Download Rachel M. Dudek - Leonard Lab

Engineering Novel Sense and Response Mechanisms
into Mammalian Cells for Synthetic Immunobiology
Devices
Research Proposal
for partial fulfillment of the requirements for
Degree of Doctor of Philosophy
Rachel M. Dudek
Department of Chemical and Biological Engineering
Northwestern University
Evanston, IL
December 9th, 2011
Dudek PhD proposal
Table of Contents
SUMMARY ................................................................................................................................................. 3
BACKGROUND AND SIGNIFICANCE ................................................................................................. 4
MOTIVATION AND APPROACH ......................................................................................................... 4
TUMOR MICROENVIRONMENT (TME) ............................................................................................. 5
CYTOKINE SIGNALING IN THE TME ................................................................................................ 6
MATRIX METALLOPROTEINASES (MMPs) IN THE TME .............................................................. 6
SYNTHETIC BIOLOGY CELLULAR SENSORS AND DEVICES ...................................................... 7
SIGNIFICANCE AND IMPACT ............................................................................................................. 9
PRELIMINARY RESULTS ...................................................................................................................... 9
MESA design overview ............................................................................................................................ 9
TLR5 MESA receptor design ............................................................................................................. 11
Preliminary TLR5 MESA feasibility testing and characterization ......................................................... 13
Investigating the role of the ectodomain in promoting or inhibiting background signaling .................. 16
RESEARCH DESIGN AND METHODS ............................................................................................... 19
AIM 1 ...................................................................................................................................................... 19
RATIONALE...................................................................................................................................... 19
EXPERIMENTAL DESIGN AND METHODS ................................................................................ 20
ANTICIPATED OUTCOME AND ALTERNATIVE DIRECTIONS .............................................. 23
AIM 2 ...................................................................................................................................................... 24
RATIONALE...................................................................................................................................... 24
EXPERIMENTAL DESIGN AND METHODS ................................................................................ 25
ANTICIPATED OUTCOME AND ALTERNATIVE DIRECTIONS .............................................. 28
AIM 3 ...................................................................................................................................................... 29
RATIONALE...................................................................................................................................... 29
EXPERIMENTAL DESIGN AND METHODS ................................................................................ 30
ANTICIPATED OUTCOME AND ALTERNATIVE DIRECTIONS .............................................. 34
RESEARCH TIMELINE ......................................................................................................................... 34
REFERENCES .......................................................................................................................................... 35
2
Dudek PhD proposal
SUMMARY
The immune system is a complex multicellular network capable of coordinating
behaviors among its members to maintain a delicate balance between inflammation and tolerance
within the human body. Diseases like cancer can co-opt this intricate biochemical and
multicellular network to promote their own survival, creating a microenvironment of immune
dysfunction that enables the disease to progress resulting in suffering, costly treatments, and, in
many cases, mortality. If we could rationally program tumor-targeting mechanisms into immune
cell “devices” to detect and disrupt the immune dysfunctional and invasive tumor
microenvironment (TME), we could potentially allow beneficial immune responses to gain the
upper hand and eliminate the cancer. Synthetic biology has given rise to a number of cellular
sensor and circuit technologies; however, none of these are able to detect species exclusive to the
extracellular environment, a capability crucial for discriminating the TME from normal tissue.
To address this need, I am developing a Modular Extracellular Sensory Architecture (MESA) to
specifically target extracellular molecules such as those characterizing the tumor
microenvironment. The MESA couples an extracellular dimerization event (induced by binding
of receptor ectodomains to their cognate ligand) to an intracellular proteolytic cleavage event,
liberating a transcription factor and enabling it to transcribe a gene of interest. I propose to
optimize the kinetic parameters of this signaling platform by directed evolution. I will
incorporate the optimized kinetics and generalize the MESA approach to build a synthetic
pseudo-cytokine (“pseudokine”) platform to model and study multi-input and orthogonal input
sensing and responding. In parallel, I propose to develop a complementary sensing modality
distinct from but able to interface with the MESA.
The specific aims of my research proposal are:
3
Dudek PhD proposal
AIM ONE
To eliminate excessive background signaling by the MESA by optimizing the proteolysis
kinetics of the protease on its cleavage sequence. I propose to generate a cleavage sequence
variant by directed evolution that will be cleaved by the protease chain only when it is in a stable
receptor-dimer-ligand, eliminating background signaling in the diffusion-limited regime.
AIM TWO
To engineer an orthogonal “pseudokine” network consisting of a) MESA receptors that form
stable homodimeric and heterodimeric complexes upon cooperative binding of a synthetic
ligand, and b) a synthetic secretable ligand capable of dimerizing the receptors. I will use this
platform to enable synthetic intercellular communication for ultimate applications in the
coordination of multicellular networks.
AIM THREE
To develop a sensor for matrix metalloproteinases to complement the MESA approach and
enable multi-input discrimination of the tumor microenvironment. I propose to link extracellular
cleavage of a peptide substrate for MMP-2 to intracellular nuclear translocation of a transcription
factor to result in a sensitive and specific MMP sensor able to interface with the MESA system.
BACKGROUND AND SIGNIFICANCE
MOTIVATION AND APPROACH
The immune system is a powerful multicellular network that constantly senses and
responds to inflammation-provoking infectious threats and tolerance-inducing endogenous
stimuli to maintain a state of healthy homeostasis within the host organism. Pathogens, cancers,
and autoimmune diseases can co-opt or suppress immune mechanisms, creating a
microenvironment of immune dysfunction characterized by unhealthy chronic inflammation or
immune repression. In cancer in particular, immune evasion is considered an emerging hallmark
of carcinogenesis.[1] Thus an attractive direction for novel cancer therapy has been to engineer
immune cells to overcome this immune evasion and attack the cancer. Cellular therapies are
attractive avenues because they are minimally toxic (compared to radiation and chemotherapy)
and have the potential for interfacing with the native immune system and establishing
immunological memory, preventing return of the cancer and eliminating the need for recurrent
administrations of therapy. Reprogrammed dendritic cells [2], [3], [4] and natural killer (NK)
4
Dudek PhD proposal
cells [5] have been attempted clinically and met with modest results, and a recent clinical trial
with T cells autologously programmed to express a chimeric antigen T-cell receptor proved to
be unexpectedly successful, with 2 out of 3 patients proceeding to complete remission.[6]
Limitations of cellular immunotherapy approach are the dependence on endogenous
cellular mechanisms, the requirement for specific tumor antigens, and the limitation to single
input detection and single output response. An immune cell engineered to recognize a novel
stimulus but to respond in an endogenous manner is still subject to regulation by the
immunological network surrounding it, potentially accounting for the failure of many autologous
cellular immune therapies. Moreover, there are as many cancer antigens as there are cancers,
and for some no specific antigen has been defined. What if we could couple antigen sensing by
an immune cell to a completely novel effector function or multiple effector functions, to free it
from regulation by a diseased immunological network? And what if we could couple the
effector function(s) to the sensing of a motif (or motifs) common to multiple cancers, such as the
immunologically dysfunctional microenvironment itself?
An overarching goal of my lab is to engineer novel functions into mammalian immune
cells to generate novel diagnostics and therapeutics for diseases mediated by immune
dysfunction, such as autoimmune disease, allergy, and cancer. We see the cell as a device,
capable of sensing inputs, executing logical computation operations, and performing a desired
output function [7]. Robust sensing of stimuli of interest is of critical importance before
processing and effector response can come into play, therefore I will focus on the development
of novel biosensors for integration into cellular devices using the tools of synthetic biology.
TUMOR MICROENVIRONMENT (TME)
It is now well recognized that cancers establish a tumor microenvironment (TME)
5
Dudek PhD proposal
permissive to their growth that entails heterotypic signaling from the tumor-associated stroma
and a complicated relationship with both tumor-antagonizing and tumor-promoting immune
cells. [1] Tumor-associated immune cells secrete cytokines such as interleukin 10 (IL-10) that
are involved in the wound-healing response and promote immune tolerance such as anergy and
regulatory phenotypes, as well as matrix metalloproteinases (MMPs) that are mediators of the
inflammatory response but in cancer serve to degrade the stromal matrix surrounding the tumor,
promoting invasion and releasing growth factors that drive tumor growth.
CYTOKINE SIGNALING IN THE TME
Cytokines are messenger molecules for the innate and adaptive immune system. Cells
release cytokines to respond to a variety of cellular stresses including infection, injury, and
inflammation and exhibit diverse roles in stimulating the immune response while minimizing
damage. In healthy individuals, a balance is maintained between inflammatory and tolerogenic
cytokine signaling. In cancer the tumor functions as an unresolved injury in the host [8], with
inflammatory cytokines promoting further tissue destruction and tolerogenic cytokines involved
in the wound-healing response suppressing beneficial immune responses and preventing clearing
of the tumor. Thus it is of interest to use this imbalanced cytokine profile as a sensory input
characteristic of the TME.
