University of Southern California Dissertations and Theses

Targeting LFA-1/ICAM-1 interaction using ICAM-1 binding elastin-like polypeptide

(USC Thesis Other)

Targeting LFA-1/ICAM-1 interaction using ICAM-1 binding elastin-like polypeptide

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content
Targeting LFA-1/ICAM-1 interaction using ICAM-1 binding elastin-like
polypeptide

By

Adrianna Giselle Vega

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

May 2020

Copyright 2020 Adrianna Giselle Vega

ii
Dedication

I want to dedicate this thesis to my family and friends. I would like to thank my siblings Alexis,
Xavier and Gabriela for being my support system and encouragement throughout my academic
career. I share my successes with them as they are an intrinsic part of the motivation that drives
me. I also want to thank my cousin Liz who has helped both personally and educationally with
her advice and invaluable contributions. Additionally, I would have not gotten this far without
the love and support of my mother. She would give me the shirt off of her back if it, in any way,
resulted in my success. I am indebted to her and the rest of my family for always pushing me
past my own limitations and reminding me that my dreams are achievable. I also dedicate this
thesis to my dad, who led by example and showed me that no obstacle is insurmountable. He
emphasized the importance of education and hard work. Gracias por sus consejos y amor. Sin
usted, nada de esto sería posible. Estar sin usted ha sido lo más difícil que yo he vivido, pero sé
que me está apoyando desde el cielo. Lo logramos abuelito.

iii

Acknowledgements

First and foremost, I would like to thank Dr. Sarah Hamm-Alvarez for her guidance and support
throughout my time in her lab. She has created a collaborative style environment which has been
essential to my success. She has allowed me to benefit from her knowledge and wisdom in
preparation of my thesis. I also want to thank Dr. MacKay and Dr. Okamoto, who have taken the
time to join my committee and have contributed greatly to this process. I would also like to thank
Dr. Maria Edman and Srikanth Janga for their direction and willingness to help. They, along with
my lab members Christina, Hao, Rim, Min, Yanni and Shruti always lent a helpful hand and
answered any of my many questions. Last, I want to especially thank Yaping, who has mentored
me throughout my program. She has assisted me every step of the way and has gone above and
beyond to ensure my success. I am profoundly grateful and appreciative of everyone who
supported me in this journey.

iv
Table of Contents
Dedication ....................................................................................................................................... ii
Acknowledgements ........................................................................................................................ iii
List of Figures ................................................................................................................................. v
Abbreviations ................................................................................................................................. vi
Abstract ......................................................................................................................................... vii
1. Introduction ................................................................................................................................. 1

2. Materials and Methods ................................................................................................................ 9
2.1. Materials ............................................................................................................................... 9
2.2. Animals ................................................................................................................................ 9
2.3. ELP Purification ................................................................................................................. 10
2.4. Cells .................................................................................................................................... 11
2.5. Splenocyte isolation and labeling ....................................................................................... 11
2.6. In vitro static adhesion ....................................................................................................... 12
2.7. CD4+ T cell isolation ......................................................................................................... 14
2.8. Mixed lymphocyte reaction (MLR) ................................................................................... 14
2.9. IL-2 ELISA ........................................................................................................................ 16
2.10. Statistics ........................................................................................................................... 17

3. Results ....................................................................................................................................... 18
3.1. IBP-SI inhibits splenocyte adherence ................................................................................ 18
3.2. Temperature dependent inhibition ..................................................................................... 21
3.3. IBP-SI blocks LFA-1/ICAM-1 costimulatory activation ................................................... 22

4. Discussion ................................................................................................................................. 25

Summary ....................................................................................................................................... 30

References ..................................................................................................................................... 31

v

List of Figures
Figure 1. Schematic of Sjögren’s syndrome pathology
Figure 2. Schematic of LFA-1 Structure
Figure 3. LFA-1 can be chemically induced by phorbol-12-myristate-13-acetate
Figure 4. IBP-SI shows dose response inhibition compared to controls
Figure 5. IBP-SI lowers splenocyte adhesion
Figure 6. IBP-SI nanoparticles at 37° C inhibit splenocyte adherence better than soluble IBP-SI
at 18° C.
Figure 7. IBP-SI reduces proliferation and IL-2 production by blocking costimulatory processes

vi
Abbreviations

APC Antigen presenting cell
CFSE 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester
DED Dry eye disease
IBP-SI ICAM-1 Binding Peptide-S48I48
ICAM-1 Intercellular adhesion molecule-1
LFA-1 Lymphocyte function-associated antigen 1
MCC Moth cytochrome c
MHC Major histocompatibility complex
MLR Mixed lymphocyte reaction
PBMC Peripheral blood mononuclear cell
PMA Phorbol-12-myristate-13-acetate
SI S48I48
SS Sjögren’s syndrome
TCR T cell receptor

vii
Abstract

Autoimmune diseases are an increasing health care concern. Patients are often prescribed
immunosuppressants or steroids that can subdue symptoms, but not cure the disease.
Immunosuppressants have shown to be effective for suppressing an overactive immune system,
however, increase the chance of infection, cause nausea and overall tiredness or weakness.
Treatment for a common autoimmune disease Sjogren’s syndrome includes eye drops along with
immunosuppressants. Therefore, advancement in treatment that can target the underlying causes
that lead to the burdensome symptoms while being specific and safe are needed. Sjogren’s
syndrome is an autoimmune disorder classified by dry eye and mouth, resulting from lacrimal
and salivary gland inflammation and subsequent deterioration by infiltrating lymphocytes.
Recent therapies have targeted lymphocyte function-associated antigen-1 (LFA-1) binding to
intercellular adhesion molecule-1 (ICAM-1) due to their roles in the migration of leukocytes to
inflamed tissues as well as immune cell costimulatory processes. Using an elastin-like
polypeptide S48I48 fused to a mICAM-1 binding peptide, this thesis shows that protein-based
nanoparticles can mitigate LFA-1/ICAM-1 binding. This biodegradable and low immunogenic
nanoparticle reduced splenocyte adhesion in vitro to ICAM-1 on bEnd.3 monolayer in a dose-
dependent fashion with an IC50 of 81.2 µM and 62.7 µM, on two different assays. IBP-SI was
also able to reduce proliferation of CD4+ T cells to 26.1% and lower IL-2 production by 72.9%
by interfering with APC and T cell co-stimulation in a mixed cell assay. Therefore IBP-SI has
potential in vivo application to deplete lymphocyte migration and proliferation, thereby reducing
inflammation.

