptide. In addition, the MGE antiserum appeared to inhibit EGFR
phosphorylation, although the pre-bleed showed inhibition of the receptor to some extent as well
(data not shown).
Figure 3-18: FACS data for rabbit polyclonal antiserum against MGE peptide bound to A431 cells. Cells were
labeled overnight at 4 oC with rabbit pre-bleed or post-peptide immunization bleed, followed by goat anti-rabbit-PE.
Antiserum binding was competed down to pre-bleed levels using 200 μM MGE peptide, but not LYN peptide.
The rabbit which produced a response against the MGE peptide was sacrificed, and its spleen
cells were fused with a plasmacytoma cell line to generate immortalized hybridoma cells (39).
115
The hybridoma supernatants were screened for their binding to A431 cells, and three hybridomas
were selected for further subcloning (Figure 3-19).
Figure 3-19: Binding of hybridoma supernatants to A431 cells. Cells were labeled with supernatant at 4 oC for (a)
2 h or (b) overnight, followed by goat anti-rabbit-PE.
The sequences of the rabbit monoclonal antibodies secreted by each of the subcloned
hybridomas was determined (Figure 3-20). All three clones contained kappa light chains, with
high sequence similarity between antibodies 13 and 32. During the course of sequencing, it was
determined that antibody 31 was not an IgG molecule, but some other class of immunoglobulin.
Thus, the purification of antibody 31 was unsuccessful, with the antibody precipitating out of
solution. However, antibodies 13 and 32 were purified for testing on A431 cells. Both antibodies
showed unusual labeling, with a large negative peak of cells even after overnight incubation with
the antibody (Figure 3-21a), although this binding could be competed by excess MGE peptide. It
was determined that antibody 13 only labeled dead cells, as shown by co-staining with 7-AAD
(Figure 3-21b). The antibody may be recognizing a different protein which is only accessible for
binding once the cell membrane has been compromised, or it may only be able to bind to slightly
degraded EGFR in dead cells.
116
VH-FR1
VH-CDR1
VH-FR2
13 QSVEESGGRLVTPGTPLTLTCTVS GFSLSTHGMI WVRQAPGEGLEWIG
31 ------------------------ -----SV--S -------K-----32 ------------------------ ----N-Y--- -----------Y--
VH-CDR2
IINSNHYTA
---NYGRAY
VV-TD-T-Y
VH-FR3
VH-CDR3
13 YANWAKGRVTISKTSTTVDLKMTSLTTEDTAIYFCGR RGNFAFNL
WGQGTLVTVSS
31 --S-----F---R--------I--P------T---A- IYPDDSIGNL ----------32 --S-V---F-----------------AA---T----- -S------ ----------VL-FR1
VL-CDR1
13 AQVLTQTASSVSAAVGGTVTISC QSSQSVYNNNWLA
31 DI-M---PA--E---------K- -A-ERIG-ALA
32 -R-----P-P------------- ---E---R-----
VL-FR2
WYQQKAGQPPNLLIY
-----P----K--------P----K----
VL-CDR2
QASKLAS
G--T-T-------
VL-FR3
VL-CDR3
13 GVPSRFSGSGSGTHFTLTISGVQCDDAATYYC QGEFSCSIADCVA FGGGTEVVVK
31 ------K------Q------DLE-A------- -SYYASGSRIYGYD ---------32 -------------Q------------------ ------RSG--I- ---------Figure 3-20: Sequences of rabbit monoclonal antibodies.
Figure 3-21: Flow cytometry data for rabbit monoclonal antibody 13 binding to A431 cells. (a) Cells were labeled
with 133 nM antibody 13 overnight at 4 oC, and binding was competed with 200 μM MGE peptide. (b) Cells were
labeled with 133 nM antibody 13 overnight and then labeled with 7-AAD, which stains dead cells.
The antibodies were cloned into an scFv format and displayed on the surface of yeast.
Titrations with MGE peptide indicated that the clones bound the peptide with affinities of ~25
nM. However, the clones did not bind 404SG or EGFR domain IV secreted by yeast (provided
by Ben Hackel). In addition, the clones were negative for binding to EGFR on the surface of
A431 cells as assayed by cell monolayer panning. Therefore, it appeared that the rabbit had
produced antibodies which bound to the MGE peptide, but not to EGFR.
117
3.4
Discussion
In this work, we have continued efforts to engineer antibodies against epitopes in
domains II and IV of EGFR. To do so, we used previously designed peptide mimics of EGFR
loops as antigens. However, many of the antibodies we selected were specific for the peptide
mimcs, but not the full EGFR protein (such as the clones against peptides CRN and MGE). This
suggests that the peptides may not accurately represent the conformations of the EGFR loops.
However, it has been shown that rabbits are capable of producing antibodies against the MGE
peptide that also bind EGFR on the surface of cells (38). Another possibility is that the some of
the peptides are accurate mimics, but the antibody clones which we happened to select bound the
peptides in such a manner that would not allow EGFR protein binding.
Another issue that arose during antibody engineering was inconsistency in antigen
quality. The yeast 404SG EGFR ectodomain mutant, even when affinity purified against a
conformation-specific antibody, showed variation between batches. In addition, it was later
discovered that the EGFR ectodomain mostly adopts a closed or tethered conformation when in
solution (40), thus masking many of the targeted domain II and IV epitopes. The addition of EGF
to 404SG did not appear to improve binding, presumably because these epitopes were also
masked in the dimerized EGFR. In an attempt to untether the EGFR ectodomain, mutants
directed at disrupting the domain II/domain IV tether were secreted (Y246D, H566F,
Y251A/R285S, and K585A). However, these mutants did not show increased binding to the
antibodies, and it was later published that the domain II/domain IV tether does not contribute
significantly to maintaining the receptor in the autoinhibited state (40). Finally, since domain IV
is unresolved in the EGFR dimer crystal structure (24), it may be inherently disordered and thus
difficult to bind.
118
In order to overcome these problems for selections against EGFR domain IV in the
future, one possibility is to use the EGFR ectodomain mutant Y251A/R285S plus EGF as an
antigen. This mutant has been shown to adopt the extended conformation, but not dimerize, upon
addition of EGF ligand (40). An isolated EGFR domain IV could also be used, but it would need
to be extensively characterized for proper folding. Finally, if the receptor could be constrained in
such a way as to prevent dimerization, such as C-terminal immobilization on a surface, EGF
could be added to untether the receptor.
119
3.5
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122
Chapter 4: Mathematical modeling of high affinity EGF binding
4.1
Background
Equilibrium titrations of EGFR on the surface of cells reveal two populations of EGFR
which appear to bind EGF with high and low affinity (1). The two populations become apparent
at low EGF concentrations, where the binding signal is higher than one would expect based on
simple receptor-ligand equilibrium. An alternate representation of equilibrium titration data is a
Scatchard plot, where bound ligand divided by free ligand is plotted versus bound ligand (2).
High affinity EGF binding thus manifests itself as a concave-up Scatchard plot. The high affinity
receptors comprise ~5-10% of the total population with an apparent EGF affinity in the range of
100-300 pM, while the remaining receptors bind EGF with an affinity of 2-15 nM (3).
Although ligand binding is the main driving force for receptor dimerization, a small
number of preformed dimers have been observed on the surface of cells in the absence of ligand.
These preformed dimers have been studied using imaging (4-6) and biochemical (7,8)
techniques, but they are only basally phosphorylated (7). This suggests that there may be inactive
and active conformations of the dimer, with ligand binding inducing a conformational change or
rotation leading to activation (4,9). In addition, early experiments suggested that purified EGFR
dimers bound EGF with a higher affinity than isolated monomers (10,11). Introduction of a
cysteine residue in the extracellular juxtamembrane region of EGFR led to the formation of
covalent dimers and an increase in the proportion of high affinity receptors (12). Imaging
techniques have also found that EGF bound EGFR dimers faster than monomers (13).
Taken together, this experimental evidence suggested that EGFR dimers may be
responsible for the high affinity population of receptors seen on the cell surface. Subsequent
123
mathematical modeling of ligand binding and dimerization events using this experimental
information was able to produce concave-up Scatchard plots; however, the model was unable to
adequately fit experimental data (14). Expansions to this model which incorporate heterogeneity
in receptor density on the cell surface can sufficiently fit experimental data (14,15). Additional
models have been developed where receptor binding to a hypothetical third site leads to the
experimentally observed high affinity EGF binding. These models can either adequately fit
experimental data without modeling dimerization events (16) or produce concave-up Scatchard
curvature while modeling dimerization and monomer untethering events (17). Overall, there is
still a lack of consensus on the origin of the apparent high affinity population of EGFR.
In previous work by Alejandro Wolf-Yadlin, rabbit polyclonal antisera against the LYN
and MGE peptides (see Figure 3-6 and Section 3.1) were characterized. The LYN peptide
mimics the EGFR domain II (DII) dimerization arm, and the MGE peptide represents an EGFR
domain IV (DIV) epitope. Based on the epitopes of the rabbit polyclonal antibodies (pAbs), they
were expected to inhibit receptor homo- and heterodimerization. Accordingly, it was found that
both pAbs inhibited EGFR phosphorylation at tyrosine 1173 and trans-phosphorylation of Her2
(18). This inhibition was not caused by the blockade of ligand binding, as the pAbs and EGF
could simultaneously bind mammalian cells expressing EGFR at saturating ligand
concentrations. However, pretreatment of cells with the pAbs led to a blockade of EGF binding
to only the high affinity receptor population, as revealed by equilibrium titrations (18).
124
In the present thesis work, we have used this experimental result to develop a
mathematical model describing high affinity EGF binding. The equilibrium model for receptor
dimerization and ligand binding assumes that the high affinity EGF binding component is due to
inactive preformed dimers and that the antibodies inhibit receptor homo- and heterodimerization.
The model can successfully reproduce the previous experimental data with only three fit
parameters and can produce a concave-up Scatchard plot for a large range of parameter values.