MATRIX METALLOPROTEINASES (MMPs) IN THE TME
To date 26 MMPs have been identified, and many have been implicated as regulators of
the tumor microenvironment and mediators of the immune response [8] [9]. MMPs are
expressed by the tumor associated stroma, fibroblasts, and immune cells and are overexpressed
in tumors, where they function to degrade the extracellular matrix (ECM), promoting invasion
and metastasis and liberating growth factors and cytokines such as transforming growth factor β
6
Dudek PhD proposal
(TGF-β) and vascular endothelial growth factor (VEGF), that contribute to tumor growth and
vascularization. [9], [10] The pleiotropic activity of MMPs makes them an appealing but
elusive therapeutic target; while in theory MMP inhibitors could prevent the cascade of
tumorigenic activity set off by MMPs, in practice, once this cascade has started, inhibiting the
MMPs is not sufficient to halt its progression. However, MMPs may serve as a convenient
biomarker for activating an antitumor cellular therapeutic specifically in the TME, and this
activity need not be related to MMP function. Finally, coupling MMP sensing to cytokine
imbalance sensing would result in improved discrimination of the TME versus healthy tissue,
improving efficacy and reducing potential off-target effects.
SYNTHETIC BIOLOGY CELLULAR SENSORS AND DEVICES
Synthetic biology has yielded a variety of sensors and circuits able to recognize a diverse
array of inputs, including small biomolecules such as vitamins [12], [13] and metabolites [14],
chemical species such as acetaldehydes and nitric oxide [15], [16], and environmental conditions
such as hypoxia [17], [18], as well as cytoplasmic molecules [19]. Mainly, these have been
developed for coordinating orthogonal cellular communication for development of coordinated
network behavior. However, these recognition events are limited to intracellular species or small
diffusible extracellular species able to cross the cell membrane. Technology for translating
exclusively extracellular recognition events into orthogonal intracellular circuits is not currently
available. This completely unmet challenge is particularly pressing for the development of
diagnostic and therapeutic cell devices for synthetic immunobiology, which will entail the
probing and manipulation of intercellular communication by cytokines, chemokines, and cell-cell
contacts, which are exclusive to the extracellular milieu.
7
Dudek PhD proposal
An enabling technology that lends inspiration for extracellular sensor design is the Tango
assay developed by Barnea et al [20]. This assay enables a transient receptor ligation to be
amplified into a stable signal as illustrated in Figure 1.
Figure 1. Schematic of Tango assay. [20] A transcription
factor (tTA) is tethered to a receptor of interest (here a G protein
coupled receptor, GPCR) by the cleavage site for a protease
(here, the tobacco etch virus, TEV, protease). The TEV protease
itself is fused to the adaptor molecule for the receptor (here
arrestin), so that ligation of the receptor by its ligand recruits the
adaptor, enabling the TEV to cleave its cleavage site and liberate
the tTA. Upon translocation to the nucleus, tTA binds to a tTA
responsive promoter consisting of the tet response element
(TRE) upstream of a minimal promoter, leading to transcription
of a reporter gene.
This assay still relies on endogenous cellular mechanisms for recruitment of the adaptor
molecule, and therefore the effector function is not strictly orthogonal and is still subject to the
cell’s regulatory mechanisms. Thus the design is useful in its current form for its intended
purpose (detection of transient ligand-receptor interactions and identification of ligands for
orphan receptors) but the concept of coupling an extracellular binding event to a strictly
orthogonal intracellular proteolysis and signal transduction will be further developed for the
application of cellular device biosensing.
Finally, enabling the sensing of protein-level extracellular cues also provides a
mechanism for allowing cell-to-cell communication between synthetic cellular device
components via “pseudokines” (synthetic orthogonal proteins used in signaling applications
analogous to the way the body uses cytokines) for coordinating multicellular synthetic networks.
In addition to developing orthogonal synthetic communication for in vivo cellular therapeutics in
the long term, such a sense-and-respond multicellular pseudokine network would offer
opportunities for investigating biology by designing novel analogous systems.
8
Dudek PhD proposal
SIGNIFICANCE AND IMPACT
In summary, the goal of my research is to generate cellular devices capable of sensing
multiple extracellular environmental inputs such as the physiological cytokine and proteinase
profile of the TME and translating these into an orthogonal intracellular response. I am a coinventor of a synthetic biology technology my lab is developing (along with Dr. Leonard,
provisional patent filed) called the Modular Extracellular Sensor Architecture (MESA) that will
enable cellular devices to sense stimuli in the extracellular environment and translate them into
intracellular processes. To achieve this goal, I am working to augment the MESA platform by
optimizing its signal to noise ratio, to implement it for multicellular synthetic communication,
and to complement it by developing a biosensor that utilizes a different sensing modality.
PRELIMINARY RESULTS
MESA design overview
I am developing the MESA system as a surface expressed cellular biosensor consisting of
receptor fusion proteins (see Figure 2). MESA consists of two complementary receptor chains
and works by ligand-induced dimerization. The target chain (TC) is a fusion protein consisting
of an ectodomain (EC, may be the ligand-binding domain of a receptor for a ligand of interest, an
antibody fragment, etc.) and a transmembrane (TM) domain connected to an intracellular
transcription factor (TF) by a tether of linker residues and the cleavage sequence for a specific
protease. The protease chain (PC) is a fusion of the same ectodomain, transmembrane and linker
domains, and the protease for the cleavage sequence. Ligand binding to one receptor chain
ectodomain recruits the complementary chain into a receptor-dimer, ligand-stabilized complex.
The dimerization event brings the protease into contact with its cognate cleavage sequence on the
9
Dudek PhD proposal
target chain, which it then cleaves, freeing the transcription factor to translocate to the nucleus
and enable transcription of a gene of interest (GOI).
Target
A
Protease
B
Ectodomain (EC)
Transmembrane
domain (TM)
Linker
Cleavage sequence (CS)
Protease
Transcription factor
(TF)
Figure 2. MESA a) schematic and b) mechanism. The target and protease chains dimerize cooperatively upon
binding their cognate ligand. This ligand-induced dimerization juxtaposes the protease with its cleavage
sequence on the target chain, which tethers a transcription factor. The resulting proteolysis of the cleavage
sequence tether releases the transcription factor to bind its response element in the nucleus, enabling transcription
of a gene of interest such as a reporter gene.
For the MESAs that I have constructed to date, I have utilized the tet transactivator (tTA)
molecule for the transcription factor and the tobacco etch virus (TEV) N1a protease and its
cognate cleavage sequence (CS). Cleavage of the CS by the TEV protease chain results in
translocation of the tTA to the nucleus to bind to a tet-response element (TRE) driving
expression of a green fluorescent protein (GFP) reporter gene [21]. The contributions of the
structural components of the MESA to device function are summarized in Table 1 below.
Table 1: Components of MESA and their roles in device function
Domain
Tunable parameters
Trade-offs
Ectodomain
Size, ligand specificity
Steric hindrance vs. affinity
Target chain linker
Length, rigidity
Conformational freedom vs. local concentration
Protease chain linker
Length, rigidity
Conformational freedom vs. local concentration
Protease cleavage
sequence (CS)
Affinity (KM), catalytic
turnover rate (kcat) of protease
Overactive proteolysis (high background) vs.
underactive proteolysis (low signal)
Since MESA development has become a collaborative project within my lab, I will be
specifically focusing on the contributions of the ectodomain and CS cleavage kinetics and less so
10
Dudek PhD proposal
on linker lengths. The receptor ectodomain dictates the kinetics of receptor complex formation,
which is linked to the kinetics of the proteolytic cleavage and signal output. TEV is a stringent
protease that recognizes the consensus sequence E-X-X-Y-X-Q (G/S) [22] and does not cleave
promiscuously [23]. However, it has been shown to tolerate a number of substitutions in its
canonical cleavage sequence (E-N-L-Y-F-Q / S/G) in the terminal, or P1’, residue as well as
multiple nonconsensus and consensus sequence residues with reduced catalytic efficiency (see
Table 2) [24], [25].
Table 2: Example modified TEV N1a protease CS variants and their resulting affinities (K M), catalytic turnover
rates (kcat), and catalytic efficiency (kcat/KM). Mutated residues appear in red.
Sequence
ENLYFQ / S
ENLYFQ / G
ENAYFQ / S
ENLYFQ / L
ENLFFQ / S
ETVFFQ / S
KM (mM)
0.043 ± 0.006
0.087 ± 0.017
>0.5
0.240 ± 0.047
0.456 ± 0.050
>0.5
kcat (s-1)
0.194 ± 0.007
0.268 ± 0.025
ND
0.014 ± 0.001
0.161 ± 0.007
ND
kcat/ KM (s-1mM-1)
4.51 ± 0.65
3.08 ± 0.67
0.027 ± 0.001
0.06 ± 0.01
0.35 ± 0.041
0.007 ± 0.001
Barnea et al have utilized this ability to tune the TEV/CS cleavage kinetics to optimize
the signal-to-noise ratio for the Tango assay, achieving a high signal to noise ratio using the L
residue in the P1’ position [20]. Thus we have reason to believe that a CS with kinetic
parameters amenable to the MESA application is possible, and this will likely be different from
that for the Tango assay since the latter requires discrimination between ligated and unligated
random diffusion interaction in the three dimensional space of the cytoplasm whereas the former
takes place in the two dimensional space of the cell membrane.