1
1. Introduction

Autoimmune diseases are characterized by abnormalities of the immune system, resulting in an
inappropriate response to self-antigens, increased inflammation and local to systemic
consequences, differing in severity (Shi et al., 2013). Living with an autoimmune disease causes
high patient morbidity and increased health-care costs due to life-long treatment. Autoimmune
diseases are intensely studied yet remain highly misunderstood (Anaya et al., 2016). Sjögren’s
syndrome falls into that category, difficult to diagnose and even more so to treat. SS is the third
most common autoimmune disease, affecting an estimated 4 million people worldwide. Women
predominate in diagnosis, outnumbering men nine to one (Fox, 2005). Similar to other
autoimmune diseases, the current treatment for Sjögren’s syndrome includes
immunosuppression, leaving patients dangerously susceptible to disease. Sjögren’s syndrome
(SS) is recognized as keratoconjunctivitis sicca, dryness of the conjunctiva and cornea, and
xerostomia, dryness of the mouth (Fox et al.,2019). The disorder is classified into two parts,
primary and secondary. Both classifications include the lacrimal and salivary gland lymphocytic
infiltration. However, secondary Sjögren’s syndrome includes the concurrent diagnosis of
another autoimmune disease such as rheumatoid arthritis (Vitali et al., 2002). Currently, the gold
standard criteria for Sjögren’s syndrome requires at least two out of the following three: positive
serum anti-SSA and/or anti-SSB; Ocular staining score > 3; or focus score > 1 focus/4 mm
2
from
labial salivary gland biopsy exhibiting focal lymphocytic sialadenitis (García-Carrasco et al.,
2012).

2

Figure 1. Schematic of Sjögren’s syndrome pathology
Disruption to resting epithelium by viral, genetic or hormonal causes initiate disease. Cycle of
disease is shown as both humoral and cellular (Voulgarelis and Tzioufas, 2010).

Sjögren’s syndrome is an idiopathic disease, making the pathophysiology difficult to elucidate.
Viral, hormonal and environmental triggers are the leading explanations for disease onset
(Rosenblum et al., 2015). Whatever the cause may be, the effect is a dysregulation in the
glandular epithelium. The epithelial layer is important in normal immune responses during
lymphocyte homing and migration. After the spontaneous disruption, an increase in chemokines
and adhesion molecules on the epithelial surface is observed, such as intercellular adhesion
molecule-1 (ICAM-1) (Gao et al, 2003). Thereafter, there is an increase in recruitment of
lymphocytes to the lacrimal and salivary glands from peripheral circulation. Dendritic cells and
CD4+ T cells comprise the majority of leukocytes in the glands in the earlier stage of SS.
Proinflammatory cytokines such as IL-1bg, IFN-g and TNF-a further activate T cells and
epithelial cells in the microenvironment, creating a vicious cycle of disease (Figure 1). An
increase in B-cell activating factor (BAFF) production by increased stimulatory IFN-a, produced

3
by activated dendritic cells and T cells, correlates to an increase in B cells in the glands.
Meanwhile, through apoptosis, presentation of autoantigens by epithelial cells induce the
production of autoantibodies anti-Ro and anti-La by local B cells (García-Carrasco et al., 2012).
The role in presentation of self-antigens, activation of B cells by local secretion of BAFF and
increase of adhesion molecules further solidifies the active participation of epithelial cells in
disease perpetuation. Minimizing lymphocytic infiltration by targeting the upregulated adhesion
molecule ICAM-1 could potentially halt the cascade of events that follow. Furthermore,
disrupting local T cell activation and proliferation could potentially minimize proinflammatory
cytokines which are important for the continuation of disease.

Intercellular adhesion molecule-1 (ICAM-1; CD54) is present on endothelial cells and is critical
for transmigration of leukocytes from blood vessels into affected tissues. ICAM-1 is heavily
upregulated in the presence of TNF-a near sites of inflammation (Wee et al., 2009). In its
adhesion pathway, one major ligand of ICAM-1 is b2 integrin leukocyte function-associated
antigen-1 (LFA-1 or aLb2 or CD11a-CD18) (Long, 2011). In its inactive form, LFA-1 is kept in
a low affinity bent position with the aI domain unable to bind to ICAM-1. Proper activation of
LFA-1 is required for tight binding to ICAM-1. Presence of P-selectins and E-selectins on
endothelium arrest leukocytes and initiate low-affinity binding to ligands on cell surface such as
PSGL-1 (P-selectin glycoprotein ligand-1) (Tinoco et al., 2016). Inside-out signaling, either by
PSGL-1 or chemokine receptor engagement, causes LFA-1 extension and activation by talin-1 or
both talin-1 and Kindlin-3, respectively (Lefort and Ley, 2012). The conformational change of
LFA-1 can increase affinity to ICAM-1 nearly 10,000-fold, which results in tighter binding.

4

Figure 2. Schematic of LFA-1 Structure
In a low affinity bent position, the I-domain is tucked toward the membrane and inaccessible to
binding. Upon conformational changes, LFA-1 increases its affinity for its receptor by making
the I-domain available for binding (Plugfelder et al., 2017).

This receptor-ligand pair is not only necessary for diapedesis, but also co-stimulatory processes
required for T cell activation. Antigen presentation to TCR by MHC complexes also results in
inside out signaling, in which the salt bridge containing an a and b tail of LFA-1 is disrupted
causing the ligand to undergo a conformational change. This makes the aI and metal ion-
dependent adhesion site (MIDAS) accessible to ICAM-1 (Walling and Kim, 2018). Downstream
signals by antigen presentation activate phospholipase C and calcium signaling, leading to the
activation of GTPase RAP1. Thus, the recruitment of protein talin-1 to the b2 cytoplasmic tail,
followed by actin binding to tail, results in the structural shift. Alternatively, LFA-1 ligand is
able to become active in the presence of phorbol esters and divalent cations (Schwartz, 2002;
Woska et al., 2001). An upregulation of LFA-1 on the cell surface is not observed but rather its
conformational change from inactive to active high affinity form.

5
Antigen presenting cells (APC) such as dendritic cells, B cells and macrophages, interact with T
cells connecting innate and adaptive immunity. APC forms an antigen/MHC complex that is then
presented to the T cell receptor on the T cell surface. Typically, MHC I class presents
endogenous antigens to CD8+ cytotoxic cells, while MHC II presents foreign entities to CD4+
TH cells (Gaudino and Kumar, 2019). Both TCR binding and co-stimulatory binding has been
shown to regulate TH cell differentiation. TH2 cells primarily secrete IL-4, IL-5 and IL-10 while
TH1 cells secrete proinflammatory cytokines IL-2, IFN-g and lymphotoxin (Salomon and
Bluestone, 1998). Thus, the first signal T cells encounter is antigen specific, but complete
activation is dependent on a two-signal model. The second signal, or co-stimulatory signal, is
required for full T cell activation, proliferation, differentiation and cytokine release. Failure to
provide this co-stimulatory signal will result in cell anergy and subsequently, cell death. For
proper activation, the TCR-CD3 complex must couple and cluster in order to send sufficient
inside out signaling to costimulatory ligands (Minguet et al., 2007). Upon activation, co-
stimulatory binding occurs between B7 and CD28, ICAM-1 and LFA-1 and CD40 and CD40L
(Gaudino and Kumar, 2019). Full T cell activation is observed upon the binding of the co-
stimulatory triad, whereas significant decline in activation is shown by limiting binding to just
one or two co-stimulatory pairs (Hodge et al., 2006). LFA-1 has been reported to induce IL-2
production, which in turn enhances T-cell proliferation (Manikwar, 2012). LFA-1/ICAM-1
interaction has been highly implicated in adhesion as well as activation, making it a target of
interest in autoimmune diseases.