125
4.2
Results
4.2.1
Model formulation and output
Previous experiments revealed that pAbs against either EGFR DII or DIV eliminated
high affinity EGF binding (18). Based on the epitopes of the pAbs and their ability to inhibit
receptor phosphorylation, we hypothesized that the pAbs were blocking receptor dimerization.
Therefore, it appeared that receptor dimerization was involved in high affinity EGF binding;
specifically, it was hypothesized that preformed receptor dimers were the high affinity binding
component. We developed an equilibrium ligand binding and dimerization model incorporating
these hypotheses to test whether such a model could be consistent with the experimental data.
The molecular events described by the model are indicated in Figure 4-1. Reactions 1-6 detail
EGF binding to and dimerization of the extended EGFR monomer, the scheme proposed in (14).
Reactions 7-8 indicate the tethered monomer converting to the untethered state and binding to
ligand as modeled in (17). Finally, reactions 9-11 are necessary to describe the analogous
heterodimerization events, since the cells used in the experiments, human mammary epithelial
cells (HMECs) (18), also express HER2.
126
Figure 4-1: Model schematic. The equilibrium binding of EGF ligand to monomeric extended EGFR is represented
by reaction 1. Reactions 2 and 3 represent the binding of EGF to unoccupied and singly occupied preformed EGF
dimers, respectively. Reactions 4-6 represent dimerization of occupied and unoccupied EGFR extended monomers.
Reaction 7 represents the tethered EGFR monomer becoming untethered, and reaction 8 represents EGF binding to
tethered EGFR. Reactions 9-10 represent heterodimerization with HER2, and reaction 11 represents EGF binding to
a preformed heterodimer. Each reaction is shown with its relevant equilibrium dissociation constant value.
When the HMECs are pretreated with either the DII or DIV pAb, both dimerization and
tether formation could potentially be inhibited. In this case, the reactions outlined in Figure 4-1
would simplify to only the first reaction, which corresponds to a single-site binding model
characteristic of simple receptor-ligand binding equilibrium. Then, the fractional EGF binding
yEGF is described by the equation
y EGF =
ymax L
,
L + K1
(1)
127
where L is the free ligand concentration. The data for cells pretreated with pAb were first fit to
this simple equation varying two parameters: the single-site equilibrium binding constant K1 and
the maximum binding signal ymax. As seen in Figure 4-2a, the data fit the single-site binding
model well for both pAbs, giving K1 = 6.04 nM (68% confidence interval [4.95, 7.37] by support
plane analysis (19)) for the DII pAb and K1 = 7.34 nM (68% confidence interval [6.05, 8.90]) for
the DIV pAb. This value for the dissociation constant is consistent with previous estimates for
the low affinity EGFR population (3,16), and is also consistent with surface plasmon resonance
measurements for soluble untethered EGFR ectodomain (C-terminal truncation starting at
residue 502) binding to immobilized EGF (20). The pAb pretreatment titration data is also
plotted in Figure 4-2c,d as Scatchard plots. Although the data points below 100 pM bound EGF
may appear to give a slightly concave-down Scatchard plot, this is likely due to experimental
error at these low concentration points. While Scatchard representations are useful for
visualization of data, the transformation distorts errors, especially at low ligand concentrations
(21). It appears from the data that the signals from these low concentration points may be below
the sensitivity of the experimental binding measurement using flow cytometry. Therefore, we
believe that the data is best fit by a single-site binding model (Figure 4-2a), which predicts linear
Scatchard plots.
128
A
0.7
B
1
0.8
Bound/Free EGF
Fractional binding
0.6
0.6
0.4
0.5
0.4
0.3
0.2
0.2
0.1
0
0.1
1
10
100
EGF concentration (nM)
1000
D
0.2
0.1
0
0
0.1
0.2
0.3
0.4
Bound EGF (nM)
0.5
0.6
Bound/Free EGF
C
Bound/Free EGF
0
0.01
0
0.1
0.2
0.3
0.4
Bound EGF (nM)
0.5
0.6
0
0.1
0.2
0.3
0.4
Bound EGF (nM)
0.5
0.6
0.2
0.1
0
Figure 4-2: Fit of model results to experimental data from (18). (a) Data from HMECs titrated at 4 oC using the
indicated concentrations of Alexa488-EGF without (filled diamonds) or with pretreatment of 2 μM DII (open
squares) or DIV (open triangles) pAb. Best fits of the model to the experimental data are shown in solid lines for no
pAb and DII pAb pretreatment and in dotted lines for DIV pAb pretreatment. (b) Scatchard representation of
titration data for no pAb pretreatment (diamonds). Best fit of the model is indicated by the solid line. The data point
indicated in grey in (a) and (b) was not used for fitting the model. (b, inset) Residual errors between the model and
experimental data are plotted for no pAb pretreatment. Titration data for DII pAb pretreatment (c) and DIV pAb
pretreatment (d) are shown in Scatchard representation. Best fit of the model is indicated by the solid line. All error
bars indicate standard deviation of at least 3 replicates.
Next, the full model shown in Figure 4-1 was used to fit the EGF titration data without
pAb pretreatment. All reactions were described by mass action relations through equilibrium
dissociation constants, with the following reactions corresponding to those diagrammed in Figure
4-1:
129
R + L ←⎯→
C
K1
D0 + L ←⎯
⎯→ D1
K2 / 2
D1 + L ←⎯
⎯→ D2
2 K3
R + R ←⎯→
D0
K4
C + R ←⎯
⎯→ D1
K5 / 2
C + C ←⎯→
D2
K6
(2)
T ←⎯→
R
K7
T + L ←⎯→
CT
K8
R + H ←⎯
⎯→ H 0
K9 / 2
C + H ←⎯
⎯→ H 1
K10 / 2
H 0 + L ←⎯
⎯
→ H1
K11
From these reactions, the following relations were derived:
K1 =
RL
C
K2 =
2 D0 L
D1
C=
RL
K1
(3)
D0 =
K 2 D1 2 K 2 R 2 L R 2
=
=
⇒
2L
2 K1 K 5 L K 4
D1 L
2 R 2 L2
R 2 L2
D2 =
=
=
⇒
2 K 3 2 K1 K 3 K 5 K12 K 6
DL
K3 = 1
2D2
K5 =
K2K4
K1
(4)
K6 =
K3K5
K1
(5)
K4 =
R2
D0
D0 =
R2
K4
(6)
K5 =
2CR
D1
D1 =
2CR 2 R 2 L
=
K5
K1 K 5
(7)
K6 =
C2
D2
D2 =
C2
R 2 L2
= 2
K 6 K1 K 6
(8)
K7 =
T
R
T = K7 R
(9)
130
K8 =
TL
CT
CT =
TL K 7 RL
=
K8
K8
(10)
K9 =
2 RH
H0
H0 =
2 RH
K9
(11)
K10 =
2CH
H1
H1 =
2CH 2 RHL
=
K10
K1 K10
(12)
K11 =
H0L
H1
H1 =
K K
H 0 L 2 RHL 2 RHL
=
=
⇒ K10 = 9 11
K1
K11 K 9 K11 K1 K10
(13)
Using a mass balance on total Her2 receptors (HT = 0.075 nM in the experiments) yields an
expression for the concentration of Her2:
H T = H + H 0 + H1
HT = H +
H=
(14)
2 RH 2 RHL
+
K9
K1 K10
(15)
H T K1 K 9 K10
K1 K 9 K10 + 2 K1 K10 R + 2 K 9 RL
(16)
Likewise, a total EGFR balance yields an equation relating the concentration of EGFR (RT = 0.5
nM in the experiments) to free ligand, L:
RT = R + C + 2 D0 + 2 D1 + 2 D2 + T + CT + H 0 + H 1
RT = R +
(17)
K RL
RL 2 R 2 4 R 2 L 2 R 2 L2
+
+
+ 2
+ K7 R + 7
+K
K1 K 4 K1 K 5 K1 K 6
K8
2 RH T K1 K 9 K10
2 RLH T K1 K 9 K10
+
K 9 (K1 K 9 K10 + 2 K1 K10 R + 2 K 9 RL ) K1 K10 (K1 K 9 K10 + 2 K1 K10 R + 2 K 9 RL )
131
(18)
To solve for R, Equation 18 was transformed to the form:
AR 3 + BR 2 + CR + D = 0
(19)
where
A=
(
)
4 K12 K 5 K 6 + 2 K1 K 4 K 6 L + K 4 K 5 L2 (K1 K10 + K 9 L )
K12 K 4 K 5 K 6
(20)
⎛ 2
⎛
4L
2 L2 ⎞
L K7 L ⎞
⎟⎟
⎜
B = K1 K 9 K10 ⎜
+
+
+ 2 ⎟⎟ + 2(K1 K10 + K 9 L )⎜⎜1 + K 7 +
K
K
K
K
K
K
K
1 5
1
8 ⎠
1
6 ⎠
⎝
⎝ 4
(21)
⎛
L K7 L ⎞
⎟⎟ + 2(K1 K10 + K 9 L )(H T − RT )
C = K1 K 9 K10 ⎜⎜1 + K 7 +
+
K
K
1
8 ⎠
⎝
(22)
D = − RT K1 K 9 K10
(23)
From Equation 19, R can be calculated and then used to compute the total amount of bound
ligand by summing the different ligand-bound species as a function of L:
bound = C + D1 + 2 D2 + CT + H 1
(24)
⎞
H T K1 K 9 K10
RL 2 R 2 L 2 R 2 L2 K 7 RL 2 RL ⎛
⎜⎜
⎟
bound =
+
+ 2
+
+
K1 K1 K 5 K1 K 6
K8
K1 K10 ⎝ K1 K 9 K10 + 2 K1 K10 R + 2 K 9 RL ⎟⎠
(25)
Nonlinear least squares fit of the model parameters to experimental data was performed
using MATLAB (Mathworks) function lsqnonlin. Despite what appears to be many free
parameters, thermodynamic and physical constraints reduce the number of fit parameters to
three. Representative values for model parameter K1 were determined earlier from fitting the
pAb pretreatment data to a single-site binding model, and therefore K1 was set equal to 7 nM. K2
represents the binding constant for the high affinity preformed dimers and was left as a free
132
parameter for data fitting. K3 represents the affinity of binding a second EGF to a singly
occupied
EGFR
dimer.