TLR5 MESA receptor design
I engineered the first generation of MESA receptors using Toll-like Receptor 5 (TLR5) as
the ectodomain with linker and CS combinations as summarized in Table 3. Plans were already
underway in my lab to generate cytokine receptor MESAs using the IL-10 receptor, therefore
11
Dudek PhD proposal
TLR5 was chosen as an alternative receptor system to generalize the approach. Like the IL-10
receptor, TLRs exhibit ligand-induced dimerization [26], the mechanism for triggering MESA
activity. TLR5 is an innate immune sensor for bacterial flagellin, and it is the only TLR that
binds a protein ligand. Finally, as part of an ongoing collaboration I have been part of with the
lab of Dr. Cynthia Collins at Rensselaer Polytechnic Institute in interkingdom communication, I
was interested in utilizing a receptor ectodomain capable of sensing a ligand that could be
secreted from bacterial cells for potential development of the MESA platform in that direction.
Table 3: First generation MESA receptors
Ectodomain
TLR5
TLR5
Transmembrane
TLR5
TLR5
Linker length CS P1’ residue
2, 5, 10
S, G, L
2, 4, 6
--
Signaling domain
tTA target
TEV protease
A naming convention I will employ in referring to these receptors is the name of the signaling
domain, followed by the number of linkers, followed by (where applicable) the P1’ residue of the
CS; for example, protease chain with 4 linkers is “protease 4”, and target chain with two linkers
and G for the P1’ CS residue is “target 2G”.
MESA cloning methods
The first 665 amino acid residues of the TLR5 receptor (corresponding to the ectodomain
and transmembrane region [27]) were cloned out of the pUNO-hTLR5 vector and the appropriate
number of linker residues as per Table 3, and SfoI and NotI restriction sites were added to the 3’
end by PCR. A BamHI site was added to the 5’ end. These fragments were subsequently
digested with BamHI and NotI and ligated into the pAAV vector to form an intermediate
plasmid named pAAV-TLR5ectm. The tTA domain was removed from the pCL-CTIG plasmid
[28] and fused to the appropriate cleavage sequence by PCR, flanked by NaeI and NotI
restriction sites. The intermediate backbone plasmid was then digested with SfoI and NotI and
ligated into the digested pAAV-TLR5ectm, forming a scarless glycine junction between the
12
Dudek PhD proposal
blunt ends left by SfoI and NaeI and ensuring in-frame insertion of the signaling domain. The
same strategy was used to excise the TEV N1a protease catalytic domain from the pRK1043
plasmid [29]. All constructs were confirmed by sequencing.
Preliminary TLR5 MESA feasibility testing and characterization
Objective and rationale
Goals of the initial feasibility study were: 1) to assess whether the TLR5 MESAs could
induce signaling as measured by reporter activity and whether a difference would be observed
between reporter activity in the ligated vs. unligated state, and 2) to begin to map out the design
space in terms of how the tunable parameters (see Table 1) would affect MESA signaling. For
simplicity, I explored only the two alternate wildtype cleavage sequences, a single linker length
on the target chain, and two linker lengths on the protease chains. Combinations of target and
protease chains were transiently transfected into HEK 293 FT cells along with a reporter plasmid
containing a tet-responsive promoter driving GFP expression (pBI-MCS-EGFP [30]) as
summarized in Figure 3. DsRed was cotransfected as a transfection control. The reporter was
transfected by itself as a negative control and the reporter along with a plasmid expressing free
tTA were cotransfected as a positive control. Additionally, each target chain was expressed by
itself, to assess whether protease-independent mechanisms such as degradation or recycling of
the receptors would induce signaling. Finally, as an additional control, I cotransfected each of
the target chains with a plasmid expressing free TEV protease to ensure that the protease was
active and the CS cleavable. Reporter activity in response to tTA cleavage is reported in Figure
3 below, quantified as mean GFP fluorescence intensity.
Results and discussion
The signal output, or reporter activity, of the constructs tested is represented in Figure 3.
13
Dudek PhD proposal
Figure 3: Reporter activity of live transfected cells for MESA feasibility study and design space characterization.
HEK 293 FT cells were plated in 24 well plates at a density of 105 cells per well and transiently transfected by the
calcium phosphate method, stimulated with 10-100 ng/mL of flagellin (fla) 18-24 hours post transfection, and
harvested 18 hours later and analyzed by flow cytometry. Cells were gated as live based on light scatter and as
transfected based on DsRed fluorescence intensity. Reporter activity is quantified as mean GFP intensity
normalized to the positive control. Each experimental condition was performed in triplicate, with the reported MFI
corresponding to the mean and the error bars corresponding to the standard deviation of the three experiments.
The data indicate that the reporter exhibits tight on and off behavior in the presence and
absence of tTA, respectively, that the TEV is active, and that the CS is cleavable. Moreover, no
significant difference in reporter activity is observed between the two CS and two PC linkers
tested, or between the ligated and unligated cases.
The lattermost observation is the most surprising, especially since this trend has been
conserved between different trial runs and for all linker length and CS variants tested (data not
shown). It would be expected that addition of the ligand, flagellin, to the TLR5 MESAs would
serve to either increase signaling by stabilizing dimeric complexes and facilitating cleavage or
else decrease it by stabilizing nonproductive complexes consisting of the protease chain and a
cleaved target chain, sequestering the protease and inhibiting additional activity.
14
Dudek PhD proposal
Several explanations are possible for the observed indistinguishable signaling levels. The
most obvious is that the ligand, flagellin, had become denatured and was no longer able to induce
signaling; however, an experiment in which I added flagellin from the same stock to wildtype
TLR5-transfected reporter cells expressing an NF-κB (the downstream target of TLR5 signaling)
reporter demonstrated that the flagellin was active and able to induce signaling, ruling out this
possibility. TLR5 downregulation in response to ligation is not supported by the literature [31],
but endogenous TLR5 regulatory mechanisms are not likely to be the same for a chimeric TLR5
lacking its native signaling domain.
The most likely explanation is that the receptors do not need to be in a ligand-stabilized
complex for signaling to be induced for one of two reasons: 1) transient encounters between noncomplexed receptors during random diffusion within the membrane are sufficient to give rise to
signaling or 2) the receptors are constitutively associated, contrary to the ligand-induced
dimerization paradigm. The latter is contrary to most current thought on TLR5 signaling, but
evidence of its possibility has been observed [32]. The diffusion-limited scenario implies that
the cleavage kinetics of the TEV protease are too fast to distinguish between transient random
encounters and stable ligated complexes, whereas the constitutive association scenario implies
that there is some degree of affinity between the TLR5 ectodomains in the absence of ligand.
In conclusion, to achieve inducible MESA signaling, background must be eliminated
either by decreasing the proteolytic activity of TEV protease, reducing affinity between
ectodomains, or both. The next experimental step is to investigate whether transient diffusive
encounter or constitutive complex formation is responsible for background signaling.
15
Dudek PhD proposal
Investigating the role of the ectodomain in promoting or inhibiting background signaling
Objective and rationale
The TLR5 MESA is a convenient system for differentiation between diffusive encounters
and constitutive complexes because the TLR5 ectodomain should have opposite effects in each
scenario. A bulky horseshoe-shaped structure, >600 amino acid long and about 80 Å in
diameter, the TLR5 ectodomain should prove a steric hindrance to random encounters in the
diffusion-limited regime. Therefore, in this regime substituting the ectodomain of one of the
MESA dimer pairs with a smaller ectodomain should increase background. However, if affinity
exists between the ectodomains causing constitutive complexes, then substituting the ectodomain
of one of the MESA dimer pairs should attenuate background signaling. To test this reasoning, I
performed crossover experiments in which I cotransfected a combination of my TLR5 MESA
target chains with protease chains utilizing the IL10 receptor β chain as an ectodomain. The
IL10 receptor was chosen because it had already been developed into a MESA in my lab, and
because the ectodomain (shown in Figure 4) is much smaller than that of TLR5. TLR5 and the
IL10 receptor are not known to have any affinity for each other, therefore they are suitable
elements for investigating the potential roles of sterics vs. affinity in background signaling.
Figure 4: Comparison of the TLR5 and
IL10Rβ ectodomains. Predicted structure of
porcine TLR5 [33] is depicted alongside
solved IL10Rβ ectodomain structure [34]. The
larger TLR5 ectodomain may be s teric
hindrance to protease chain activity in the
diffusion-limited regime.