LFA-1/ICAM-1 has been highly targeted for treating inflammation. Antibodies have been an
important tool in understanding the properties of each conformation of the LFA-1 ligand as well

6
as the binding domain. For instance, researchers found blocking the I domain of LFA-1
interfered with cell-surface adhesion between LFA-1 and ICAM-1. The effect of anti-LFA-1 on
alternative sites resulted in minimal blocking (Stanley and Hogg, 1997). Additionally, teams
have developed antibodies specific for low affinity and high affinity LFA-1 states and
demonstrated the lack of binding in the bent conformation (Wang et al., 2009). Therapies for
autoimmune diseases such as rheumatoid arthritis and psoriasis have shown promise using anti-
ICAM-1 and anti-LFA-1 antibodies (Kavanaugh et al., 1996). Similarly, the administration of
these antibodies has been effective in reducing allograft rejection (Isobe et al., 1992). Though the
use of antibodies targeting different variations of LFA-1/ICAM-1 interaction is highly specific,
their use as therapeutic agents poses numerous challenges. Antibodies are expensive to produce
and can elicit immunogenicity. Efforts to develop small molecules to inhibit binding of ICAM-1
and LFA-1 have also occurred. For instance, lovastatin has been observed to bind to the MIDAS
motif of the aL I-domain of LFA-1 after undergoing hydrolysis of its lactone ring (Kallen et al.,
1999). Use of lovastatin in mice has been successful in reducing the lymphocytic infiltration into
the retina when given parenterally. Lowered concentrations of IFN-ã and IL-10, correlating to
altered TH1 and TH2 profiles was also observed (Gegg et al., 2005). Nevertheless, lovastatin as
other statins come with a long list of systematic side effects such, including headaches, muscle
pain, nausea, abdominal pain and more. The front runner for optical treatment of Sjögren’s
syndrome and DED is lifitegrast, which is described as a potent tetrahydroisoquinoline derived
LFA-1/ICAM-1 inhibitor (Zhong et al., 2011). An FDA approved ophthalmic solution of 5% had
shown strong reduction of Jurkat T-cells attachment to ICAM-1 in vitro, with an IC50 below 3
nM. A statistically significant concentration dependent inhibition of proinflammatory cytokines
from TH1 and TH2 cells from human PBMC was also observed (Murphy et al., 2011). However,

7
side effects such as blurred vision, irritation and change in taste have been commonly observed.
It is reasonable that a new treatment with specific targeting and minimal side effects could be
developed.

Biopolymers have long been a topic of interest in regard to drug delivery. More specifically,
elastin-like polypeptides (ELP) have emerged in the realms of customizable drug delivery
vehicles, specific targeting and increased drug loading. ELPs are repetitive polypeptides
mimicking that of endogenous elastin. They contain a pentapeptide repeat of (Val-Pro-Gly-X-
Gly)n, where “X” is a guest residue that can be any amino acid of choice and n is also variable
(Hassouneh et al.,2012). Targeting proteins or carriers can be linked to terminal ends of ELPs
without relying on bioconjugate chemistry. These biopolymers exhibit a unique thermo-
responsive reversible self-association at temperatures dependent on the chemical and physical
properties of guest residues and molecular weight. Additionally, they are biocompatible,
biodegradable and have negligible immunogenicity. ELPs are practical on the manufacturing
front because they are easily purifiable. Due to their temperature sensitive phase shifts, they are
soluble below transition temperature and come out of solution above the transition temperature,
simplifying purification based on heating and cooling (Baniuk et al., 2018). They are also rather
inexpensive compared to antibody production.

Using this technology, ELP (Val-Pro-Gly-Ser-Gly)48(Val-Pro-Gly-Ile-Gly)48 or simply S48I48
(SI) had been constructed, showing transition temperatures around 27°C (T1) and 75°C (T2)
(Shah et al., 2012). Together with a novel murine ICAM-1 binding peptide (IBP) incorporated at
the N terminus (Bélizaire et al., 2003), IBP-SI can theoretically phase shift enclosing the more

8
hydrophobic isoleucine end inward, displaying the targeting peptide on the outer layer. IBP-SI
was developed as an ICAM-1 targeted nanoparticle (Hsueh, 2017). While this construct was
characterized from a biophysical standpoint, and it was shown to undergo ICAM-1 mediated
endocytosis, the goal of my thesis was to test whether this ICAM-1 targeted nanoparticle had the
potential to block the function of ICAM-1. Specifically, I determined whether this nanoparticle
could prevent leukocyte adhesion to an endothelial cell monolayer, reflective of successful
ICAM-1/LFA-1 binding. I further tested whether it was capable of preventing co-stimulation,
thereby decreasing lymphocyte proliferation and cytokine concentration in a mixed cell assay.

Deleted: -

9
2. Materials and Methods

2.1. Materials
bEnd.3 cells and Dulbecco’s Modified Eagle’s Medium for cell culture was from ATCC
(Manassas, VA). CH27 B cells were a kind gift from Dr. Xie (University of Southern
California). RPMI 1640, penicillin/streptomycin, 0.25% (w/v) Trypsin-0.03% (w/v) EDTA and
L-Glutamine were from Cellgro (Manassas, VA). MojoSortÔ Mouse CD4 T cell negative
isolation kit, 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester kit (CFSE), Alexa Fluor
700 anti- mouse CD3 and ELISA MAXÔ Deluxe Set Mouse IL-2 kit was from Biolegend (San
Diego, CA). Recombinant mouse TNF alpha protein (ab9740) was from Abcam (Cambridge,
UK). Moth cytochrome c (88-103) was from GenScript (Piscataway, NJ). TBÒ powder growth
media was from MO BIO Laboratories (Carlsbad, CA). AcrodiscÒ Units with MustangÒ E
Membrane filters were from Pall (New York, NY). Clear 24-well flat bottom plates, black 96-
well flat bottom plates and clear 96-well round bottom plates were from Corning Inc. (Corning,
NY). 70 µm filter was from Corning Inc. (Corning, NY).

2.2. Animals
C57BL/6 breeders were purchased from Jackson laboratories. Animals were bred at the
University of Southern California (USC) vivarium. Female mice older than 10 weeks were used
in this study. The spleens were removed after intraperitoneal injection of anesthesia (55 mg
ketamine and 14 mg xylazine per kilogram of body weight) and cervical dislocation.

10

2.3. ELP Purification
DNA from previously developed IBP-SI and SI was transfected into Shuttle T7 Escherichia coli.
E. coli was spread on agar plate and incubated upside down at 37°C overnight. The following
day, one colony was added to a 250 mL flask containing 50 mL autoclaved growth media with
0.1 mg/mL ampicillin. The flasks were shaken at 30 The flask was put in shaking incubator at
250 rpm, 30°C overnight. The following day, the 50 mL culture was split into two 1L autoclaved
growth media, also containing antibiotics. Both flasks were shaken at 250 rpm at 30°C. Cultures
were shaken until OD600 was between 0.4-0.8, thereafter 400 µL of 200 mM IPTG was added.
Flasks were shaken overnight at 25°C, 250 rpm. The following day, cultures were spun down at
4000g for 20 min. The supernatant was discarded, and the pellet was resuspended in 1XPBS.
Then, the resuspended media was sonicated for 10 min at amplification 10 with 20 sec on and 10
sec off intervals. Nucleic acid debris was precipitated out by 0.05% PEG. Again, the culture was
spun down, collecting the supernatant. The solution was placed in water bath of 37°C and a
maximum of 5M NaCl was added. Following, several rounds of centrifugation at 37°C,
collecting and resuspending pellet in 1XPBS, and centrifugation at 4°C, collecting supernatant,
was performed until no visible debris pellet formed. Protein concentrations were determined by
measuring 280 nm absorbance and calculated using Beer-Lambert’s law with extinction
coefficient of 1285 M
-1
cm
-1
. ELP samples were filtered with AcrodiscÒ Units with MustangÒ E
Membrane (Pall).