Previously,
it
has
been
shown
that
for
an
equilibrium
dimerization/ligand-binding model such as this one, K3 must be greater than K2 to produce a
concave-up Scatchard plot (14). Also, if K3 were equal to K2, the dimerization of bound and
unbound monomers (reaction 5) would lead to the creation of many more high affinity sites than
are experimentally observed. To reduce the number of fit parameters, as a simplifying
assumption K3 was therefore set equal to K1, the binding constant for the low affinity binding
site. K4 represents the dimerization or cross-linking coefficient, and was left as a free parameter.
Three of the equilibrium constants are not independent; K5, K6, and K10 can be derived as a
function of the other parameters (see Equations 4, 5, and 13). K7 was set to 30 according to the
estimated energy of untethering (17,20,22), and K8 was set to 400 nM as the affinity of EGF for
EGFR domain III (17,23). K9, the EGFR/HER2 heterodimerization constant, was set equal to the
EGFR homodimerization constant K4. Modeling and experiments have shown that homo- and
heterodimerization occur with comparable affinities, with the prevalence of heterodimers
observed in many cell types arising from a combination of relative expression levels and
differential endocytic trafficking properties (24). Finally, K11 was set equal to K2, the high
affinity interaction, since increasing expression of HER2 has been shown to increase the fraction
of receptors in the high affinity state (25). The EGFR and HER2 receptor concentrations, RT and
HT, respectively, were calculated as the total average concentration in the sample based on the
average number of receptors per cell and number of cells per labeling volume. Therefore, for
modeling of the EGF titration without pAb pretreatment, a total of three parameters were varied
to fit the data: K2, K4, and the maximum binding signal.
133
4.2.2
Model characterization
As seen in Figure 4-2, the model fits the EGF titration data well. The residual errors
between the model and the data are randomly distributed around zero, indicating an adequate fit
(Figure 4-2b, inset). The model and experimental data are also shown in Scatchard format in
Figure 4-2b. One data point, indicated in gray, was determined to be an outlier and not used for
the model fitting. One possible explanation is that the reaction was under depleting ligand
conditions, leading to a lower signal than expected. Nevertheless, if this data point was used in
the analysis, the fit parameters were not affected by more than twofold, and the model fit was
only slightly worse (data not shown). The best-fit parameters from the model were K2 = 110 pM
for high affinity EGF binding to preformed dimers and K4 = 13 pM for the dimerization constant.
This K2 value is consistent with previous estimates for the high affinity EGFR population (3,16).
For EGFR on the surface of cells, the value of K4 depends on the total receptor concentration
used in the model and is normally within an order of magnitude of RT (17), as it is in this case.
Also, K4 and RT being within an order of magnitude is required if both receptor monomers and
dimers are present in the absence of ligand, which has been experimentally observed (4-7).
Although K3 was fixed and set equal to the binding constant for the low affinity site K1 (7 nM),
allowing K3 to vary as a free parameter yielded similar results, with K3 = 18 nM, indicating a low
affinity binding site.
For the HMECs, the model predicts that in the absence of ligand, the EGF receptors are
distributed as 81% tethered monomers, 6% preformed homodimers, 10% preformed
heterodimers with HER2, and 3% untethered monomers. Half of the homodimers plus the
heterodimers, ~13% of the receptors in this case, are thus predicted to be in a high affinity state.
134
While the percentage of receptors in the high affinity state will vary according to cell line and
EGFR and HER2 expression levels, this percentage is within the generally reported range (3,6).
In order to assess the parameter space of the model, a support plane analysis (19) was
performed (Figure 4-3a). The analysis yielded a defined confidence interval when the product of
the high affinity binding constant and the receptor dimerization constant, K2K4, was used as a
lumped parameter. K2K4 was varied, and the remaining parameters, K4 and maximum signal,
were re-fit at each of the values of K2K4. This was repeated until the resulting error exceeded an
acceptable value as judged by the appropriate F-statistic for the system, yielding a 68%
confidence interval of [4.6x10-4, 2.4x10-3] for K2K4. Next, we sought to determine how well the
model can capture the experimentally observed concave-up Scatchard behavior of EGF binding.
K2 and K4 were varied over 9 orders of magnitude, and the concavity of the resulting Scatchard
plot was determined. For the model, a concave-up Scatchard plot is obtained for a wide range of
values of K2 and K4 (Figure 4-3b).
135
(a)
χ2
2
χ min
K2K4 (nM2)
(b)
K4 (nM)
101
10-1
10-3
10-5
10-7
10-5
10-3
10-1
101
K2 (nM)
Figure 4-3: Model characterization. (a) Support plane analysis of parameter space of the model. The product K2K4
was varied, and the remaining parameters K4 and maximum signal were re-fit at each value of K2K4. The y-axis
shows the error at the corresponding value of K2K4 divided by the minimum error (when all 3 parameters are
allowed to vary). The dotted line indicates the 68% confidence interval. (b) Parameter space giving a concave-up
Scatchard plot. Parameters K2 and K4 were varied, and the shaded area indicates parameter values for which the
resulting Scatchard plot was concave-up. Scatchard plots were considered concave-up if all calculated points were
below the straight line connecting the two endpoints of the curve.
136
4.3
Discussion
Since pretreatment of cells with the DII and DIV pAbs eliminated the high affinity EGF-
binding component typically seen with cell-surface EGFR, we speculated that pAb binding
inhibited receptor homo- and heterodimerization and that dimers may represent that high affinity
binding mode. To probe this idea, we formulated an equilibrium model where preformed dimers
were the high affinity component, and the model (Figure 4-1) was successfully fit to the
experimental data (Figure 4-2). Further analysis of the model demonstrated its appropriateness to
the system, as a wide variety of parameter values could yield a concave-up Scatchard plot
(Figure 4-3b). The features of the model that produce concave-up Scatchard plots are: [a] high
affinity preformed dimers and [b] negative cooperativity of EGF binding to the dimer. Therefore,
in this model, the first EGF binds an unoccupied preformed dimer with high affinity, and the
second EGF binds with low affinity to the singly occupied dimer. There exists experimental
evidence that preformed dimers possess a higher affinity for EGF; dimers stabilized by a
juxtamembrane disulfide bond on the surface of living cells show an increased percentage of
high affinity receptors (12), and dimers observed by FRET also appear to be high affinity (4).
While there is mathematical support for negative cooperativity in EGF binding (14), there is also
experimental support in that disulfide-stabilized EGFR dimers still display both high and low
affinity binding to EGF (9,12). One possible structural interpretation of these model features is
that the preformed dimer holds EGFR domains I and III in an ideal orientation for ligand
binding, with correct positioning of the domains while allowing enough flexibility for the ligand
to enter the binding site, thus leading to high affinity binding. However, once the first EGF binds
to the preformed dimer, this could potentially change the positions of the two EGFR molecules
with respect to each other, restricting the motion of the unoccupied EGFR site and making it
137
more difficult for the second EGF to bind. It has been shown that ligand binding stabilizes an
additional EGFR dimerization interface C-terminal to the DII dimerization arm that is not
present in the absence of ligand (20), and this interface could mediate such a change.
Although wild-type EGFR molecules form dimers on the surface of cells in the absence
of ligand, receptors lacking the cytoplasmic domain through partial truncation or substitution
with the EpoR cytoplasmic domain do not form these predimers (7). Mutants lacking part of the
cytoplasmic domain also give rise to linear Scatchard plots of a single low affinity binding site
(26), but can still dimerize in the presence of ligand (27). Taken together, these data are
consistent with our model of preformed receptor dimers as the high affinity component and
suggest that the cytoplasmic domain contributes to stabilizing the EGFR dimer, especially in the
absence of ligand. Recent modeling and experiments on EGFR with a cytoplasmic domain
truncation suggest that the low affinity receptors represent interconverting tethered and extended
receptor conformations (28), which is also the case in our model. Likewise, deletion of the
domain II dimerization arm (29) or mutations in this region (30) result in a loss of high affinity
binding. A V583D mutation designed to promote receptor untethering increases the percentage
of high affinity sites (30), which is consistent with our model. However, the same study found
that the mutation did not increase the amount of dimers in the absence of ligand, although the
sensitivity of the immunoblotting assay may have been limited. Further interpretation of
mutation results is difficult because many mutations designed to inhibit receptor tethering may
also inhibit dimerization with opposing effects. In addition, fibroblasts in which α1,6fucosyltransferase has been deleted (leading to a loss of protein core fucosylation) show no high
affinity EGF binding. These cells also have substantially lower EGF-induced EGFR
phosphorylation (31), suggesting a possible defect in receptor dimerization, and this data is
138
consistent with the hypothesis that receptor dimers are the high affinity component. Finally, an
N579Q mutant designed to eliminate glycosylation at this site leads to both an elevated level of
preformed dimers in the absence of ligand and a larger fraction of receptors in the high affinity
state (32).
Our model is similar to that of Wofsy et al., which also incorporates high affinity
preformed dimers with negative cooperativity (14). However, in that model both binding events
to the dimer were still higher affinity than binding to the monomer, and the best fit parameters
led to less than 1% of EGFR in preformed dimers in the absence of ligand. Although able to
produce concave-up Scatchard plots, the authors were unable to successfully fit this model to
their data, even though this model incorporated two essential elements of our model. The
experiments in Wofsy et al. used membrane vesicles with a C-terminal truncation of EGFR
starting at residue 1023 in their experiments, which may have affected the results. A separate
study showed that membrane vesicles only contained low affinity EGFR (33), demonstrating
variability in membrane vesicle composition. The parameters in the Wofsy et al. model could not
be uniquely determined, so certain parameters were fixed while others were fit. It is possible that
some relatively recent experimentally determined constants not available to the authors at the
time could have aided in the convergence of their model in a more restricted parameter space.