~80 Å
~32 Å
16
Dudek PhD proposal
Results and discussion
Comparing the homotypic (TLR5 target + TLR5 protease) and heterotypic (TLR5 target
+ IL10Rβ protease) signaling levels summarized in Figure 5 seems to argue that, while diffusive
encounter is likely the dominant mechanism of background signaling, it may not be the only one.
Figure 5. TLR5 and IL10Rβ MESA crossovers to assess the role of the TLR5 ectodomain as a steric
hindrance, attenuating background signaling, or a constitutive dimerizer, increasing background. HEK 293 FT
cells were plated in 12 well plates at a density of 3x10 5 cells per well and transiently transfected by the
calcium phosphate method. Media was changed 18hours post transfection, and cells were harvested 18 hours
later and analyzed by flow cytometry. Cells were gated as live based on light scatter and as transfected based
on DsRed fluorescence intensity. Reporter activity is quantified as mean GFP intensity normalized to the
positive control. Each experimental condition was performed in triplicate, with the reported MFI
corresponding to the mean and the error bars corresponding to the standard deviation of the three experiments.
Abbreviations: TC: target chain, LD: linker domain, P1’: P1’ residue of cleavage sequence, PC: protease chain
For the TLR5 target 2S, background signaling is observed to be high when it is
coexpressed with either homotypic protease chain and even higher when coexpressed with each
of the three heterotypic protease chains. Generally, signaling appears to increase for longer
protease chain linker lengths (the dip in signaling for the TLR5 target 2S and IL10Rβ protease 6
is likely due to noise, as it was not observed in a repeat experiment). This supports the random
diffusion model of background signaling. The TLR5 target 2L concurs, as it exhibits the same
trends, albeit at lower background levels given its slower cleavage kinetics.
17
Dudek PhD proposal
The TLR5 target 10S chain experiments are confounding, as they exhibit the opposite
trend, with homotypic background being higher and heterotypic background being lower. The
same trend appeared again in an independent repeat experiment. This suggests that, at longer
linker lengths on the target chain, the drop in local concentration of the CS-tTA signaling moiety
dominates over the gained conformational flexibility, so that the combination of transience and
low local concentration prohibits the proteolytic reaction from proceeding in the diffusionlimited regime. Also implied is that in this conformationally-flexible, local concentration low
regime, weak association between the ectodomains may be required to stabilize background
signaling. If this is the case, it is also possible that for shorter linker lengths, the association
between TLR5 ectodomains actually restrains them in a conformation where they cannot signal
as a complex, so that only the random diffusive encounters can give rise to signaling. Thus
while the data may not directly or strongly support the constitutive associated model, they
suggest it is a possibility.
In conclusion, it is clear from the feasibility study and the background signaling
characterization study that elimination of background signaling will be necessary for robust
sensing using the MESA platform and that the major source of background is random encounter
of target and protease chains by diffusion. A promising strategy for eliminating background in
the diffusion-limited regime is randomization of the CS and screening for a variant that operates
in a kinetic window able to discriminate between diffusive and stable ligated interactions,
motivating my Aim 1. Moreover, since receptor-ligand association kinetics are essentially
coupled to proteolysis kinetics in the MESA platform and these will also play a role in
determining the signal to noise ratio, it will be advantageous to investigate signaling dynamics
for a variety of ectodomain-ligand pairs, especially ones with established association and
18
Dudek PhD proposal
dissociation rates. This will be enabled by my Aim 2. Finally, it will be worthwhile to develop
alternative sensing-signaling modalities in addition to the dimerization-induced mechanism,
which I will pursue in my Aim 3.
RESEARCH DESIGN AND METHODS
AIM ONE
To eliminate excessive background signaling by the MESA by optimizing the proteolysis
kinetics of the protease on its cleavage sequence. I propose to generate a cleavage sequence
variant by directed evolution that will be cleaved by the protease chain only when it is in a stable
receptor-dimer-ligand, eliminating background signaling in the diffusion-limited regime.
RATIONALE
The TEV protease was chosen because it is a well characterized protease with high
stringency for its cognate cleavage sequence. While its catalytic efficiency is in a suitable range
for the Tango application where diffusion is in three dimensions and capturing of transient
interactions is desirable, is not so well suited to the MESA application, where diffusion is in two
dimensions (increasing the rate of transient chance encounters) and it is desirable to filter out
transient interactions and capture only stable longer-lasting complex formations in the signal
output. It has been shown that mutating various residues in the TEV CS can alter the catalytic
efficiency of proteolytic cleavage. By large-scale diversification and screening, it is theoretically
possible to search the entire number of potential cleavage sequence variants that will still be
recognized and cleaved by TEV at some lesser efficiency and discover one or more variants with
a catalytic efficiency able to discriminate between a substrate that it encounters transiently and a
substrate that it encounters conjugated to a receptor dimer pair.
A library of MESA target chains expressing randomized TEV cleavage sequences will be
generated. These will first be expressed in a negative selection cell line to eliminate signalers in
the diffusion-limited regime, and then in a positive selection cell line to recover dimerization
19
Dudek PhD proposal
regime signalers. Finally, constructs that pass the screens will be recovered, fused to the TLR5
ectodomain, and validated by cotransfection with the TLR5 protease chain and reporter.
EXPERIMENTAL DESIGN AND METHODS
A library of target chains with CD4 for an ectodomain and randomized cleavage
sequences will be assembled by PCR. CD4 domains dimerize constitutively [35], therefore
transfection of each CD4 target into a cell line stably expressing the CD4 homotypic protease
chain will serve as a positive screen to see if the dimerized pair can result in signaling for the
given CS variant (see Figure 6). For the negative screen, the CD4 target test chain will be
transfected into a cell line stably transfected with an example heterotypic ectodomain for which
it has no affinity, such as the red fluorescent protein mCherry. Any signaling between the
heterotypic pairs will be due to diffusive encounters, since the CD4 and mCherry are not known
to associate.
B
A
Figure 6. Screening methodology for CS variant library.
The CS variants will be expressed in target chains with
CD4 as an ectodomain. A) In the negative selection
round, CD4 target test chains will be transfected in a cell
line expressing and mCherry TEV chain, so that all
signaling will be exclusive to the diffusion-limited
regime. Signaling cells will be rejected. B) In the
positive selection round, the stably expressed TEV chain
will have the CD4 ectodomain, which will constitutively
dimerize with the CD4 target chain. All CD4 target
chains that signal in the positive but not the negative
selection line will be specific for the dimerization regime.
Library creation
The CD4 ectodomain will be cloned into the existing MESA target construct by
staggered extension PCR (StEP) [36] to give the plasmid pAAV-CD4-target and will serve to
discriminate between dimeric complexes and random diffusive encounters in the selection steps
(detailed in the following sections). A library of randomized cleavage sequences will be
encoded by commercially supplied oligonucleotide primers with random base pairs encoding
20
Dudek PhD proposal
four of the six amino acids in the sequence. The random CS variants will be incorporated into
the CD4 target chain by round the world PCR (RTW PCR), as illustrated in Figure 7.
Figure 7. Schematic of round the world PCR addition of randomized cleavage sequence library and linkers to
CD4 target chain. A sense primer encoding the linker residues, randomized cleavage sequence (‘x’ represents
an amino acid encoded by random base pairs) and an overlapping region of tTA and an antisense primer
encoding residues of the transmembrane domain will be added to a PCR reaction with the pAAV-CD4-target
plasmid. The result will be a library of linearized plasmids with each of the random CS added on. This PCR
reaction product will be digested with DNase, to remove all the template plasmids (which will be methylated),
and then ligase will be added to circularize the plasmid library.
The wildtype TEV CS is E-N-L-Y-F-Q- (G/S), and the consensus sequence has been
defined as E-X-X-Y-X-Q-(G/S). However, alterations in up to 3 independent residues (including
consensus residues) have been demonstrated to be cleavable by the TEV protease with reduced
catalytic efficiency (see Table 2). For the initial library, to obtain good coverage but avoid
sequences the protease may fail to recognize, I will randomize the nonconsensus and P1’
residues to obtain ~1.7x107 variants. Additionally, I will space these from the membrane with
linker lengths of 0, 2, 5, and 8 glycine-serine residues, increasing the size of the initial library to
~6.7x107. Since a practical upper limit for library screening by the proposed method is on the
order of 108, this size library should give sufficient coverage while remaining feasible.
Selective cell lines
The negative selection cell line will be an HEK 293 FT cell line stably expressing TEV
protease chains fused to mCherry as an ectodomain and the tTA responsive reporter (see Figure
6). This stable cell line will be generated using lentiviral transduction and antibiotic selection.