11

2.4. Cells
bEnd.3 cells were cultured in Dulbecco’s Modified Eagle’s Medium, 10% fetal bovine serum,
1X penicillin/streptomycin and 2 mM L-Glutamine in 37°C 5% CO2 incubator. The media was
replenished every three days. Removal of cells for plating was done by discarding media and
adding 2 mL of 0.25% (w/v) Trypsin-0.03% (w/v) EDTA for 5 minutes. Fresh culture media was
added to the cells and gently aspirated. Cells were spun down at 437 g for 5 minutes and
resuspended in fresh culture media. bEnd.3 cells were aliquoted into appropriate plate and left to
adhere overnight for in vitro static adhesion. The following day, media is replaced with fresh
media containing TNF-a at 10 ng/mL and left to incubate overnight to overexpress ICAM-1
(Anderson and Siahaan, 2003).

CH27 cells were kept in sterile filtered RPMI 1640 supplemented with 2 mM L-Glutamine, 1X
penicillin/streptomycin and 10% FBS in 37°C 5% CO2 incubator. The media was changed every
two days by spinning down cell suspension at 70 g for 5 minutes and resuspending in fresh
media.

2.5. Splenocyte isolation and labeling
Spleens were removed from female C57BL/6 mice. Spleens were crushed through a 70 µm filter
and washed with red blood cell lysing buffer (0.15 M NH4Cl, 1 mM NaHCO3, 0.1 mM EDTA)
until homogenous solution. Cells were spun down at 436 g for 10 minutes and resuspended in 1X

12
PBS. Cells were then counted via TC20 automated cell counter (Bio-Rad). Splenocytes were
then stained with 2 µM CFSE and left to incubate at 37°C 5% CO2 for 20 minutes, away from
light. The remaining free label in solution was quenched with 5 mL of bEnd.3 media containing
10% FBS and then spun down at 436 g for 5 minutes. The CFSE stained splenocytes were then
activated with 10 ng/mL PMA in pre-warmed culture media to induce LFA-1 activation (Strazza
et al., 2014).

2.6. In vitro static adhesion
IBP-SI at varying concentrations (10 µM-170 µM) and SI (10 µM-170 µM) was diluted in
adhesion solution (PBS with 0.5% BSA, 2 mM MgCl2, 1 mM CaCl2) and incubated with 10
6

CFSE-labeled, PMA-induced splenocytes. Both were co-incubated on ICAM-1 overexpressing
bEnd.3 cell monolayer on clear 24-well tissue culture plated, with a confluency above 80%, at
37°C 5% CO2 for 45 minutes. Nonadherent cells were removed by gentle aspiration, followed by
adding 250 µL of wash solution (1X PBS, 2 mM MgCl2, 1 mM CaCl2). Washes were repeated
twice. Adherent cells were trypsizined and spun down by centrifugation. The cell pellet was then
resuspended in FACS buffer (1X PBS, 0.5% BSA) and read on FITC channel on LSR2 flow
cytometer. Analysis was performed on FlowJo software v10.6.2. Each treatment was done in
triplicate wells, thrice. Data is shown as percentage of fluorescence intensity per log
concentration relative to non-ELP treated wells, defined as 100%, as shown as follows:
% normalized = (treated)/ (non-ELP treated) *100% (Eq.1)

For a temperature sensitive analysis, bEnd.3 cells in culture media were incubated at various
temperatures (4°C, 15°C, 18°C and 20°C) for 2 hours in 75 cm
3
flasks. After incubation, cells

13
were observed under microscope and cell viability was assessed. Aliquots of media supernatant
were collected and analyzed using automated cell counter. In vitro static adhesion was also
performed for IBP-SI (60 µM) at 18°C 5% CO2 and IBP-SI (60 µM) at 37°C 5% CO2 in clear
24-well tissue culture plates coated with ICAM-1 induced bEnd.3 cells with same procedure as
previously mentioned above. Splenocyte adherence was analyzed using similar methods.
Temperature threshold for chosen ELP concentration was determined by equation:
Tt = m*log [CELP] + b (Eq. 2)
m = slope
[CELP] = ELP concentration
b = transition temperature at 1 µM

Similarly, black 96-well tissue culture flat bottom plates were also pre-cultured with TNF-a
stimulated bEnd.3 cells. A clear 96-well flat bottom plate was also seeded in conjunction to
ensure confluency above 80%. IBP-SI, SI (10 µM-170 µM) and Lifitegrast (0.00001-1 µM) were
individually co-incubated with 10
5
CFSE-labeled, PMA-induced splenocytes in adhesion
solution on mICAM-1 overexpressing bEnd.3 monolayer for 45 minutes at 37°C 5% CO2. Non-
adherent cells were gently aspirated, and each well was washed with 50 µL of wash solution,
twice. Following, 100 µL of wash solution was added to each well. All treatments were
performed in triplicates, on three separate occasions and fluorescence was measured with
excitation at 485 nm and emission at 535 nm using a plate reader SpectraMax iD3 (Molecular
Devices) and Softmax Pro (Molecular Devices). Data is shown as percentage of fluorescence
intensity per concentration relative to non-treatment control of PMA-induced CFSE-labelled

14
splenocytes on ICAM-1 upregulated bEnd.3 monolayer, defined as 100%, following the same
equation as previously mentioned (Eq.1).

2.7. CD4+ T cell isolation
Spleen from C57BL/6 female mice was removed, passed through 70 µm filter and washed using
pre-warmed 1XPBS. Homogenous cell solution was spun down at 436 g for 10 min and
resuspended with 1 mL of 1X PBS. Cells were then counted using TC20 automated cell counter.
Biotin-antibody cocktail (anti- CD8a, CD11b, CD11c, CD19, CD24, CD45R/B220, CD105, I-
A/I-E (MHC II), TER-119/Erythroid, TCR-gd) at 10 µL per 10
7
cells in 100 µL of buffer was
added to homogenous cell suspension and incubated on ice for 15 minutes. Following, magnetic
streptavidin nanobeads was added at 10
7
cells in 100 µL of buffer to the cell solution containing
anti-body cocktail followed by another 15-minute incubation on ice. The cell solution was then
left to rest for 5 minutes in magnetic stand (eBioscience) at room temperature. The unlabeled cell
solution was dispensed and counted on automated cell counter. Cells were then labeled with
CFSE, as previously described.