Wofsy et al. thus concluded that other factors such as heterogeneity in receptor density could be
responsible for the concavity of the Scatchard plot, and successfully modeled this hypothesis
(14). Likewise, Mayawala et al. also recently modeled heterogeneous EGFR density with success
(15). While receptor heterogeneity likely exists on the cell surface (34), it is unclear how the
binding of an antibody to either EGFR domain II or IV could lead to a loss in this heterogeneity
to produce a linear Scatchard plot as observed in previous experiments (18). Thus, heterogeneity
139
may not be the cause, or at least not the sole cause, of observed Scatchard concavity. Finally, our
model is not inconsistent with models which include a hypothetical third binding partner that
leads to high affinity EGFR sites (16,17). Klein et al. modeled that only receptor dimers could
bind to the hypothetical partner, and thus our observation that putatively dimerization-blocking
antibodies result in loss of high affinity binding could be consistent with this model (17).
However, we believe that the utility of our model is in its ability to reproduce experimental data
by incorporating known EGFR molecular events and parameters based on experiments, without
the need to introduce any additional mechanisms not yet independently verified.
A ligand-binding and dimerization model for soluble EGFR ectodomain has also been
developed in the past (23). It is difficult to compare receptor dimerization constants measured in
solution with those on the surface of cells, since receptors restricted to the membrane lose
rotational and translational degrees of freedom (35) and the cytoplasmic domain may also
contribute to dimerization (see above). Since we have modeled equilibrium binding only, kinetic
parameters and predictions are beyond the scope of the model. However, a spatially distributed
Monte Carlo-based simulation that utilized kinetic parameters determined that at low EGF
concentrations, the predominant ligand-binding mode is binding to preformed dimers (36), which
is consistent with the model presented here. An additional kinetic model based on experimental
imaging data found that preformed EGFR dimers bound EGF faster than monomeric sites, but
their mathematical model suggested positive cooperitivity in EGF binding (37). Also beyond the
scope of the model are higher-order EGFR and HER2 oligomers, which experiments on the
surface of cells suggest may exist (38).
EGFR antibodies that lead to altered Scatchard plots have previously been described.
Monoclonal antibody 108 was shown to reduce the number of high affinity EGFR receptors, as
140
the pAbs did; however, unlike the pAbs, 108 both partially inhibited EGF binding and was
unable to block signaling at saturating EGF concentrations (39). Conversely, another monoclonal
antibody, 2E9, appeared to block binding to only low affinity EGFR; however, 2E9 competed
with EGF binding almost completely at saturating concentrations of EGF, and EGFR was still
able to signal when cells were treated with a 90% saturating amount of 2E9 (40). This is not
surprising, considering that a calcium signaling response has been demonstrated for a few as 300
molecules of bound EGF (13). While these antibodies have been reported to alter Scatchard
plots, they still appear to at least partially compete with EGF binding at saturating
concentrations; thus, their mechanisms of action are unclear.
Physiologically, the formation of high affinity preformed dimers in the absence of ligand
could aid the cell in responding quickly to a ligand stimulus. These dimers on the cell surface
would be poised to signal once ligand is present, without needing to wait for two ligand-bound
monomers to diffuse together. We hypothesize that the function of the autoinhibited EGFR
conformation is to limit the number of extended monomers that can form dimers in the absence
of ligand, thus preventing a high level of basal signaling.
141
4.4
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144
Appendix 1: Sequences
1.1
EGFR ectodomain amino acid sequence (1-621)
LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAV
LSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQ
CSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADS
YEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPE
NRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHAL
CSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTL
VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS
1.2
EGFR 273-621 DNA sequence
CGTAATTATGTGGTGACAGATCACGGCTCGGCCGTCCGAGCCTGTGGGGCCGACAGCTATGAGATGGAGGAAGACGGCGTCCGCAAGTGTAAGAAGT
GCGAAGGGCCTTGCCGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAA
CTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGACCCACAGGAACTGGAT
ATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCA
TACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGA
TGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATT
ATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACT
GCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTG
CATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGAC
GGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGT
GCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCC
Note: WT cysteine 283 mutated to alanine (truncation at 273 causes unpaired cysteine)
N-terminal c-myc tag included in above sequence
1.3
404SG DNA sequence
CTGGAGGAAAAGAAAGTTTGCCAAGGCACGAGTAACAAGCTCACGCAGTTGGGCACTTTTGAAGATCATTTTCTCAGCCTCCAGAGGATGTTCAATA
ACTGTGAGGTGGTCCTTGGGAATTTGGAAATTACCTATGTGCAGAGGAATTATGATCTTTCCTTCTTAAAGACCATCCAGGAGGTGACTGGTTATGT
CCTCATTGCCCACAACACAGTGGAGCGAATTCCTTTGGAAAACCTGCAGATCATCAGAGGAAATATGTACTACGAAAATTCCTATGCCTTAGCAGTC
TTATCTAACTATGATGCAAATAAAACCGGACTGAAGGAGCTGCCCATGAGAAATTTACAGGAAATCCTGCATGGCGCCGTGCGGTTCAGCAACAACC
CTGCCCTGTGCAACGTGGAGAGCATCCAGTGGCGGGACATAGTCAGCAGTGACTTTCTCAGCAACATGTCGATGGACTTCCAGAACCACCTGGGCAG
CTGCCAAAAGTGTGATCCAAGCTGTCCCAATGGGAGCTGCTGGGGTGCAGGAGAGGAGAACTGCCAGAAACTGACCAAAATCATCTGTGCCCAGCAG
TGCTCCGGGCGCTGCCGTGGCAAGTCCCCCAGTGACTGCTGCCACAACCAGTGTGCTGCAGGCTGCACAGGCCCCCGGGAGAGCGACTGCCTGGTCT
GCCGCAAATTCCGAGACGAAGCCACGTGCAAGGACACCTGCCCCCCACTCATGCTCTACAACCCCACCACGTACCAGATGGATGTGAACCCCGAGGG
CAAATACAGCTTTGGTGCCACCTGCGTGAAGAAGTGTCCCCGTAATTATGTGGTGACAGATCACGGCTCGTGCGTCCGAGCCTGTGGGGCCGACAGC
TATGAGATGGAGGAAGACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT
CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTC
CTTCACACATACTCCTCCTCTGGACCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTCTTTGCTGATTCAGGCTTGGCCTGAA
AACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCGGCCTGAACA
TAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTG
GAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTG
TGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTC
TGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACG
GGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTG
GTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGA
ATGGGCCTAAGATCCCGTCC
Note: Contains four mutations from wild-type EGFR to allow expression in yeast: A62T, L69H,
F380S, S418G
145
1.4
VL sequences of selected clones from LYN-A4 and LYN-A5
A4-8
A5-2
A5-3
A5-4
A5-5
A5-6
A5-8
A5-9
VL-FR1
NFMLTQPHSVSESPGKTATISC
ETTLTQSPAFLSATPGDKVDISC
ETTFTQSPAFMSATPGDKVNIFC
ETTLTQSPAFMSATPGDKVNISC
QPVLTQSPSASGTPGQSVTISC
NFMLTQPHSVSESPGETVTISC
NFMLTQPHSVSESPGKTVTISC
ETTFTQSPAFMSATPGDKVNISC
A4-8
A5-2
A5-3
A5-4
A5-5
A5-6
A5-8
A5-9
VL-CDR2
EDNQRPS
EATTLVP
EATTLVP
EATTLVP
SNNQRPS
EDHQRPS
ENNQRPS
DATTLVP
VL-CDR1
TRSSGSIASNYVQ
KASQDIDDDLN
KASQDIDDDVS
KASQDIDDDMN
SGSGSNIGNNKVN
TGSSGSIASNSVQ
TGSSGSIATNYVQ
KASQDIDEDVN
VL-FR2
WYQQRPGGAPTTVIY
WYQQKPGEAAVFIIQ
WYQQRPGEAPIFIIQ
WYQQKPGEAAIFIIQ
WYQQLPGTAPKLLIY
WYQQRPGSAPRTVIY
WYQQRPGSAPTTVIY
WYQQKPGEAPIFIIQ
VL-FR3
GVPDRFSGSIDSSSNSASLTISGLRTEDEADYYC
GIPPRFSGSGYGTDFTLTINNIESEDAAYYFC
GTPPRFSGSGYGTDFTLTIDNIESEDAAYYFC
GIPPRFSGSGYGTDFTLTINNIESEDAAYYFC
GVPDRFSGSKSGTSASLAISGLQSEDEADYYC
GVPDRFSGSIDTSSNSASLTISGLRTEDEGDYYC
GVPDQFSGSIDSSSNSASLTISGLKTEDKADYYC
GIPPRFSGSGHGTDFTLTIDNIESEDAAYYFC
VL-CDR3
QSYDRSNKAV
LHHDDLPLT
LQDDSFPLT
LQHDNFPWT
AAWDDSLNGYV
RSYDSSHTV
QSYDSSXLXV
LQHDNFPPYT
FGGGTQLTILSGIL
FGQGTKLEIKSGIL
FGQGTKLEIKSGIL
FGQGTRVEIKSGIL
FGTGTKLTVLSGIL
FGGGTQLTVLSGIL
XXXGTKXXVLXXIX
FGQGTKLEIKSGIL
Note: A4-8, A5-2, A5-3, A5-4, A5-6, A5-8, A5-9: LYN-1 VH
A5-5: LYN-2 VH
1.5
VL sequences of selected clones from CRN-A3
1
2
3
4
5
6
7
8
9
10
VL-FR1
NFMLTQPHSVSESPGKTVTISC
EIVLTQSPGTLSLSPGERVTLSC
DIQVTQSPSSLSASVGDRVTITC
ETTLTQSPGTLSLSPGERATLSC
ETTLTQSPGTLSLSPGERATLSC
NFMLTQPRSVSESPGKTVTISC
ETTLTQSPAFMSATPGDKVNISC
EIVLTQSPATLSLSPGERATLSC
DIRVTQSPSSLSASVGDRVTITC
ETTLTQSPGTLSLSPGDRVTISC
1
2
3
4
5
6
7
8
9
10
VL-CDR2
ENNQRPS
GASTRAT
DATRVES
DASSRAT
GASSRAT
EDNQRPS
ESTVLLP
GASSRAT
AASSLQS
EATTLVP
VL-CDR1
TGSSGSIATNYVQ
RASQTVSSSYLA
RASQSISSYLN
RASQSVGGSYLA
RASQGVAGSDLA
TGSSGSIASNYVQ
KASQDIDDDMN
RASQSFTGNYLA
RASQSINTYLN
KASHDVDDDLN
VL-FR2
WYQQRPGSAPTTVIY
WYQQKTGQAPRLLMY
WYQQKPGTAPKLLIY
WYQKKTGQAPRLLIY
WYQQKPGQAPRLLIY
WYQQRPGSAPTIVIH
WYQQKPGEAPILILQ
WYQQKPGQAPRLLIY
WYQQKPGKAPKLLIY
WYQQKPGKAPVLIIR
VL-FR3
GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GVPSRFSGSGSGTDFTLTISGLQVEDVATYYC
GIPDRFSGSGSGTDFTLTINRVEPEDFAMYYC
GIPDRFSGSGSGTDFSLTISRLEPEDFAVYYC
GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
GIPPRFSGSGYGSDFTLTINNMQSEDAAYYFC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYHC
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
GIPPRFSGSGYGTDFTLTINNMESEDAAYYFC
VL-CDR3
QSYDSSNHAV
QQYGSSRWT
QRGYNTPRT
QQYGSSPVT
QQFDTVTWT
QSYDSSSRWV
LQHDSFPFT
QQYGTSPT
QQSYSSPSL
LQHDSPPYS
146
FGGGTQLTVLSGIL
FGQGTKVEIKSGIL
FGQGTKVDIKSGIL
FGQGTRLKIKSGIL
FGQGTKVEIKSGIL
FGGGTKLTVLSGIL
FGQGTKLEIKSGIL
FGQ
FTFGPGNKVDIKSG
FGQGTKLEIRSGIL
1.6
Selected clones from CAH-A6
1
2
4
5
VH-FR1
EVQLVESGGGLVQPGRSLRLSCAAS
QVQLKQSGPGLVNPSQTLSLTCAIS
QVQLVESEGGVVQPGRSLRLSCAAS
QVQLQQSGPGLVKPSQTLSLTCAIS
VH-CDR1
GFSFADYAMH
GDSVSSDSAA
GFSFSRFAMH
GDSVSSNSAAWN
VH-FR2
WVRQVPGKGLEWVS
WNWIRQSPSRGLEWLG
WVRQAPGKGLEWVA
WIRQSPSRGLEWLG
1
2
4
5
VH-FR3
YADSVKGRFTISRDNAKNTVYLEVNNIRPEDTALYYCAK
YTESVKSRIIINPDTSKNQFSLQLNSVTPEDTALYYCAR
YADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCAR
YAASVRSRITINPDTSKNQFSLQLNSVTPDDTAMYYCAR
VH-CDR3
VRDPNIEAFDV
FLRGTFDI
HYDSSGRDH
SRSSSPIDY
VH-CDR2
SISWNSGIKG
RTYYRSKWYNE
VISYDGSNKF
RTAYRSKWNSD
WGQGTMVTVSS
WGQGSMVTVSS
WGQGTLVTVSS
WGQGTLVTVSS
Linker
VL-FR1
VL-CDR1
1
GILGSGGGGSGGGGSGGGGS EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLG
2 ASTKGPSGILGSGGGGSGGGGSGGGGS NFMLTQPHSVSESPGKTVTISC TGSSGSIATNYVQ
4
GILGSGGGGSGGGGSGGGGS EIVMTQSPGTLSSSPGERATLSC RASKDVSSQFLA
5
GILGSGGGGSGGGGSGGGGS EIVLTQSPGTLSLSPGERVTLSC RASQRVSSSYVA
1
2
4
5
VL-CDR2
GASSRAT
ENNHRPS
ETSTRAT
GASSRAT
1.7
VL-FR3
GIPDRFSGSGSGTDFALTISRLEPEDFAVYYC
GVPDRFSGSIDSSSNSASLTISGLKTEDEAGYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
VL-CDR3
QQHDRSSWT
QSSDNDFHAV
QQYGRSPRT
QQYDSSPRT
VL-FR2
WYQQKPGQAPRLLIY
WYQQRPGSAPTTVIY
WYQQKPGQAPRLLIY
WYQQKPGQAPRLLIY
FGQGTKVEIKSGIL
FGGGTQLTVLSGIL
FGQGTKVEIKSGIL
FGQGTKLEIKSGIL
VL sequences of selected clones from MGE-A4
2
3
4
7
VL-FR1
DIRVTQSPSSLSASVGDRVTITC
QSVLTQPASVSGSPGQSITISC
QSVLTQPPSVSGSPGQSITISC
NFMLTQPHSVSESPGKTVAISC
2
3
4
7
VL-CDR2
AASSLQS
EVNKRPS
EVNKRPS
EDKERPS
VL-CDR1
RASQSISNYLN
TGTSSDVGGYDYVS
TGTSSDVGNYNLVS
TRSSGSIASNYVQ
VL-FR2
WYQQKPGKAPKLLIH
WYQQHPGKTPKRMIY
WYQRHPGKAPKLMIY
WYQQRPGSSPTTVIY
VL-FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
GVSNRFSGSKSGNTASLTISGLQAEDEADYYC
GISHRFSGSKSGNTASLTISGLQAED
GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
VL-CDR3
QQSYSMPFN FGPGTK
TSYRSGSSYV FGTGTKLTVLSGIL
QSYDGTNTV
FGGGTQLTVLSGIL
Note: All clones MGE-2 VH
1.8
VL sequences of selected clones from LVW-A3
1
2
3
4
5
7
8
9
VL-FR1
DIRVTQSPSSLSASIGDRVTITC
NFMLTQPHSVSESPGKTVTISC
NFMLTQLHSVSGSPGKTITISC
ETTLTQSPAFLSATPGDKVTISC
NFMLTQPHSVSESPGKTVTISC
NFMLTQPHSVSESPGKTVTISC
NFMLTQPHSVSESPGKTVTISC
NFMLTQPHSVSESPGKTVTISC
1
2
3
4
5
7
8
9
VL-CDR2
AASSLQD
EDNQRPS
EDKQRPS
EATTLVP
EDNQRPS
DDDQRPS
EDNQRPS
ENNQRPS
VL-CDR1
RASQTIGSYLN
TGSSGSIASNYVQ
TRSSGSIATNYVQ
KASLDIDDDVN
TRSSGSIASNYVQ
TGSSGSIADKYVQ
TGSSGSITINYVQ
TGSSGSIASNYVQ
VL-FR2
WYQHRPGKAPKLLIY
WYQQRPGSAPTTVIY
WYQQRPGRAPSTVIY
WYQQKPGQAPIFIIQ
WYQQRPGRAPTTIIF
WYQQRPGSAPRNVIF
WYQQRPGSAPTTVIY
WYQQRPGSAPTTVIY
VL-FR3
GVPSRFSGGGSGTDFTLTISNLQPEDFTTYYC
GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
GIPPRFSGSGYGTDFTLTINNIQPEDAAYYFC
GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
GVPDRFSGSIDTSSNSASLIISGLKTEDEADYYC
GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
GVPDRFSGSIDSSSNSASLTISGLKTEDEAEYYC
VL-CDR3
QQSYGXXRDV
QSYDNSNPAV
QSYDSSNYV
LQHDNFPYT
QSYDSSNAV
QSFDSSNKTV
QSYDSSNQGV
QSYDSSNHAV
Note: All clones LWV-2 VH
147
RXRDQGGXSNPGIL