Briefly, mCherry and the reporter will each be cloned into separate lentiviral vector plasmids
with different antibiotic resistance cassettes (e.g. neomycin and puromycin) and these will be
21
Dudek PhD proposal
transfected into cells along with the necessary packaging plasmids and the virions harvested
from the culture media and purified, if necessary, by ultracentrifugation. Viral titer will be
determined by quantitative real-time PCR (qRT-PCR) [37]. HEK 293 FT cells will be
transduced with the virus and selected by addition of the selective antibiotics to the culture
medium after an appropriate recovery period. Lentiviral transduction is desirable to ensure
integration of the plasmids into the cells’ genomes, to prevent loss of the plasmids over time and,
as a result, false negatives.
The positive selection cell line will be generated in a similar manner to the negative
selection line using the CD4 ectodomain for the target chain plasmid. Since CD4 domains
dimerize constitutively, these constructs will be selective for complex-mediated signaling.
Viral packaging of library
The library will be packaged into adeno-associated virus (AAV) for viral transduction
into the selective cell lines. Briefly, the pAAV plasmids encoding the library will be
cotransfected with AAV packaging plasmids pXX2 and pHelper [38] into AAV 293 cells (a
strain of HEK 293 cells with optimized AAV packaging efficiency), and after 3 days (or when
necrosis is evident) cells will be harvested, lysed, and centrifuged, and the supernatant containing
the AAV particles will be recovered. Titer will be determined by qRT-PCR. AAV is an
attractive vector for expressing the library for screening because it can package a maximum
genome of 4.7 kilobases [39] (genomes larger than 4.7 kb being truncated randomly [40]),
ensuring that each viral particle is essentially representative of a single library variant (library
constucts will be ~3 kb). Also, AAV genomes can be rescued from the transduced cells and
further manipulated. The packaged virus library will be transduced into the selective cell lines
for screening of the library constructs.
22
Dudek PhD proposal
Screening / directed evolution
In the negative selection screen, the CD4-target encoding virions will be transduced into
the mCherry-protease cell line at a low multiplicity of infection (MOI) to obtain a single AAV
infection per cell. The cells will be subjected to fluorescence assisted cell sorting (FACS) and
cells gated as positive for GFP expression (indicative of reporter activity due to constitutive
signaling) will be eliminated while GFP negative cells will be retained. The viral genomes will
be rescued from these cells by coinfection with helper adenovirus (to return AAV stable
integrants to the episomal state), followed by lysis, heating killing of the adenovirus, and
extraction and purification of the plasmid DNA. These recovered genomes will be transduced
into the positive selection cell line. Cells with active reporters will be selected and their
genomes rescued and sequenced.
Validation of screen-selected CS
I will clone any cleavage sequences selected by this screening method into the TLR5 and
IL-10 target chains and assay for reporter activity as in the preliminary data section.
ANTICIPATED RESULTS AND ALTERNATIVE DIRECTIONS
I anticipate that a thorough search of the potential CS variants for the TEV protease will
yield a variant with a catalytic efficiency that will give a suitable signal to noise ratio for the
MESA application, as selected by the screen and demonstrated in the TLR5 model system.
In the event that repeated rounds of diversification and screens do not yield a cleavage
sequence with the desired properties, an alternative may be directed evolution of the protease
itself. However, since this is a larger space to sample, this approach would be more timeconsuming and not amenable to an exhaustive search. An alternative protease to the TEV is the
tobacco vein mottling virus (TVMV) protease, which is also well-characterized and has different
23
Dudek PhD proposal
affinity and catalysis kinetics [25]. Additionally, mechanisms for conformational change-based
activation as opposed to dimerization-based activation may be explored.
AIM TWO
To engineer an orthogonal “pseudokine” network consisting of a) MESA receptors that form
stable homodimeric and heterodimeric complexes upon cooperative binding of a synthetic
ligand, and b) a synthetic secretable ligand capable of dimerizing the receptors. I will use this
platform enable synthetic intercellular communication for ultimate applications in the
coordination of multicellular networks.
RATIONALE
To enable the eventual goal of multi-input, multi-output cellular devices, it will be
beneficial to enable communication between the devices in a manner analogous to the native
immune system’s intercellular communication by cytokines. However, these pseudokines must
be orthogonal to native signaling mechanisms to avoid crosstalk with endogenous pathways.
Furthermore, the pseudokine platform may provide a useful tool for modeling the interplay
between cytokines in vitro.
The pseudokine receptors will be built using the previously characterized peptide-binding
protein domains PDZ and SH3 [41] as the synthetic ectodomain fused to the transmembrane
domain of the T cell CD28 molecule [42] and the MESA signaling domains described in the
Preliminary Results. PDZ is a subunit of the murine α-syntrophin protein (comprising residues
77-171) and has been shown to bind the small peptide epitope GVKESLV with high affinity (Kd
= 8 µM) [43]. SH3 comprises residues 134-190 of the mouse Crk viral oncogene product and
adapter protein [44] and binds the peptide PPPALPPKRRR with a Kd of 0.1 µM. Both proteins
function as adaptor proteins facilitating protein-protein interactions in vivo, and have been
utilized by Dueber et al [41] intracellularly as scaffolding proteins for engineering modular
frameworks for cellular signaling circuits. I intend to express them on the surface to detect a
24
Dudek PhD proposal
synthetic ligand comprising their peptide recognition sequences complexed to a fluorescent
protein.
EXPERIMENTAL DESIGN AND METHODS
Receptor design and construction
I will build a library of synthetic receptors as summarized in Table 4 below.
Table 4: Synthetic receptor constructs for pseudokine network
Tag
FLAG
HA
FLAG
HA
Ectodomain
SH3
SH3
PDZ
PDZ
TM
Cd28
Cd28
Cd28
Cd28
Linker (no. residues)
2,4,6
2,4,6
2,4,6
2,4,6
Signaling domain
pcs+tTA
TEV
pcs+tTA
TEV
The murine Ig κ-chain V-J2-C signal peptide will be fused to the N-terminus of each
construct to signal trafficking of the synthetic receptors to the cell surface, but will be cleaved in
processing and not present on the final constructs. StEP will be used to assemble the domains,
and the final PCR product will be digested with appropriate restriction enzymes and ligated into
the pAAV vector under control of the CMV promoter. An AAV expression plasmid was chosen
as the vector because it is suitable for both transient transfection and for AAV viral packaging as
described in Aim 1. All constructs will be verified by sequencing.
Ligand design and construction
To induce homodimeric and heterodimeric complexes of synthetic receptors, synthetic
ligands consisting of the peptide ligands of the SH3and PDZ domains (PPPALPPKRRR and
GVKESLV, respectively) connected by an appropriate number of glycine-serine linker residues
will be constructed as summarized in Table 5. Synthetic ligand constructs will be tethered to
GFP, and the entire complex will be fused to the murine Ig κ-chain V-J2-C signal peptide
secretion tag for secretion outside the cell into the culture medium [45].
25
Dudek PhD proposal
Table 5: Synthetic ligand “pseudokines”
Dimer
induced
PDZ-PDZ
SH3-SH3
PDZ-SH3
Fusion
partner
GFP
GFP
GFP
Linkers
Ligand 1
Linkers
5,10
5,10
5,10
GVKESLV
5,10
PPPALPPKRRR 5,10
GVKESLV
5,10
Ligand 2
GVKESLV
PPPALPPKRRR
PPPALPPKRRR
The resulting synthetic receptors and ligands are represented in Figure 8 below.
B
A
SH3
PDZ
SH3 ligand
PDZ ligand
Figure 8. Proposed A) synthetic receptors and B) pseudokine ligands. Receptors may be expressed as
homodimeric or heterodimeric pairs and ligated using the appropriate peptide “dimerizer” pair
complexed to GFP. Purple ectodomain tags are FLAG tags (for detection of surface expression of target
chains) and orange tags are HA tags (for the detection of protease chains).
Expression analysis
Receptors will be transiently transfected into HEK 293 FT cells and surface expression
will be confirmed using fluorescently conjugated antibodies against the FLAG and HA epitope
tags. Likewise, the ligand pseudokines will be expressed in HEK 293 FT cells, and GFP
secretion will be confirmed visually and, if necessary, by performing Western Blot on the culture
media using anti-GFP antibodies.
Association analysis
To confirm that the pseudokine receptors do not associate and induce signaling in the
absence of ligand, combinations of target and protease chains will be transiently cotransfected
with a cyan fluorescent protein (CFP) reporter plasmid in the absence of ligand and fluorescence
will be monitored by microscopy and quantified by flow cytometry. Alternatively, a luciferase
reporter may be used in the event of problematic spectral overlap between CFP reporter and GFP
26
Dudek PhD proposal
ligand (which may also be avoided by using a yellow fluorescent protein, or YFP, ligand) or for
more high-throughput monitoring. In the event of high background, association (or lack thereof)
between unligated receptor domains may be detected by immunoprecipitation of either the target
or protease chain by its epitope tag and immunoblotted with antibodies against the epitope tag
for the complementary chain. Association analyses will be conducted for a series of
concentrations of the receptors to determine at what concentrations overexpression may mimic
association.