2.8. Mixed lymphocyte reaction (MLR)
CH27 cells were counted using TC20 automated cell counter and pelleted down at 70 x g for 10
minutes. They were then resuspended in CH27 media containing MCC peptide (15 µM) and
incubated for 30 minutes at 37°C 5% CO2. CFSE labeled CD4 T cells in 1XPBS, as previously
mentioned, were counted and aliquoted into 1.7 mL tubes at 10
5
cells per treatment and pelleted

15
down. The CD4 T cell pellets were then resuspended in dilutions of IBP-SI (200 µM) and SI
(200 µM) in CH27 cell media. In a clear 96-well round bottom tissue culture plate, 50 µL of
CH27 cells containing MCC and 50 µL of ELP containing CD4 T cell solution were co-
incubated at 37°C 5% CO2 for 5 days. Non-ELP treated CD4 T cells were also incubated 1:1 to
CH27 B cells with MCC at 37°C 5% CO2 for 5 days. A final concentration of 10
5
CH27 B cells
and 10
5
CD4 T cells were used. ELP concentrations above that of IC50, IBP-SI (100 µM) and SI
(100 µM), were also chosen. Each treatment was done in triplicate wells. After 5 days, each well
was collected into separate 1.7 mL tubes, counted and spun down at 5000 rpm. The supernatant
from each well was collected and stored at -80°C for further investigation. Each pellet was
reconstituted by Alexa Fluor 700 anti-mouse CD3 anti-body at 0.5 µg per million cells diluted in
1XPBS. After 30 minutes incubation on ice, cells were pelleted down at 20°C 5000 rpm for 8
minutes and resuspended in FACS buffer. Samples were read on both channels FITC, for CFSE
labelled cells, and APC-Cy7, for Alexa Fluor 700 anti-mouse CD3 labelled cells, on LSR2 flow
cytometer, while analysis was done on FlowJo software v10.6.2 using biology proliferation
settings. Proliferation is defined as the change from non-proliferated cells (generation 0) by day
0 analysis to observed proliferation of day 5 analysis. Data is shown as percentage proliferation
for each treatment normalized to total proliferation of non-ELP treatment control, defined as
100%, as shown below:
% proliferation (normalized) = (% proliferation from treated/ % proliferation from untreated)
*100% (Eq. 3)

16
2.9. IL-2 ELISA
Supernatant collected from each sample in mixed lymphocytic reaction was analyzed by ELISA
for IL-2 protein concentration. Triplicate wells from MLR were pooled to one sample. On Day 1,
100 µL of 1X Mouse IL-2 ELISA MAXÔ Capture Antibody solution (Biolegend) was added to
each treatment well on clear 96-well plate. The plate was sealed and left overnight at 4°C. The
following day, each well was washed with 300 µL of wash solution (1X PBS, 0.05% Tween-20)
four times, followed by 200 µL blocking agent (1X Assay Diluent A; Biolegend). The plate was
incubated at room temperature for 1 hour while shaking. Each well was washed with 300 µL
washing solution four times, followed by adding 100 µL of 1:300 diluted unknown samples
(non-ELP treated control, IBP-SI treated, and SI treated supernatant from mixed lymphocyte
reaction) and 100 µL of mouse IL-2 standard (125 pg/mL-1.95 pg/mL) with 2 hour incubation at
room temperature on shaker. After incubation, wells were washed four times again and 100 µL
of Detection Antibody solution (BioLegend) was added with a one hour incubation on shaker.
The wells were washed again four times and 100 µL of Avidin-HRP solution (Biolegend) was
added with 30 minute incubation period on shaker. Next, the wells were washed five times,
soaking for 1 minute per wash, and 100 µL of TMB Substrate (Biolegend) was added. The plate
was incubated in the dark for 30 minutes without shaking. Stop solution (Biolegend) was added
to each well followed by immediate analysis on plate reader. Absorbance was measured as 570
nm subtracted from 450 nm using SpectraMax iD3 (Molecular Devices) and Softmax Pro
(Molecular Devices). IL-2 standards and absorbance values were log transformed to make
standard curve. IL-2 concentrations from experimental samples were calculated using an
equation derived from the standard curve. Data was compared to non-treatment control
concentration in pg/mL.
Commented [1]: t
Commented [AV2R1]:
Commented [AV3R1]:
Deleted: normalized to percent IL-2 concentration relative to
non-ELP treated control, defined as 100%.

17
2.10. Statistics
All experiments were performed a minimum three times. Data is shown as mean ± SD. Means of
two groups was compared via unpaired two-tailed student’s t-test and statistically significant p
value was defined as 0.05 and below. Means of three groups were compared by one-way
ANOVA and statistically significant below p value 0.05.

Deleted: each

18
3. Results

3.1. IBP-SI inhibits splenocyte adherence
An important function in immune response is the migration of lymphocytes to the site of injury.
LFA-1/ICAM-1 interaction is largely known to aid in the movement of leukocytes across the
endothelium. In order to assess the potential of IBP-SI in disrupting said interaction between the
two receptors, an in vitro model using bEnd.3 cells grown in a monolayer and stimulated by
TNF-a to induce ICAM-1 expression (Bélizaire et al.2003) and CFSE-labelled splenocytes was
used. The I-domain of LFA-1 remains tucked in toward the cell membrane, out of reach for
receptor binding. In order to overcome this and activate the receptor, isolated splenocytes were
treated with phorbol-12-myristate-13-acetate to ensure activation and reduce variability of
binding efficiency. Splenocytes served as a source of LFA-1, which was chemically activated
using PMA and resulted in higher potential splenocyte adhesion (Figure 3) (p=0.009).

Figure 3. LFA-1 can be chemically induced by phorbol-12-myristate-13-acetate
Splenocytes labelled with CFSE were treated with and without PMA prior to incubation on TNF-
a induced bEnd.3 cell monolayer. After 45 min incubation at 37°C 5% CO2, fluorescence from
Deleted: ICAM-1 overexpressing
Deleted:
Deleted: s
Deleted: ,
Commented [4]: reference
Deleted: ,

19
adherent splenocytes was measured on plate reader with 485 nm excitation and 535 nm emission.
Data is reported as mean±SD. **p<0.01

IBP-SI showed a dose response inhibition of splenocyte adherence with an IC50 of 62.7 ± 4.8 µM
(Figure 4A) using flow cytometry assay. Similarly, IBP-SI also showed a dose response
inhibition on the plate reader assay with an IC50 of 81.2 ± 1.7 µM (Figure 4B). SI without the
mICAM-1 binding protein showed no dose response inhibition via plate reader assay (Figure
4C). Similarly, a commercially available LFA-1 antagonist, Lifitegrast, showed no dose response
(Figure 4D). For further investigations, a concentration slightly above the IC50 of 100 µM IBP-
SI was chosen.

Deleted: ì
Deleted: ìM
Deleted: ìM

20
Figure 4. IBP-SI shows dose response inhibition compared to controls
A) An IC50 for IBP-SI for splenocyte adherence was determined by incubating varying
concentrations of IBP-SI (10-170 µM ) with CFSE-labelled PMA-induced splenocytes on
ICAM-1 upregulated bEnd.3 monolayer with confluency above 80%. Post incubation, cells were
trypsinized and read on the FITC channel of flow cytometer. B) IC50 of IBP-SI was determined
by plate reader assay. After incubation, non-adherent cells were washed away. Fluorescence
from adherent cells were read at 485 nm excitation and 535 nm emission. Both SI (C) and
Lifitegrast (D) at varying concentrations were analyzed using plate reader assay in similar
fashion to IBP-SI (B). Samples were normalized to non-ELP treated control, defined as 100%.
Each data point is reported as mean±SD.