FGGGTQLTVLSGIL
FGTGTKLTVLSGIL
FGQGTKLEVKSGIL
FGGGTQLTVLSGIL
FGRSTQLTVLSGIL
FGRRTKLTVLSGIL
FGGGTQLTVLSGIL
1.9
1
3
5
6
7
10
1
3
5
6
7
10
Selected clones from CRN-B4
VH-FR1
VH-CDR1
VH-FR2
VH-CDR2
QVQLQQWVPGLLKPSETLSLTCGVS
QVQLQQWGPGLLEPSETLSLTCGVS
QVQLQQWGPGLLKPSETLSLTCGVS
QVQRQQWGPGPLKPSETLFLTCGVS
QLQQWGPGLLKPSETLSLTCGVS
GGSLSGYYWS
GGSLSGYYWS
GGSLSGYYWS
GGSLSGYYWS
GGSLSGYYWS
WIRQSPGKELEWIG
WIRQSPGKELEWIG
WIRQSPGKELEWIG
WVRQSPGKELEWIG
WIRQSPGKELEWIG
EINQGGSTN
EINQGGSTN
EINQGGSTN
DINQGGSTN
EINQGGSTN
VH-FR3
VH-CDR3
YNPSLRSRVTISVDTSKKQLSLKVNSVTASDTAVYYCAR
YNPSLRSRATISVDTSKKQLSLKVNSVTASDTAVYYCAR
YNPSLRSRVTISVDTSKKQLSLKVNSVTASDTAVYYCAR
YDPSLRSRVATSAGTSKKQLSLKVDPATASDTAAYYCAR
YNPSLRSRVTISVDTSKKQLSLKVNSVTASDTAVYYCAG
ETFRGSNCFDS
ETFRGSNCFDS
ETFRGSNCFDS
EIFRGSNRFDS
ETFKGSNCFDS
Linker
1
3
5
6
7
10
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGPGGGGGGGGGSGGGGP
GILGSGGGGSGGGGSGGGGS
1
3
5
6
7
10
VL-CDR2
GASTRAT
GASTRAT
GASTRAT
GASTRAT
GASTRAT
GASTRAT
VL-FR1
EIVLTQSPGTLSLSPGERVTLSC
EIVLTQSPGTLSLSPGERVTLSC
EIVLTQSPGTLSLSPGERVTLSC
EIVLTQSPGTLSLSPGERVTLSC
EIVLTQSPGALSLSPGERVTLSC
EIVLTQSPGTLSLSPGERVTLSC
VL-FR3
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GIPDRFSGSGSGTDFTLTISRLEPEDLAVYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GIPDRFSGSGSGTDFTLTISRLEPENLAVYHC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
WGQGTLVTVSS
WGQGTLVTVSS
WGQGTLVTVSS
WGQGILVAASS
WGQGTLVTVSS
VL-CDR1
RASQTVSSSYLA
RASQTVSSSYLA
RASQTVSSSYLA
RASQTVSSSYLA
RASQIVGSSYLA
RASQTVSSSYLA
VL-CDR3
QQYGSSRWT
QQYGSSWWT
QQYGSSRWT
QQYGSSRWT
QQYGSSRWT
QQYGSSRWT
VL-FR2
WYQQKTGQAPRLLMY
WYQQKTGQAPRLLMY
WYQQKTGQAPRLLMY
WYQQKTGQAPRLLMY
WCQRETGQAPRLLTY
WYQQKTGQAPRLLMY
FGQGTKVEVKSGIL
FGQGTKVEIKSGIL
FGQGTKVEIKSGIL
FGQGTKVEIKSGIL
LGQGTKVETKPGIL
FGQGTKVEIKSGIL
1.10 CRN-B4-2
QVQLQQWGPGLLKPSETLSLTTVIYEDNQRPSAVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYHSSIVVFGGGTKLTVLSGIL
1.11 CRN-B4-4
GGGSGGGGSAGILEQKLISEEDL
1.12 CAH-B4-1
VH-FR1
VH-CDR1
VH-FR2
VH-CDR2
EVQLVESGGGLVQPGRSLRLSCAAS GFSFADYAMH WVRQVPGKGLEWVS SISWNSGIKG
VH-FR3
VH-CDR3
YADSVKGRFTISRDNAKNTVYLEVTNIRPEDTALYYCAK VRDPNIEAFDV WGQGTMVTVSS
Linker
VL-FR1
VL-CDR1
VL-FR2
GILGPGGGGSGGGGSGGGGS EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLG WYQQKPGQAPRLLIY
VL-CDR2 VL-FR3
VL-CDR3
GASSRAT GIPDRFSGSGSGTDFALTISRLEPEDFATYYC QQANSFPYT FGQGTKLEIKSGIL
148
1.13 Selected clones from MGE-B6
1
2
3
4
5
6
7
8
9
10
VH-FR1
QVQLQEPGPGLARPSETLSLTCTVS
QVQLQESGPGLVKPSETLSLTCTVS
QVQLQESGPGLVKPSETLSLTCTVS
QVQLQESGPGLVKPSETLSLTCTVS
QVQLQESGPGLVKPSETLSLTCTVS
QVQLQESGPGLVKPSETLSLTCTVS
QVQLQESGPGLVKPSETLSLTCTVS
QVQLQESGPGLVKPSEALSLTCAVS
QVQLQESGPGLVKPSETLSLTCTVS
QVQLQESGPGLVKPSETLSLTCTVS
VH-CDR1
GGPIGDSDYCWG
GGSISNSDYYWG
GGSISNSGYYWG
GGSISNSDYYWG
GGSISNSDYYWG
GGSISNSDYYWG
GGSISNSDYYWG
GGSISNSDYYWG
GGSISNSDYYWG
GGSISNSDYYWG
1
2
3
4
5
6
7
8
9
10
VH-FR3
YNPSLKNRVAMSVGTSKDQFSLRLGPVAATDTAVYYCAR
YNPSLKSRVAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
YNPSLKSRVAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
YNPSLKSRVAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
YNPSLKSRAAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
YNPSLKSRVAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
YNPSLKSRVAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
YNPSLKSRVAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
YNPSLKSRVAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
YNPSLKSRVAMSVDTSKNQFSLKLSSVTAADTAVYYCAR
1
2
3
4
5
6
7
8
9
10
Linker
GILGSGGGGSGGGGSGGGGS
GILGSGGGGGGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGPGGGGS
GILGSGGGGSGGGGSGGGGP
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
1
2
3
4
5
6
7
8
9
10
VL-CDR2
EDKERPS
EVNKRPS
EVNKRPS
EVNKRPS
ATSSLQR
EVNKRPS
EVNKRPS
EVNKRPS
ATPSLQR
EVNKRPS
VH-FR2
WIRQPPGKGLRWVG
WIRQPPGKGLRWIG
WIRQPPGKGLRWIG
WIRQPPGKGLRWIG
WIRQPPGKGLRWIG
WIRQPPGKGLRWIG
WIRQPPGKGLRWIG
WVRQPPGKGLRWIG
WIRQPPGKGLRWIG
WIRQPPGKGLRWIG
VH-CDR3
RGANSWFFDL
RGANSWFFDL
RGANSWFFDL
RGANSWSFDL
RGANSWFFDL
RGANSWFFDL
RGANSWFFDL
RGANSWFFDL
RGANSWFFDL
RGANSWFFDL
VL-FR1
NFMLTQPHSVSESPGKMAAISC
QSVLTQPPSVSGSPGQSITISC
QSVLTQPASVSGSPGQSITISC
QSVLTQPASVSGSPGQSITISC
DIRVTQSPSSLSASVGDRVTITC
QSVLTQPASVSGSPGQSITISC
QSVLTQPASVSGSPGQSITISC
QSVLTQPPSVSGSPGQSITISC
DIQVTRSPSSPSASVGGRVTITC
QSVLTQPASVSGSPGQSITISC
VL-FR3
GVPDRLSGSTDSSSNSASLTISGLKPEDGADYYC
GVSNRFSGSKSGNTASLTISGLQAEDEADYYC
GISHRFSGSKSGNTASLTISGLQAKDEGDYYC
GASNRFSGSKSGNTASLTISGLQAEDEADYYC
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
GVSNRFSGSKSGNTASLTISGLQAEDEGDYYC
GVSNRFSGSKSGNTASLTISGLQAEDEADYYC
GISHRFSGSKSGNTASLTISGLQAEDEGDYYC
GVPSGSSGSGSGADSTLTASSLQPEDFATCHC
GVSNRFSGSKSGNTASLTISGLQAEDEADYYC
VH-CDR2
GINCYGTTC
SINYYGTTY
SINYYGTTY
SINYYGTTY
SINYYGTTY
SINYYGTTY
SINYYGTTY
SINYYGTTY
SINYYGTTY
SINYYGTTY
WGRGTLVTVSS
WGRGTLVTVSS
WGRGTLVTVSS
WGRGTLVTVSS
WGRGTLVTVSS
WGRGTLVTVSS
WGRGTLVTVSS
WGRGTLVTVSS
WGRGTLVTVSS
WGRGTLVTVSS
VL-CDR1
TRSSGSIASNYVQ
TGTSSDVGGYDYVS
TGTSSDVGNYNLVS
TGTGSDVGGYDYVS
RASQSISSSLN
TGTSSDVGGYDYVS
TGTSSDVGGYDYVS
TGTSSDVGNYNLVS
RASPSTSSSLS
TGTSSDVGGYDYVS
VL-CDR3
QPYDRNNRAV
TSYRSGSSYV
CSYTSTGGTV
TSYRSGSSYV
QQSYNTPRT
CSYTSTGGTV
TSYRSGSSYV
CSYTSTGGTV
QQSYSTPRT
TSYRSGSSYV
149
VL-FR2
WYQQRPGSSPTTVIY
WYQQHPGKTPKRMIY
WYQRHPGKAPKLMIY
WYQQHPGKTPKRMIY
WYQQKPGKAPNLLIY
WYQQHPGKTPKRMIY
WYQQHPGKTPKRMIY
WYQRHPGKAPKLMIY
WYQQEPGKASDLLTY
WYQQHPGKTPKRMIY
FGGGTQLTVLSGIL
FGTGTKLTVLSGIL
LGGGTQLTVLSGIL
FGTGTKLTVLPGIL
FGQGTKVEIKSGIL
LGGGTQLTVLSGTL
FGTGTKLTVLSGIL
LGGGTQLTVLSGIL
SGQGARMETKSGIL
FGTGTKLTVLSGIL
1.14 Selected clones from LVW-B4
1
2
3
5
8
9
1
2
3
5
8
9
VH-FR1
VH-CDR1
VH-FR2
VH-CDR2
QTTLEEPGPTVVKPAQTLTPTCTFP GSSLSTSGVGVG WIRQSPGKALEWLA PISWDGDKR
QTVLKEPSPTAARPTQALTLTCASS GLSLSTSGVGTD RAHQPPGRALEWPA LISRDDGKR
QIILKESGPTVVRPTQTLTLTCTSF GLSPSTSGVGVG WARQPPGRALEWLA LISWGDGKR
QTTLKESGSTTAKPIQTPTLACTFS GFSLSTSGVGAG WIRRLPGKAPEWPA LISWGDDKR
VH-FR3
VH-CDR3
YSPSLRSRPTITEDTSKNQVVLTMTNMDPVDTATYFCAH LTSFVDVIELRTGWCFDA WGRGTLVTVSS
HSPSPKGRLTITKGTSKNQAVLAVASMDPVGTAACPWAH PVSSVGAVELRTGWCSDT WGRGTLVTVPS
YSPSLKSRLPITKDTSKDQVALTATNMDPVDTATYFCAY PINFADAVELRTGWRFDV WGRGTLVTASS
YSPSLKSRPTITKDTSKSQVILTMTNMDPVGTATYFCTH LINFVDATERRTGWRSDV WGRGTLVTASS
1
2
3
5
8
9
Linker
VL-FR1
GILGSGGGGSGGDGSGGGGS NSTPTQPHSVSESPGKTVTISR
GTLGSGGGGSGGGSSGGGGP DFMLTQPHPVSESPRRTVTVSC
DIRVTQSPSSLSASVGDRVTITC
ETAFTQSPAFMSATPGDKVNIFC
GTLGSGGGGSGGGGPGGGGP NFMLTQPRAVSESPGKMATASC
GILGSGGGGSGGGGSGGGGS NFVLTQPHSVSESPGKAVTISY
1
2
3