To confirm association of receptors in the presence of ligand, supernatant from the
ligand-producing sender cells will be added to the culture medium of the receptor-transfected
receiver cells and reporter activity monitored as previously described. Serial dilutions of the
ligand-conditioned media will be added to different wells of receiver cells to titrate the receptor
response and determine the sensitivity to ligand concentration. Association of the ligand with
the receptors will also be confirmed by immunoblot.
In vitro pseudokine signaling network
Having confirmed the ability of the receiver cells to respond to sender cells, the two
populations will be cocultured on a single plate (see Figure 9) and reporter output will be
observed by microscopy and flow cytometry.
Figure 9. Pseudokine signaling network.
Sender cells produce secretable GFP
tethering the ligands for homodimeric
(not shown) and heterodimeric receptor
pairs on receiving cells to induce
signaling measurable by reporter activity.
Subsequently, I will create a model of multicellular negative feedback (Figure 10). In
this model, the sender cells will be transfected with SH3 MESAs modified to use TetR-KRAB (a
27
Dudek PhD proposal
transrepressor counterpart of tTA fused to the Krueppel-associated box) [46] in place of tTA, as
well as the PDZ ligand fused to YFP under the control of a strong constitutive promoter with a
TRE (so that release of the TetR-KRAB will silence instead of induce it). The receiver cells will
express the PDZ MESA receptors (with tTA as before) and the SH3 ligand fused to CFP under
the control of a minimal promoter and the TRE (like the reporter previously described, so that
tTA binding induces expression). These senders and receivers will be cocultured and observed
over by microscopy to determine the time course of YFP expression and secretion (sending), the
appearance of CFP (receiving, signal processing, and output of secondary sender) and YFP
decline (repression of initial sender signal). It will also be interesting to observe whether the
network exhibits fluctuation, with CFP diminishing in response to YFP decline and subsequent
YFP recovery.
Figure 10. Pseudokine negative feedback signaling network. YFP tethering the PDZ ligand will be constitutively
produced by cells expressing the SH3 receptors. Ligation and dimerization of the PDZ receptors will induce
expression of the SH3 ligand tethered to CFP. Ligation and dimerization of the SH3 receptors will repress
production of the YFP-PDZ ligand fusions, which will in turn cease to stimulate SH3 ligand production, in a
negative feedback model.
ANTICIPATED OUTCOMES AND ALTERNATIVE DIRECTIONS
I anticipate that the platform will be able to produce inducible homodimeric and
heterodimeric complexes in the presence of synthetic ligand and demonstrate the ability to signal
in a paracrine fashion. This will be a useful system for developing strategies for coordinated
28
Dudek PhD proposal
multicellular function in a mammalian host, testing logical gate processing, and modeling
cytokine signaling.
Alternative directions include the use of different receptor/ligand pairs (such as the GBD
domain from the Dueber 2003 study and its peptide ligand, or single chain antibodies), or
secretion of the peptide ligand dimers tethered to a different protein or alone. Another
alternative direction is synthesis of all-in-one receptors with both a protease domain and a target
domain on each chain, so that all interactions are productive and it will be possible to perform
directed evolution on the receptors for enhanced ligand affinity or reduced background.
AIM THREE
To develop a sensor for matrix metalloproteinases to complement the MESA approach and
enable multi-input discrimination of the tumor microenvironment. I propose to link extracellular
cleavage of a peptide substrate for MMP-2 to intracellular nuclear translocation of a transcription
factor to result in a sensitive and specific MMP sensor able to interface with the MESA system.
RATIONALE
While detection of cytokine-type inputs capable of dimerizing MESAs will be important
for targeting cell devices to the tumor microenvironment, it is useful to develop a secondary
sensing modality to be coupled, ultimately, into the same device. Such bimodal sensing will
have improved specificity for the TME, particularly if it is able to incorporate a different class of
molecule. Since MESA signaling is mediated by proteolytic cleavage, it is an intuitive next step
to extend design principles from the MESA to generate a cleavage-activated sensor for
metalloproteinases.
MMP-2 was chosen because it is one of the best characterized proteinases overexpressed
in tumors [8]. Physiologically, MMP-2 has multifaceted roles in cancer progression including
mesenchymal cell invasion [47], proteolytic cleavage and activation of the growth factor TGF-β1
[48], angiogenesis and vascularization [49], metabolic disregulation, and generation of the
29
Dudek PhD proposal
premetastatic niche [50]. Thus it is present in many stages of cancer pathology and can be used
to differentiate between the TME and healthy tissue.
EXPERIMENTAL DESIGN AND METHODS
I am developing two strategies for implementing the MMP-2 sensor, summarized in
Figure 11 below. Both designs will be constructed and tested in parallel, as both present unique
advantages and disadvantages.
Figure 11. Strategies for construction of a transmembrane MMP-2 sensor. A. Pull-through design, where cleavage
of the CS by the MMP removes the extracellular anchor, allowing the payload to pull the transmembrane domain and
optional positively-charged cell-penetrating residues through the membrane and translocate to the nucleus. B.
Quench-release model, where cleavage liberates the cell-penetrating peptide from a positively-charged quencher
peptide, allowing it to associate with the negatively-charged membrane and tow the payload into the cytoplasm
where it may subsequently translocate to the nucleus. ECA: Extracellular anchor, HA: HA epitope tag, CS:
cleavage sequence, FLAG: FLAG epitope tag, CPP: cell-penetrating peptide, PL: payload, QP: quencher peptide
The pull-through method (Figure 11A) is attractive because the payload (a transcription
factor as in the MESA system, or a fluorescent protein such as mCherry for initial
characterization experiments) is protected on the inside of the cell but runs the risk of failing to
pull through the transmembrane residues and therefore not activating. Data from other members
of the lab indicate that fusion receptors with only FLAG for an ectodomain are not stable on the
cell surface, but it is unclear whether this is due to unsuccessful trafficking to the membrane or
successful trafficking but subsequent diffusion back into the cell, due to insufficient anchoring
by the minimal ectodomain in the plasma membrane. Therefore, I will incorporate positively
30
Dudek PhD proposal
charged arginine residues in varying increments between the FLAG tag and transmembrane
domain to determine if this will aid in pulling the FLAG and transmembrane domain into the
cytoplasm without pulling the entire receptor ectodomain through. The quench-release method
builds on the MMP-2 imaging method employed by Jiang et al [8], in which a positively-charged
poly-arginine cell-penetrating peptide (CPP) was fused to a fluorophore along with the MMP-2
cleavable peptide PLGLAG and a positively charged poly-glutamate quenching peptide. In the
absence of MMP-2, the quenching peptide associated with the CPP and inhibited it from
electrostatically associating with the cell membrane, but the presence of MMP-2 caused cleavage
of the tether, permitting the CPP and fluorophore cargo to enter cells. In the proposed
application, this signaling modality will be generated by the cell’s own transcriptional machinery
(rather than synthesized) and expressed on the cell surface by endogenous mechanisms. While
this approach has the benefit of restricting CPP activity except in the presence of MMP-2, it has
potential liabilities in terms of cleaved constructs not getting back into the cell, or the payload
becoming denatured in the process or cleaved by the extracellular MMP-2. These and other
potential failure modes will be investigated and where possible circumvented.
Device construction
Planned MMP-2 sensor constructs are summarized in Tables 6 and 7 below. Again, the
murine Ig κ-chain V-J2-C signal peptide will serve to direct surface expression. Constructs will
be fused together using StEP and ligated into pAAV for later transfection or transduction.
Table 6: Planned pull-through constructs
ECA
PDZ or SH3
PDZ or SH3
PDZ or SH3
PDZ or SH3
Tag 1
HA
HA
HA
HA
CS
PLGLAG
PLGLAG
PLGLAG
PLGLAG
Tag 2
FLAG
FLAG
FLAG
FLAG
CPP
0R
2R
4R
6R
TM
CD28
CD28
CD28
CD28
PL
tTA, mCherry
tTA, mCherry
tTA, mCherry
tTA, mCherry
ECA: Extracellular anchor, HA: HA epitope tag, CS: cleavage sequence, FLAG: FLAG epitope tag, CPP: cellpenetrating peptide, TM: transmembrane domain, PL: payload.
31
Dudek PhD proposal
Table 7: Planned quench-release constructs
PL
tTA, mCherry
tTA, mCherry
tTA, mCherry
tTA, mCherry
CPP
9R
9R
9R
9R
CS
PLGLAG
PLGLAG
PLGLAG
PLGLAG
QP
9R
8R
7R
6R
ECA
PDZ or SH3
PDZ or SH3
PDZ or SH3
PDZ or SH3
TM
CD28
CD28
CD28
CD28
ICA
PDZ or SH3
PDZ or SH3
PDZ or SH3
PDZ or SH3
ECA: Extracellular anchor, CS: cleavage sequence, CPP: cell-penetrating peptide, TM: transmembrane domain, PL:
payload, QP: quencher peptide, ICA: intracellular anchor. Epitope tags included as diagrammed in Figure 11,
omitted here for clarity.