Using the same in vitro adhesion model, IBP-SI in both flow cytometer and plate reader assay
showed a reduction in splenocyte adherence compared to non-specific SI control. IBP-SI
demonstrated an inhibition to an average of 47.1 ± 3.4% (p=0.017), while SI showed minimal
inhibition to 86.9 ± 9.6% by flow cytometry (Figure 5A). Significant depletion in adherence is
also observed by measuring percent fluorescence intensity on plate reader assay, showing IBP-SI
to hold adherence to 34.4 ± 28.7% (p=0.004) and SI at 83.8 ± 16.5% fluorescence intensity
(Figure 5b).

Figure 5. IBP-SI lowers splenocyte adhesion
IBP-SI was directly compared to SI at 100 µM concentration by incubating with CFSE, PMA-
induced splenocytes and observing fluorescence intensity, correlating to splenocyte adherence,
using both flow cytometry (A) and plate reader assays (B). A) After washing non-adherent cells
Deleted: ìM
Deleted: 8.3
Deleted: 0.3
Deleted: 3
Deleted: 76.2
Deleted: 10.8
Deleted:
Deleted: ìM

21
away, each sample was trypsinized and read on the FITC channel on flow cytometer. Data was
analyzed using FlowJo software. B) Non-adherent cells were washed away and read on plate
reader at 485 nm excitation and 535 nm emission. Percent FITC+ and fluorescence intensity are
normalized to non-ELP treated control. Each treatment was done in triplicate wells. Data is
plotted as mean±SD. *p<0.05 **p<0.01

3.2. Temperature dependent inhibition
ELP’s are soluble monomers below transition temperature and begin to form nanoparticles
above. ELP’s transition temperature varies on the properties of their guest residue, Xaa, in their
Val-Pro-Gly-Xaa-Gly backbone. The thermal transition behavior of IBP-SI and SI were
previously reported by measuring optical density at 350 nm using UV-Vis spectrophotometry
(Hsueh, 2017). SI displayed two thermal responses of 25.5°C (Tt1) and 73.8°C (Tt2). IBP-SI, on
the other hand, showed transition temperatures of 25.7°C (Tt1) and 46.9°C (Tt2). Transition
temperatures of both IBP-SI and SI have similar Tt1 given their (Val-Pro-Gly-Ile-Gly)48(Val-Pro-
Gly-Ser-Gly)48 sequence; however, IBP-SI has a much lower Tt2, giving reason to believe the
mICAM-1 binding peptide is the cause for the temperature difference. Further, a linear
relationship was observed between transition temperature and log concentration. Using this
information, 60 µM of IBP-SI was used based on Equation 1, giving a transition temperature
well above 18°C at 24.4°C. In order to determine if IBP-SI nanoparticle formation is an
important factor in effective splenocyte adherence inhibition, an in vitro adherence assay below
IBP-SI Tt1 was performed. bEnd.3 monolayer, with confluency above 80%, was first incubated at
37°C with TNF-a, for increased ICAM-1 expression. Following, bEnd.3 cells were incubated
alone at temperatures 4°C, 15°C, 18°C and 20°C for 2 hrs, to assess cell viability outside of their
optimal conditions of 37°C. The lowest temperature that maintained cell viability was 18°C. IBP-
SI (60 µM ) was co-incubated with CFSE stained PMA-induced splenocytes at both 18°C 5%
Deleted:
Formatted: Not Superscript/ Subscript
Deleted: ìM
Deleted: ɑ
Deleted: ìM

22
CO2 and 37°C 5% CO2 on ICAM-1 upregulated bEnd.3 monolayer. IBP-SI (60 µM ) at 37°C 5%
CO2 limited splenocyte adherence to an average of 57.6 ± 9.5% (p=0.02) compared to at 18°C
5% CO2 with adherence average of 94.7 ± 0.8% (Figure 6).

Figure 6. IBP-SI nanoparticles at 37° C inhibit splenocyte adherence better than soluble
IBP-SI at 18° C.
IBP-SI (60 µM ) was incubated with PMA-stimulated CFSE-labelled splenocytes on a bEnd.3
monolayer, with confluency above 80%, at both 18°C in 5% CO2 and 37°C in 5% CO2. Cells
were trypsinized, read on the FITC channel on flow cytometry and analyzed by FlowJo software.
Data is plotted as percent FITC normalized to non-ELP treatment control, defined as 100%. Each
treatment was done in triplicate wells, thrice, with data reported as mean±SD. *p<0.05

3.3. IBP-SI blocks LFA-1/ICAM-1 costimulatory activation
The interaction between LFA-1 and ICAM-1 is monumental for the migration of leukocytes;
however, it is also involved in co-stimulatory processes that regulate proliferation, cell activation
and differentiation. In order to determine the effect of IBP-SI on co-stimulation, an in vitro
mixed lymphocyte reaction assay was performed. CH27 B cells were incubated with Moth
cytochrome c (MCC), a widely used antigen, for MHC II antigen presentation to CD4+ T cell
Deleted: ìM
Deleted: ìM

23
receptor (Sagerström et al., 1993). Proliferation of CD4+ T cells was assessed by staining with a
transmembrane cell division tracker dye CFSE and fitting in FlowJo proliferation software.
Additional fluorescence peaks to generation 0 of day 0 were considered proliferated cells. After
five-day incubation, IBP-SI was able to reduce the percent proliferation of CD4+ T cells to 26.1
± 18.7% (p=0.01) compared to a non-ELP treated control, defined as 100%. Our non-specific
control, SI, showed almost no reduction of 91.2 ± 13.9% compared to non-ELP control (Figure
7A). Additionally, concentrations of IL-2 from supernatant were determined via ELISA (Figure
7B). Both IBP-SI and SI significantly reduced the concentration of IL-2, relative to non-ELP
treatment control which was regarded as 100%. Between IBP-SI and SI, however, no statistical
significance was found. IBP-SI displayed a slightly lower average, reducing IL-2 concentration
down to 598.1 ± 74.2 IL-2 pg/mL, while SI averaged 690.6 ± 265 IL-2 pg/mL, from non-
treatment IL-2 concentration of 2317 ± 731 pg/mL. Thus IBP-SI reduced IL-2 production to 27.1
± 7.0% and SI to 32.1 ± 15.6% compared to non-treatment control, defined as 100%.