5
8
9
VL-CDR2
ENNQRPS
EGERRPP
AASSLQS
EATTLVP
ETNHRPS
GDSQRPS
VL-FR3
GVPDRFFGPIDSSSNSASPTISGLKTEDEADHYC
GVPGRLSGSADHPPNSASLTVFGLKAGDGAGHYC
GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC
GTPPRFSGSGYGTDFTLTIDNIESEDAAYYFC
GAPDRFSGSIDSSSNSASLTISGLKTEDEADYHR
AAPDRLPGSADSSSDSASPPTSGLKAEDEADYYC
VL-CDR1
TRSSGSIASDYVQ
TRNSDGIAGGRAQ
RPSRSISNYLN
KASQDIDDDVS
TGSSGSIASDPVQ
ARSGGSVATNYVR
VL-CDR3
QSYDSSNHVV
QSYDGNSRAV
LQHNSLPLT
LQDDSFPLT
QSYDRSNHVV
QSHYSSTVV
150
VL-FR2
WPQQRPGSVPTTVTH
RHQQRPGSSPTTTTR
WYQQKPGKAPKLLIY
WYQQRPGEAPIFIIQ
WYQQRPGSAPTTVTH
WCQQRPGGSPTTVVY
FGGGTQPTVLSGTL
FGEGTQLTVLPGAP
FGPGTKVDIKSGIL
FGQGTKLEIKSGIL
FGGGTRLAVLSGIP
FDGGAKLTALSGIL
1.15 Selected clones from LYN-C2
1
3
5
6
7
8
10
11
12
13
VH-FR1
QVQLVQSEGGVVQPGRSLGLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
QVQLVQSEGGVVQPGRSLRLSCAAS
VH-CDR1
GFTFSSYAMH
GFTFSSYAMH
GFTFSSYAMH
GFTFSSYAMH
GFTFSSYAMH
GFTFSSYAMH
GFTFSSYAMH
GFTFSSYAMH
GFTFSSYAMH
GFTFSSYAMH
VH-FR2
WVRQAPDKGLEWVA
WVRQAPGKGLEWVA
WVRQAPGKGLEWVA
WVRQAPGKGLEWAA
WVRQAPGKGLEWVA
WVRQAPGKGLEWVA
WVRQAPGKGLEWVA
WVRQAPGKGLEWVA
WVRQAPGKGLEWVA
WVRQAPGKGLEWVA
1
3
5
6
7
8
10
11
12
13
VH-FR3
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTISRDTSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTIARDNSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
1
3
5
6
7
8
10
11
12
13
Linker
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGPGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
1
3
5
6
7
8
10
11
12
13
VL-CDR2
EDHQRPS
EDNQRPS
EDNQRPS
EDNQRPS
EDNQRPS
EDNQRPS
EDNQRPS
EDNQRPS
EDNQRPS
DATTLVP
VH-CDR2
VISYDGSNKY
VISCDGSNKY
VISYDGSNKY
AVSYDGSNKY
VISYDGSNKY
VISYDGSNKY
VISYDGSNKY
VISYDGSNKY
VISYDGSNKY
VISYDGSNKY
VH-CDR3
DEGYYGSGCIDY
DEGYYGSGCIDY
DEGYYGSGCIDY
DEGYYGSGCIDY
DEGYYGSGCIDY
DEGYYGSGCIDY
DEGYYGSGCIDY
DEGYYGSGCIDY
DEGYYGSGCIDY
DEGYYGSGCIDY
VL-FR1
NFMLTQPHSVSESPGETVTISC
NFMLTQPHSVSESPGKTVTISC
NFMLAQLHSVSESPGKTVTTSC
SFMLTQPHSAPESPGKTVTISC
NFMLTQPHSVSESPGKTVTISC
NFMLTQPHSVSESPGKTVTISC
NFMLTQPHSVSESPGKTVTISC
NFMLTQPHSVSESPGKAATISC
NFMLTQPHSVSESPGKTATISC
ETTFTQSPAFLSATPGDKVDISC
VL-FR3
GVPDRFSGSIDTSSNSASLTISGLRTEDEGDYYC
AVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
AVPDRFSGSIDSSSNSASLTISGLKTEDEAGYYC
AVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
AVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
AVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
AVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC
GVPDRFSGSIDSSSNPASLTISGLRTEDEADYYC
GVPDRLSGSIDSSSNSASLTISGLRTEDEADYYC
GIPPRFSGSGHGTDFTLTIDNIESEDAAYYFC
WGQGTLVTVSS
WGQGTLVTVSS
WGQGTLVTVSS
WGQGTLVTASS
WGQGTLVTVSS
WGQGTLVTVSS
WGQGTLVTVSS
WGQGTLVTVSS
WGQGTLVTVSS
WGQGTLVTVSS
VL-CDR1
TGSSGSTASNSVQ
TRSSGSIATNYVQ
TRSSGSIATNYVQ
TRSSGSTATNYVQ
TGSSGSIATNYVQ
TRSSGSIATNYVQ
TRSSGSIATNYVQ
TRSSGSIASNYVQ
TRSSGSIASNYVQ
KASQDIDEDVN
VL-CDR3
RSYDSSHTV
QSYHSSIVV
QSYHSSIVV
QPYDSSNPWV
QSYHSSIVV
QSYHSSIVV
QSYHSSIVV
QFYHSSIVV
QSYDRSNKAV
LQHDNFPPYT
151
VL-FR2
WYQQRPGSAPRTVIY
WYQQRPGSSPTTVIY
WYQQRPGSSPTTVIY
WYQQRPGSSPTTVIY
WYQQRPGSSPTTVIY
WYQQRPGSSPTTVIY
WYQQRPGSSPTTVIY
WYQQRPGGAPTTVIY
WYQQRPGGAPTTVIY
WYQQKPGEAPIFIIQ
FGGGTQLTVLSGIL
FGGGTKLTVLSGIL
FGGGTKLTVLPGIP
FGGGTKLTVLSGIL
FGGGTKLTVLSGIL
FGGGTKLTVLSGIL
FGGGTKLTVLSGIL
FGGGTKLTVLSGIL
FGGGTQLTILSGIL
FGQGTKLEIKSGIL
1.16 Selected clones from CRN-2B6 and CRN-C5
2B6-1
2B6-4
2B6-6
2B6-8
C5-1
C5-11
VH-FR1
QVQLQQWGPGLLKPSETLSLTCGVS
QVQLQQWGPGLLKPSETLSLTCGVS
QVQLQQWGPGLLKPSETLSLTCGVS
QVQLQQWGPGLLKPSETLSLTCGVP
QVQLQQWGPGLLKPSETLSLTCGVS
QVQLQQWGPGLLKPSETLSLTCGVS
VH-CDR1
GGSLSGYYWS
GGSLSGYYWS
GGSLSGYYWS
GGSLSGYYWS
GGSLSGYYWS
GGSLSGYYWS
VH-FR2
WIRQSPGEELEWIG
WIRQSPGEELEWIG
WIRQSPGEELEWIG
WIRQSPGEELEWIG
WIRQSPGEELEWIG
WIRQSPGEELEWIG
2B6-1
2B6-4
2B6-6
2B6-8
C5-1
C5-11
VH-FR3
YNPSLRSRVTISVDTSKKQLSLKVNSVTASDTAVYYCAR
YNPSLRSRVTISVDTSKKQLSLKVNSVTASDTAVYYCAR
YNPSLRSRVTISVDTSKRQLSLKVNSVTASDTAVYYCAR
YNPSLRSRVTISVDTSKKQLSLKVNSVTASDTAVYYCAR
YNPSLRSRVTISVDTSKKQLSLKVNSVTASDTAVYYCAR
YNPSLRSRVTISVDTSKKQLFLKVNSVTASDTAVYYCAR
2B6-1
2B6-4
2B6-6
2B6-8
C5-1
C5-11
Linker
GILGSGGSGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGS
GILGSGGGGSGGGGSGGGGP
2B6-1
2B6-4
2B6-6
2B6-8
C5-1
C5-11
VL-CDR2
GASTRAT
GASTRAT
GASTRAT
GASTRAT
GASTRAT
EDDQRPS
VH-CDR3
ETFRGSNCFDS
ETFRGSNCFDS
ETFRGSNCFDS
ETFRGSNCFDS
ETFRGSDCFDS
ETFRGSNCFDS
VL-FR1
EIVLTQSPGTLSLSPGERVTLSC
EIVLTQSPGTLSLSPGGRVTLSC
EIVLTQSPGTLSLSPGERVTLSC
EIVLTQSPGTLSLSPGERVTLSC
EIVLTQSPGTLSLSPGERVTLSC
NFMLTQPHSVSESPGKTVTISC
VL-FR3
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAMYYC
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
GVPERFSGSIDSSSNSASLTISGLKTEDEADYYC
VH-CDR2
EINRDGSTN
EINQGGSTN
EINQGGSTN
EINQGGSTN
EINRDGSTN
EINQGGSTN
WGQGTPVTVSS
WGQGTPVTVSS
WGQGTPVTVSS
WGQGTPVTVSS
WSQGTPVTVSS
WDQGTLVTVSS
VL-CDR1
RASRTVSSSYLA
RASQTVSSSYLA
RASQTVSSSYLA
RASQTVSSSYLA
RASRTVSSSYLA
TRSSGSIASNYVQ
VL-CDR3
QQYGSSRWT
QQYGSSRWT
QQYGSSRWT
QQYGSSRWT
QQYGSSRWT
QSYDSSDGWV
FGQGTKVGIKSGVL
FGQGTKVEIKSGIL
FGQGTKVEIKSGIL
FGQGTKVEIRSGIL
FGQGTKVGIKSGVL
FGGGTKLAVLSGIL
1.17 Selected clones from MGE-Fab-M6
VH-FR1
VH-CDR1
VH-FR2
1 QVQLQESGPGLVKPLETLSLTCTVS GGSISNSGYYWG WIRQPPGKGLEWIG
2 QVQLQESGPGLVKPSETLSLTCTVS GGSISNSDYYWG WVRQPPGKGLEWIG
3 QVQLQESGPGLVKPSETLPLTCTVS GGSISNSDYYWG WVRQPPGKGLEWIG
VH-CDR2
SINHYGTTY
SINYYGTTY
SINYYGTTY
VH-FR3
VH-CDR3
1 YNPSLKSRVAMSVDTSKSQFSLKLSSVTAADTAVYYCAR RRANSWSFDL WGRGTLVTVSS
2 YNPSLKSRVAMSADTSKNQFSLKLSSVTAADTAVYYCAR RGANSWFFDL WGRGTLVTVSS
3 YNPSLKSRVAMSVDTSKNQFSLKLSSATAADTAVYYCAR RGANSWLFDL WGRGTLVTVSS
VL-FR1
VL-CDR1
VL-FR2
1 DIQVTRARMETK
2 DIQVTRARMETK
3 DFMLTQPHSVSESPGKTVTISCTGS SGSIASNYVQ WYQQRPGSAPTTVIF
VL-CDR2 VL-FR3
VL-CDR3
1
2
3 EDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQS YDRSNHAVFGRGTQLTVL
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VL-FR2
WYQQKTGQAPRLLMY
WYQQKTGQAPRLLMY
WYQQKTGQAPRLLMY
WYQQKTGQAPRLLMY
WYQQKTGQAPRLLMY
WYQQRPGSFPTTVIY
Appendix 2: Protocol
Yeast panning of mammalian cell monolayers
(adapted from Wang & Shusta, J Immunol Methods, 304:30, 2005 and Steve Sazinsky)
Materials:
PBSCM (PBS w/ 1 mM CaCl2, 0.5 mM MgSO4, 0.5% BSA), sterile filtered
SDCAA, pH 3.5 (20 g/L glucose, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 14.7 g/L
sodium citrate, 4.29 g/L citric acid monohydrate), pH to 3.5, sterile filtered
Note: keep PBSCM and 6-well plate on ice during Steps 3-7.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Grow mammalian cell monolayer to confluency.