Sensor expression and surface localization
Fusion proteins and a tTA responsive reporter (as appropriate) will be transiently
transfected into HEK293 ft cells as previously described. The constructs using mCherry as the
payload will provide visual feedback as to whether the fusion proteins are being made.
However, the ability of the cell to synthesize the constructs is particularly of concern for the
quench-release design, where the association of the polycation and polyanion chains may pull the
nascent fusion protein transcript off the ribosome. In the event that mCherry is observed but no
surface expression and incomplete transcription is suspected, the cells will be lysed and assayed
for the HA tag by Western blot to confirm that transcription has not proceeded beyond the QP.
An additional epitope tag may be added to the C terminus of the complete fusion to ensure
complete transcription. For the pull-through method, the payload is attached at the C terminus,
therefore visual observation of mCherry will confirm that the sensor has been transcribed by the
cell.
Since the pull-through method will utilize an ectodomain-secretion tag system previously
characterized (see aim 2), it is likely to be expressed on the surface if correctly synthesized.
However, a concern is that the optional CPP residues may pull the ectodomain down into the
membrane, limiting the availability of the cleavage sequence. Antibody staining against the HA
epitope tag will be used to confirm that the receptor is on the surface, and surface staining
against FLAG will confirm that the CS is outside the cell membrane. The same antibody
32
Dudek PhD proposal
staining scheme will apply for the quench-release system. Fluorophore-conjugated antibodies
will be used, and cells will be analyzed by flow cytometry.
CS cleavability
MMP-2 will be obtained in plasmid encoded proenzyme form. This will be expressed in
HEK 293 FT cells along with appropriate activating enzymes and existent MESA constructs and
reporter activity observed as previously described. Alternatively, proenzyme may be obtained
commercially, activated, and incubated with cells expressing a fluorescent protein tethered to the
membrane by the cleavage sequence, and the culture media analyzed for presence of the
fluorescent protein.
Transcription factor activity post translocation
To confirm that the tTA can translocate through the cell membrane and retain activity, I
will cotransfect cells with the reporter and a construct consisting of tTA fused to the CPP and
secretion tag. As controls, I will express in separate wells 1) the reporter and a CPP-tTA fusion
as a positive control, and 2) the reporter and a secretion tagged tTA as a negative control. If the
secretion tagged CPP-tTA fusion is able to induce reporter activity but the negative control is
not, this will indicate that the secretion tagged tTA is able to leave the cell and requires the CPP
to re-enter, and that post reentry it retains its biological activity. However, if the reporter activity
of the secretion tagged CPP-tTA is low, this will indicate that the tTA is not able to reenter the
cell, whereas if negative control reporter activity is high then the tTA is not able to leave the cell.
In vitro sensing
Finally, each MMP-2 sensor will be expressed in HEK 293 FT cells by transient
transfection along with the reporter. For initial studies, the MMP-2 protease will be activated
from proenzyme form and incubated with the sensors and reporter activity observed by
33
Dudek PhD proposal
microscopy and flow cytometry. Future goals will be testing of the sensor in vivo and coupling it
to MESA sensing via logical processing; however, for the purposes of this proposal, I will focus
on construction and characterization.
ANTICIPATED OUTCOMES AND ALTERNATIVE DIRECTIONS
I anticipate that this aim will yield a sensitive and specific MMP sensor for applications
in sensing the tumor microenvironment. Moreover, this technology may be extended for the
sensing of other immunologically relevant proteases, such as those mediating allergic asthma.
Thus the diagnostic and therapeutic potential of this device is not limited to cancer.
A different protease, such as MMP-9, could also be used as the protease to be sensed by
this system. Should failure of the sensor to anchor in the membrane, or to do so but at the wrong
orientation, become a problem, an anchor protein that is more stable in the cell membrane could
be susbstituted for the Cd28 transmembrane domain, such as maltose binding protein or a seven
transmembrane receptor. Finally, if tTA loses structural integrity due to membrane crossing or
proteolysis by the MMP, another transcription factor such as PIT may be substituted, or (in the
event that proteolysis is the issue) directed evolution performed to remove the protease substrate
sequence.
RESEARCH TIMELINE
Year One
Year Two
Specific Aim One
Specific Aim Two
Specific Aim Three
34
Year Three
Dudek PhD proposal
REFERENCES
Hanahan, D., & Weinberg, R. A. (2011). Review Hallmarks of Cancer : The Next
Generation. Cell, 144(5), 646-674. Elsevier Inc. doi:10.1016/j.cell.2011.02.013
2. Wei, H., Wang, H., Lu, B., Li, B., Hou, S., Qian, W., Fan, K., et al. (2008). Cancer
immunotherapy using in vitro genetically modified targeted dendritic cells. Cancer research,
68(10), 3854-62. doi:10.1158/0008-5472.CAN-07-6051
3. Xu, Y., Darcy, P. K., & Kershaw, M. H. (2007). Tumor-specific dendritic cells generated by
genetic redirection of Toll-like receptor signaling against the tumor-associated antigen,
erbB2. Cancer gene therapy, 14(9), 773-80. doi:10.1038/sj.cgt.7701073
4. Steinman, R. M., & Banchereau, J. (2007). Taking dendritic cells into medicine. Nature,
449(7161), 419-26. doi:10.1038/nature06175
5. Li, L., Liu, L. N., Feller, S., Allen, C., Shivakumar, R., Fratantoni, J., Wolfraim, L. A., et al.
(2010). Expression of chimeric antigen receptors in natural killer cells with a regulatorycompliant non-viral method. Cancer gene therapy, 17(3), 147-54. Nature Publishing Group.
doi:10.1038/cgt.2009.61
6. Kalos, M., Levine, B. L., Porter, D. L., Katz, S., Grupp, S. a, Bagg, a, & June, C. H. (2011).
T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish
Memory in Patients with Advanced Leukemia. Science Translational Medicine, 3(95),
95ra73-95ra73. doi:10.1126/scitranslmed.3002842
7. Fritz, B. R., Timmerman, L. E., Daringer, N. M., Leonard, J. N., & Jewett, M. C. (2010).
Biology by design: from top to bottom and back. Journal of biomedicine & biotechnology,
2010, 232016. doi:10.1155/2010/232016
8. Dranoff, G. (2004). CYTOKINES IN CANCER PATHOGENESIS AND CANCER
THERAPY. Nature, 4(January). doi:10.1038/nrc1252
9. Jiang, T., Olson, E. S., Nguyen, Q. T., Roy, M., Jennings, P. a, & Tsien, R. Y. (2004). Tumor
imaging by means of proteolytic activation of cell-penetrating peptides. Proceedings of the
National Academy of Sciences of the United States of America, 101(51), 17867-72.
doi:10.1073/pnas.0408191101
10. Kessenbrock, K., Plaks, V., & Werb, Z. (2010). Matrix metalloproteinases: regulators of the
tumor microenvironment. Cell, 141(1), 52-67. doi:10.1016/j.cell.2010.03.015
11. Valastyan, S., & Weinberg, R. a. (2011). Tumor metastasis: molecular insights and evolving
paradigms. Cell, 147(2), 275-92. Elsevier Inc. doi:10.1016/j.cell.2011.09.024
12. Weber W, Lienhart C, Daoud-El Baba M, & Fussenegger M (2009) A Biotin-triggered
Genetic Switch in Mammalian Cells and Mice. Metab Eng.
13. Weber W, Bacchus W, Daoud-El Baba M, & Fussenegger M (2007) Vitamin Hregulated
transgene expression in mammalian cells. Nucleic Acids Res 35(17):e116.
14. Fung E, Wong WW, Suen JK, Bulter T, Lee SG, & Liao JC (2005) A synthetic
genemetabolic oscillator. Nature 435(7038):118-122.
15. Weber, W., Daoud-El Baba, M., & Fussenegger, M. (2007). Synthetic ecosystems based on
airborne inter- and intrakingdom communication. Proceedings of the National Academy of
Sciences of the United States of America, 104(25), 10435-40. doi:10.1073/pnas.0701382104
16. Wang WD, Chen ZT, Kang BG, & Li R (2008) Construction of an artificial intercellular
communication network using the nitric oxide signaling elements in mammalian cells. Exp
Cell Res 314(4):699-706.
1.
35
Dudek PhD proposal
17. Culler SJ, Hoff KG, & Smolke CD (2010) Reprogramming cellular behavior with RNA
controllers responsive to endogenous proteins. Science 330(6008):1251-1255.
18. Wright, C. M., Wright, R. C., Eshleman, J. R., & Ostermeier, M. (2011). A protein
therapeutic modality founded on molecular regulation. Proceedings of the National Academy
of Sciences, 108(39), 16206-16211. doi:10.1073/pnas.1102803108
19. Kramer BP, Fischer M, & Fussenegger M (2005) Semi-synthetic mammalian gene regulatory
networks. Metab Eng 7(4):241-250.