Figure 7. IBP-SI reduces proliferation and IL-2 production by blocking costimulatory
processes
Formatted: Font: (Default) Times New Roman
Formatted: Font: (Default) Times New Roman
Formatted: Font: (Default) Times New Roman
Deleted:

24
A) All treatments were assessed on day 0 and proliferation was compared between generation 0
of day 0 and day 5 by flow cytometry. FITC fluorescent intensity from CFSE-labelled CD4+ T
cells was fitted into a proliferation model by FlowJo software. Data is given as percent
proliferation compared to generation 0 peak and normalized to non-ELP treatment control’s
proliferation, defined as 100%. Each treatment was done in triplicate wells, three separate times.
Data is plotted as mean±SD. *p<0.05
B) Supernatant from mixed lymphocyte reaction was tested by ELISA for IL-2 concentration.
Concentrations were determined using a standard curve. Data is plotted as IL-2 concentration.
Data is plotted as mean±SD. *p<0.01

Deleted: percent
Deleted: normalized to non-ELP treated control, defined as
100%. …

25
4. Discussion

In the presence of chemically activated LFA-1 by PMA, which was able to significantly increase
binding of splenocytes to upregulated ICAM-1 on a bEnd.3 monolayer (Figure 3), IBP-SI
disrupted the LFA-1/ICAM-1 binding interface. IBP-SI nanoparticles were able to inhibit
splenocyte adherence in a dose-dependent fashion. Using two different assays, an IC50 of 81.2 ±
1.7 µM and 62.7 ± 4.8 µM at physiological temperature was measured using flow cytometry
(Figure 4A) and plate reader assay (Figure 4B), respectively. Non-targeting control, SI, showed
no dose dependent inhibition up to 170 µM , nearly double of IBP-SI’s reported IC50 (Figure
4C). This further validates the binding capabilities of the IBP motif. Surprisingly, lifitegrast,
commercially available as XiidraÒ, also showed no dose dependent adherence inhibition below
or above its reported IC50 3 nM (Figure 4D). Similar attachment assays were conducted to show
the ICAM-1/LFA-1 specificity of lifitegrast, however using Jurkat T cells and recombinant
human ICAM-1. In vivo data shows the ocular penetration after topical administration of 5% to
keratoconjunctivitis sicca (KSC) affected dogs to sustainably target the ocular tissues (Murphy et
al., 2011). Another study used different concentrations of lifitegrast treatment in murine models
for corneal inflammation and neutrophil infiltration. Compared to other findings that have used
dog and human models, a dose dependent relationship was not found showing 5% concentration
less effective at inhibiting neutrophil recruitment (Sun et al., 2013). LFA-1 containing both
human ɑβ chains can successfully bind to human ICAM-1, while LFA-1 with murine ɑ and
human β cannot, providing evidence for species-specific interactions (Huang and Springer,
1995). Therefore, direct comparisons using mICAM-1 binding peptide ELP with lifitegrast poses
difficulties. Both SI and IBP-SI at 100 µM , a concentration above IBP-SI IC50 were directly
Deleted: ìM
Deleted: ìM
Deleted: ìM
Deleted: ®
Deleted: ìM

26
compared, however. At physiological temperature, IBP-SI was able to significantly reduce
splenocyte adherence to an average of 47.1 ± 3.4% (p=0.017), compared to SI 86.9 ± 9.6%
(Figure 5A). Similarly, using a different assay, IBP-SI again showed significant inhibition of
attachment to 34.4 ± 28.7% (p=0.004), while SI showed minimal reduction to 83.8 ± 16.5%
(Figure 5B).

Given that IBP-SI is able to reduce attachment of high affinity conformation of LFA-1 to
upregulated ICAM-1 on bEnd.3 monolayer, there is promise that IBP-SI can deliver similar
results in vivo. Thereby reducing lymphocyte migration into inflamed tissues and subsequently
reducing inflammation. Anti-LFA-1 and anti-ICAM-1 treatment has been successful in rat
autoimmune glomerulonephritis by decreasing the flux of immune infiltrates and thereafter
reducing glomerular lesions (Hayashi et al., 1995). Similarly, MLR/Ipr mice, a Sjögrens
syndrome-like mouse model, were treated with monoclonal antibodies specific for mouse ICAM-
1 and LFA-1. Their results showed a trend to decrease in the mean ratio of lymphocytic
infiltrated area versus the total lacrimal tissue area after antibody combination therapy. They also
evaluated the importance of leukocyte migration in the progression of disease by treating mice at
earlier time points. Mice at 3 weeks of age and 8 weeks of age were injected with the anti-LFA-1
anti-ICAM-1 cocktail for 8 weeks. Results showed a significant decrease of lacrimal gland
infiltration for the mice treated at an earlier age to IgG control as well as those starting at 8
weeks of age (Gao et al., 2003). This data reiterates the importance of leukocyte migration in
disease perpetuation. Currently, there are limited methods for early diagnosis of Sjögren’s
syndrome, however this area is being heavily researched. If and when the development of early
prevention occurs, blocking LFA-1/ICAM-1 could potentially slow the progression of disease.
Deleted: 8.3 ± 0.3% (p=0.003),
Deleted: 76.1 ± 10.8%

27

ELP thermal transition properties have been advantageous in our studies. Elastin-like
polypeptides can be manipulated to phase shift at desirable temperatures upon changing its guest
amino acid, X. This enables them to be easily purified by simply heating and cooling above and
below its transition temperature. This also enables them to form unique 3D structures such as the
nanoparticle described here which has multivalent functionality for ICAM-1, with multiple
appended IBP. The thermal transition temperatures for IBP-SI are 25.7°C (Tt1), which ensures
nanoparticle formation at physiological temperature, and 46.9°C (Hsueh, 2017). The formation
of the IBP-SI nanoparticle has been shown to effectively decrease activated LFA-1 binding to
ICAM-1 to 57.6 ± 9.5% (p=0.02) at physiological temperature while IBP-SI in its monomer
form at a temperature below its transition temperature failed to significantly reduce adherence,
maintaining an average of 94.7 ± 0.8% binding compared to non-ELP treatment control (Figure
6). This evidence shows correlation between the temperature dependent ELP properties and
effectiveness in inhibition. The increase of ICAM-1 binding peptide on the surface due to
nanoparticle formation provides higher opportunity for binding to its receptor compared to its
monomer counterpart. Use of nanoparticle targeting technology in drug delivery has been
promising in other disease areas such as cancer research due to their specificity, encapsulation
possibilities and low immunogenetic properties (Rosenblum et al., 2018).

Two important steps in the pathology of Sjögren’s syndrome and DED is the flux of immune
cells and their activation, proliferation and cytokine release in the microenvironment. LFA-
1/ICAM-1 binding not only is essential in migration but also duals as a costimulatory process.

28
CH27 B cell line was incubated with Moth cytochrome c (MCC 88-103), for MHC II antigen
presentation to TCR on CD4+ T cells. The cells were incubated with IBP-SI (100 µM) or SI (100
µM) for five days. CD4+ T cell proliferation was significantly reduced to 26.1 ± 18.7% (p=0.01)
compared to non-ELP treatment control. Our non-specific ELP, SI, showed no significant
reduction of 91.2 ± 13.9% proliferation compared to non-treatment control (Figure 7A). One
group studied proliferation of CD4+ T cells using strains of LFA-1 (CD18
-
/
-
) deficient mice
compared to wild-type. In a similar mixed lymphocyte reaction, they observed drastic depletion
of CD4+ T cell proliferation in the LFA-1 deficient sample compared to control (Kandula and
Abraham, 2004). Another study showed similar importance of co-stimulation by LFA-1/ICAM-1
for complete T cell activation. Using CD18
-
/
-
together with dendritic cells, other co-stimulation
pathways were activated such as CD28 to measure proliferation and cytokine release. They
observed a decrease in proliferation as well as TH1 differentiation, showing a significant decrease
in TH1 cytokines such as IFN-g and IL-2. A trend of reduction was observed for TH2 cytokines,
yet the differences were not statistically significant (Varga et al., 2010). LFA-1/ICAM-1 is not
the only co-stimulation pathway on the immune cell surface, but overwhelming data has shown
its importance in full activation of T cells. Similarly, the supernatant on the fifth day of CH27 B
cell and CD4+ T cells for IL-2, in the presence and absence of ELP, was tested via ELISA. A
significant decrease of IL-2 concentration to 27.1 ± 7.0% was observed with IBP-SI treatment
reducing IL-2 to 598.1 ± 74.2 pg/mL compared to non-treatment control IL-2 concentration of
2317 ± 731 (Figure 7B). A slight difference was observed between IBP-SI and SI treated
samples; however, it did not reach statistical significance.