Serum starve cell monolayers overnight before panning.
Wash monolayers 3x with 1 mL PBSCM.
Wash yeast cells 2x with 1mL PBSCM:
a. Use 2x106 yeast/cm2 of monolayer cells.
b. For 6-well plates, use 2x107 cells/well.
c. Use 1 well for yeast displaying an irrelevant antibody, and 1 well for a yeastdisplayed positive control.
Resuspend cells in 1 mL PBSCM (per well) and pipette yeast slurry onto monolayer
dropwise to cover all surface area.
Gently swirl plate and incubate at 4 oC with gentle rocking for 2 h.
Wash wells 2x with PBSCM:
a. Tilt plate and aspirate yeast slurry.
b. Gently pipette 1 mL buffer onto side of well.
c. Rock gently at 4 oC for 1 min.
Add 1 mL SDCAA, pH 3.5 to each well and rock vigorously at room temperature for 20
min to elute the bound yeast from the monolayer.
Measure OD600 to determine number of cells captured. After OD measurement, yeast can
be removed from cuvette, and dilutions can be plated on SDCAA plates as an alternate
measure of cells captured.
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Office:
77 Massachusetts Ave. E19-563
Cambridge, MA 02139
(617) 258-5293
gingerc@mit.edu
Home:
345 Franklin St. #105
Cambridge, MA 02139
(409) 350-2984
Ginger Chao
Education
Massachusetts Institute of Technology, Cambridge, MA
Doctoral candidate in Chemical Engineering, Ph.D. anticipated October 2007
Minor in Biology, GPA: 4.9/5.0
Rice University, Houston, TX
Bachelor of Science in Chemical Engineering with Bioengineering Focus
Conferred Summa Cum Laude May 2002
Research
Experience
Massachusetts Institute of Technology
Graduate Research Assistant
January 2003 – present
Thesis Advisor: K. Dane Wittrup
Thesis Title: “Characterizing and engineering antibodies against the epidermal
growth factor receptor (EGFR)”
• Developed a novel method for epitope mapping antibodies using random
mutagenesis and yeast surface display
• Utilized above method to determine the epitopes of three therapeutically
relevant antibodies against EGFR
• Continued work on engineering single-chain antibodies against dimerization
epitopes of EGFR using yeast surface display and directed evolution
• Created a mathematical model for equilibrium receptor-ligand binding and
dimerization to describe the elimination of high affinity EGF binding to
EGFR through antibodies against dimerization epitopes
Pharmacia
Summer 2001
Summer Engineering Intern
Conducted laboratory experiments on catalyst immobilization and reaction
optimization to yield high enantiomeric selectivity
Rice University
Summer 1999
Undergraduate Research Assistant
Conducted research sponsored by the BP Amoco Summer Research Program,
with results included in publication:
Lee, J.T., Chou C.Y., Chao G., Pilaski D.R. & Robert M. (2005). Twodimensional diffusion of colloids in polymer solutions. Mol. Physics 103,
2897-2902.
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Publications
Chao G., Cochran J.R. & Wittrup K.D. (2004). Fine epitope mapping of antiepidermal growth factor receptor antibodies through random mutagenesis and yeast
surface display. J. Mol. Biol. 342, 539-550.
Colby D.W., Garg P., Holden T., Chao G., Webster J.M., Messer A., Ingram V.M.
& Wittrup K.D. (2004). Development of a human light chain variable domain (VL)
intracellular antibody specific for the amino terminus of huntingtin via yeast
surface display. J. Mol. Biol. 342, 901-912.
Sivasubramanian A., Chao G., Pressler H.M., Wittrup K.D. & Gray J.J.
(2006). Prediction of the mAb 806-EGFR complex structure by computational
docking followed by computational and experimental mutagenesis. Structure
14, 401-414.
Chao G., Lau W.L., Hackel B.J., Sazinsky S.L., Lippow S.M. & Wittrup K.D.
(2006). Isolating and engineering human antibodies using yeast surface
display. Nat. Protocols 1, 755-768.
Supervisory
Experience
Supervision of MIT Undergraduate Research Opportunity Program
(UROP) students:
Heather Pressler, “Mutation analysis of the antibody 806 epitope” (2004-2005)
Nick Nguyen, “Measuring anti-EGFR antibody endocytosis rates” (2007)
Teaching Assistant Experience:
Introduction to Experimental Biology, MIT (Spring 2006)
Conducted daily recitation lectures, provided instruction and management
during laboratory hours, and graded exams
Thermodynamics I, Rice University (2001-2002)
Held weekly office hours and graded homework assignments
Skills of
Interest
Recombinant DNA techniques
Protein biochemistry
Protein engineering/directed evolution using yeast surface display
Fluorescence activated cell sorting (FACS)
Mammalian cell culture
MATLAB programming
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Honors
National Defense Science and Engineering Graduate Fellowship
National Science Foundation Graduate Fellowship (declined)
David Koch Graduate Fellowship (MIT Center for Cancer Research)
Robert T. Haslam Presidential Graduate Fellow, MIT
Outstanding Chemical Engineering Seminar Award (Fall 2005)
Rice University President’s Honor Roll
Tau Beta Pi National Engineering Honor Society
Ann and Joe Hightower Award in Chemical Engineering (Rice University)
Presentations,
Posters, and
Patents
Oral Presentations:
Chao G., Olsen M.J., Wolf-Yadlin A. & Wittrup K.D. (2005). Engineering
antibodies against the epidermal growth factor receptor to block dimerization.
American Institute of Chemical Engineers (AIChE) Annual Meeting.
Chao G., Cochran J.R. & Wittrup K.D. (Sept. 2006). Epitope mapping of
antibodies against the epidermal growth factor receptor (EGFR). American
Chemical Society (ACS) National Meeting.
Posters:
Chao G., Olsen M.J., Wolf-Yadlin A. & Wittrup K.D. (2005). Engineering antiepidermal growth factor receptor (EGFR) antibodies to block receptor
dimerization. Keystone Symposium: Antibody-Based Therapeutics for Cancer.
Chao G., Wolf-Yadlin A., Olsen M.J., Lauffenburger D.A. & Wittrup K.D. (2007).
Antibodies against domains II or IV of the epidermal growth factor receptor
(EGFR) inhibit high affinity EGF binding. Keystone Symposium: Antibodies as
Drugs.
Chao G., Wolf-Yadlin A., Olsen M.J., Lauffenburger D.A. & Wittrup K.D. (2007).
Antibodies against domains II or IV of the epidermal growth factor receptor
(EGFR) inhibit high affinity EGF binding. Protein Society Annual Symposium.
Patent Applications:
Johns T.G., Scott A.M., Burgess A.W., Old L.J., Adams T.E., Wittrup K.D. &
Chao G. (2005). EGF receptor epitope peptides and uses thereof.
Wittrup K.D., Lauffenburger D.A, Wolf-Yadlin A., Olsen M.J. & Chao G. (2005).
Antibodies and peptides targeted to specific epidermal growth factor receptor
epitopes for diagnostic and therapeutic applications in human disease.
(Provisional).
Way J.C., Chao G., Cresson C., Lauffenburger D.A., Wittrup K.D. (2007).
Treatment of tumors expressing mutant EGF receptors.
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