20. Barnea, G., Strapps, W., Herrada, G., Berman, Y., Ong, J., Kloss, B., Axel, R., et al. (2008).
The genetic design of signaling cascades to record receptor activation. Proceedings of the
National Academy of Sciences of the United States of America, 105(1), 64-9.
doi:10.1073/pnas.0710487105
21. Gossen, M., & Bujard, H. (1992). Tight control of gene expression in mammalian cells by
tetracycline-responsive promoters. Proceedings of the National Academy of Sciences of the
United States of America, 89(12), 5547-51.
22. Parks, T. D., Leuther, K. K., Howard, E. D., Johnston, S. a, & Dougherty, W. G. (1994,
February 1). Release of proteins and peptides from fusion proteins using a recombinant plant
virus proteinase. Analytical biochemistry. doi:10.1006/abio.1994.1060
23. Phan, J., Zdanov, A., Evdokimov, A. G., Tropea, J. E., Peters, H. K., Kapust, R. B., Li, M., et
al. (2002). Structural basis for the substrate specificity of tobacco etch virus protease. The
Journal of biological chemistry, 277(52), 50564-72. doi:10.1074/jbc.M207224200
24. Kapust, R. B., Tözsér, J., Copeland, T. D., & Waugh, D. S. (2002, June 28). The P1’
specificity of tobacco etch virus protease. Biochemical and biophysical research
communications. doi:10.1016/S0006-291X(02)00574-0
25. Tözsér, J., Tropea, J. E., Cherry, S., Bagossi, P., Copeland, T. D., Wlodawer, A., & Waugh,
D. S. (2005). Comparison of the substrate specificity of two potyvirus proteases. The FEBS
journal, 272(2), 514-23. doi:10.1111/j.1742-4658.2004.04493.x
26. Leonard, J. N., Ghirlando, R., Askins, J., Bell, J. K., Margulies, D. H., Davies, D. R., &
Segal, D. M. (2008). The TLR3 signaling complex forms by cooperative receptor
dimerization. Proceedings of the National Academy of Sciences of the United States of
America, 105(1), 258-63. doi:10.1073/pnas.0710779105
27. Gay, N. J., Gangloff, M., & Weber, A. N. R. (n.d.). Toll-like receptors as molecular switches.
Nature, 693-698.
28. Kafri, T., Praag, H. V., Gage, F. H., & Verma, I. M. (2000). Lentiviral Vectors : Regulated
Gene Expression AND. Gene Therapy, 1(6), 516-521. doi:10.1006/mthe.2000.0083
29. Fox, J. D., Kapust, R. B., Anderson, D. E., Cherry, S., Copeland, T. D., & Waugh, D. S.
(2001). Tobacco etch virus protease : mechanism of autolysis and rational design of stable
mutants with wild-type catalytic proficiency. Protein Engineering, 14(12), 993-1000.
30. Yu, J., Zhang, L., Hwang, P. M., Rago, C., Kinzler, K. W., & Vogelstein, B. (1999).
Identification and classification of p53-regulated genes. Gene Expression, 96(25).
31. Aboussahoud, Wedad; Aflatoonian, Reza; Bruce, Chris; Elliot, Sarah; Ward, John; Newton,
Sue; Hombach-Klonisch, Sabine; Klonisch, Thomas; Fazeli, A. (n.d.). Expression and
function of Toll-like receptors in human endometrial epithelial cell lines.
32. Ivison, S. M., Khan, M. a S., Graham, N. R., Bernales, C. Q., Kaleem, A., Tirling, C. O.,
Cherkasov, A., et al. (2007). A phosphorylation site in the Toll-like receptor 5 TIR domain is
required for inflammatory signalling in response to flagellin. Biochemical and biophysical
research communications, 352(4), 936-41. doi:10.1016/j.bbrc.2006.11.132
36
Dudek PhD proposal
33. Shinkai, H., Suzuki, R., Akiba, M., Okumura, N., & Uenishi, H. (2011). Porcine Toll-like
receptors: recognition of Salmonella enterica serovar Choleraesuis and influence of
polymorphisms. Molecular immunology, 48(9-10), 1114-20. Elsevier Ltd.
34. Yoon, S.-I., Jones, B. C., Logsdon, N. J., Harris, B. D., Deshpande, A., Radaeva, S.,
Halloran, B. a, et al. (2010). Structure and mechanism of receptor sharing by the IL-10R2
common chain. Structure (London, England : 1993), 18(5), 638-48. Elsevier
35. Moldovan 2002
36. Heckman, K. L., & Pease, L. R. (2007). Gene splicing and mutagenesis by PCR-driven
overlap extension. Nature protocols, 2(4), 924-32. doi:10.1038/nprot.2007.132
37. Scherr, M. et. al. Quantitative Determination of Lentiviral Vector Particle Number by RealTime PCR. BioTechniques 31:520-526. September 2001.
38. Xiao, X., Li, J., Samulski, R. J., Xiao, X., & Li, J. (1998). Production of High-Titer
Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus
Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of
Helper Adenovirus. Microbiology, 72(3).
39. Dong, J. Y., Fan, P. D., & Frizzell, R. a. (1996). Quantitative analysis of the packaging
capacity of recombinant adeno-associated virus. Human gene therapy, 7(17), 2101-12.
doi:10.1089/hum.1996.7.17-2101
40. Wu, Z., Yang, H., & Colosi, P. (2010). Effect of genome size on AAV vector packaging.
Molecular therapy : the journal of the American Society of Gene Therapy, 18(1), 80-6.
Nature Publishing Group. doi:10.1038/mt.2009.255
41. Dueber, J. E., Yeh, B. J., Chak, K., & Lim, W. a. (2003). Reprogramming control of an
allosteric signaling switch through modular recombination. Science (New York, N.Y.),
301(5641), 1904-8. doi:10.1126/science.1085945
42. Haynes, N. M., Trapani, J. a, Teng, M. W. L., Jackson, J. T., Cerruti, L., Jane, S. M.,
Kershaw, M. H., et al. (2002). Single-chain antigen recognition receptors that costimulate
potent rejection of established experimental tumors. Blood, 100(9), 3155-63.
doi:10.1182/blood-2002-04-1041
43. Schultz, Johan; Hoffmuller, Ulrich; Krause, Gerd; Ashurst, Jennifer; Macias, Maria J.;
Schmieder, Peter; Schneider-Mergener, Jens; Oschkinat, H. (1998). Specific interactions
between the syntrophin PDZ domain and the voltage-gated sodium channels. Nature
structural biology.
44. Wu, X., Knudsen, B., Feller, S. M., Zheng, J., Sali, a, Cowburn, D., Hanafusa, H., et al.
(1995). Structural basis for the specific interaction of lysine-containing proline-rich peptides
with the N-terminal SH3 domain of c-Crk. Structure (London, England : 1993), 3(2), 215-26.
45. Flinterman, M., Farzaneh, F., Habib, N., Malik, F., Gäken, J., & Tavassoli, M. (2009).
Delivery of therapeutic proteins as secretable TAT fusion products. Molecular therapy : the
journal of the American Society of Gene Therapy, 17(2), 334-42. doi:10.1038/mt.2008.256
46. Deuschle, U., Meyer, W. K., & Thiesen, R. (1995). Tetracycline-reversible silencing of
eukaryotic promoters . These include : Tetracycline-Reversible Silencing of Eukaryotic
Promoters. Microbiology, 15(4).
47. Rupp, P.A., Visconti, R.P., Czirok, A., Cheresh, D.A., and Little, C.D. (2008). Matrix
metalloproteinase 2-integrin alpha(v)beta3 binding is required for mes- enchymal cell
invasive activity but not epithelial locomotion: a computational time-lapse study. Mol. Biol.
Cell 19, 5529–5540.
37
Dudek PhD proposal
48. Mu, D., Cambier, S., Fjellbirkeland, L., Baron, J.L., Munger, J.S., Kawakatsu, H., Sheppard,
D., Broaddus, V.C., and Nishimura, S.L. (2002). The integrin alpha(v)beta8 mediates
epithelial homeostasis through MT1-MMP-dependent activation of TGF-beta1. J. Cell Biol.
157, 493–507.
49. Littlepage, L.E., Sternlicht, M.D., Rougier, N., Phillips, J., Gallo, E., Yu, Y., Wil- liams, K.,
Brenot, A., Gordon, J.I., and Werb, Z. (2010). Matrix metallopro- teinases contribute distinct
roles in neuroendocrine prostate carcinogenesis, metastasis, and angiogenesis progression.
Cancer Res. 70, 2224–2234.
50. Kaplan, R.N., Riba, R.D., Zacharoulis, S., Bramley, A.H., Vincent, L., Costa, C.,
MacDonald, D.D., Jin, D.K., Shido, K., Kerns, S.A., et al. (2005). VEGFR1- positive
haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438, 820–
827.
38