Deleted: ìM
Deleted: ìM
Deleted: ã
Deleted: ,
Deleted: 27.1 ± 7.0%
Deleted: compared to

29
Promising in vitro data has been shown using IBP-SI to mitigate complete T cell activation and
adherence by disrupting the LFA-1/ICAM-1 interface. Future work needs to be done to observe
translational results in vivo. IBP fused to a different ELP-backbone has already been successfully
used as a targeting peptide but with a drug carrier/drug complex end (Ju et al., 2019). In vivo, we
can test the potential of IBP-SI to mimic that of drug and antibody treatment, which if successful,
would be favorable in regard to potential toxicity. Using the gold-standard criteria for Sjögren’s
syndrome, we can detect if IBP-SI has an effect on anti-Ro and anti-La production by testing
blood serum concentrations. Tear and saliva production can also be measured with IBP-SI
treatment. With the progression of the disease, a decreased flow is observed due to glandular
destruction by inflammation. Given that IBP-SI was able to reduce adherence to upregulated
ICAM-1, CD4+ T cell proliferation and IL-2 concentrations in vitro, theoretically it can be
suggested that IBP-SI affects lymphocytic migration and activation. Therefore, reduced
degradation of exocrine glands by mitigating flux could be observed by increased production of
tear and saliva flow. Also, a reduction in antibody concentration by interfering with B cell
activation could also be observed. Based on the data presented and potential future work, there is
a great need for advancement of IBP-SI research.

30
Summary
Migration and co-stimulation processes are crucial in the development and sustainment of
Sjogren’s syndrome. This thesis describes the ability of elastin-like polypeptide nanoparticles
with ICAM-1 binding peptide terminal to obstruct these processes by preventing successful
binding of LFA-1 ligand to its receptor ICAM-1. In doing so, splenocyte adherence, which is a
significant step in lymphocytic migration, was disrupted and reduced. Also, T cell proliferation
and cytokine IL-2 production was depleted by the interference of LFA-1/ICAM-1 binding.
Compared to other LFA-1 antagonists, IBP-SI is both economically and environmentally
preferable, specifically targets ICAM-1 and displays a low immune response and
biodegradability.

31

References

Anaya J.M., Ramirez-Santana C., Alzate M.A., Molano-Gonzalez N. and Rojas-Vilarraga A. The
Autoimmune Ecology. Front. Immunol. 2016. 7:139. doi: 10.3389/fimmu.2016.00139

Anderson M.E. and Siahaan T.J. Targeting ICAM-1/LFA-1 interaction for controlling
autoimmune diseases: designing peptide and small molecule inhibitors. Peptides. 2003. 24; 487-
501. doi: 10.1016/S0196-9781(03)00083-4

Bahniuk M.S., Alshememry A.K. and Unsworth L.D. High-yield recombinant expression and
purification of marginally soluble, short elastin-like polypeptides. Biotechniques. 2018. Vol. 61.
No. 6. doi: 10.2144/000114482

Bélizaire A.K., Tchistiakova L. and Alakhov V. Identification of a murine ICAM-1-specific
peptide by subtractive phage library selection on cells. Biochem Biophys Res Commun. 2003 Sep
26; 309 (3):625-30. doi: 10.1016/j.bbrc.2003.08.050

Fox R.I. Sjögren’s syndrome. Lancet. 2005. 366:321-31

Fox R.I., Fox C.M., Gottenberg J.E. and Dörner T. Treatment of Sjögren’s syndrome: current
therapy and future directions. Rheumatology. 2019. doi: 10.1093/rheumatology/kez142

García-Carrasco M., Fuentes-Alexandro S., Escárcega R.O., Salgado G., Riebeling C. and
Cervera R. Pathophysiology of Sjögren’s syndrome. Archives of Medical Research. 2006. 37;
921-932. doi: 10.1016/j.arcmed.2006.08.002

García-Carrasco M., Mendoza-Pinto C., Jiménez-Hernández C., Jiménez-Hernández M., Nava-
Zavala A. and Riebeling C. Serologic features of primary Sjögren’s syndrome: clinical and
prognostic correlation. Int J Clin Rheumtol. 2012 December; 7(6): 651-659. doi:
10.2217/ijr.12.64

Gaudino S.J. and Kumar P. Cross-talk between antigen presenting cells and T cells impacts
intestinal homeostasis, bacterial infections, and tumorigenesis. Front. Immunol. 2019. 10:360.
doi: 10.3389/fimmu.2019.00360.

Gao J., Morgan G., Tieu D., Schwalb T.A., Luo J.Y., Wheeler L.A. and Stern M.E. ICAM-1
expression predisposes ocular tissues to immune-based inflammation in dry eye patients and
Sjögrens syndrome-like MRL/lpr mice. Experimental Eye Research. 2004. 78; 823-835.

Gegg M.E., Harry R., Hankey D., Zambarakji H., Pryce G., Baker D., Adamson P., Calder V.
and Greenwood J. Suppression of autoimmune retinal disease by lovastatin does not require th2
cytokine induction. J Immunol. 2005. 174 (4) 2327-2335; doi: 10.4049/jimmunol.174.4.2327

32

Hassouneh W., MacEwan S.R. and Chilkoti A. Fusions of Elastin-like Polypeptides to
Pharmaceutical Proteins. Methods in Enzymology. Vol. 502. ISSN 0076-6879, doi:
10.1016/B978-0-12-416039-2.00024-0.

Hayashi Y., Haneji N., Yanagi K., Higashiyama H., Yagita H. and Hamano H. Prevention of
adoptive transfer of murine Sjögren’s syndrome into severe combined immunodeficient (SCID)
mice by antibodies against intercellular adhesion molecule-1 (ICAM-1) and lymphocyte
function-associated antigen-1 (LFA-1). Clin Exp Immunol. 1995; 102:360-367.

Hodge J.W., Greiner J.W., Tsang K., Sabzevari H., Kudo-Saito C., Grosenbach D.W., Gulley
J.L., Arlen P.M., Marshall J.L., Panicali D. and Scholm J. Costimulatory molecules as adjuvants
for immunotherapy. Front in Bios. 2006 January. 11; 788-803.

Hsueh A. P. Trafficking of targeted elastin-like polypeptide nanoparticles in the lacrimal gland.
2017. University of Southern California, Ph.D. dissertation. University of Southern California
Digital Library

Huang C. and Springer T.A. A binding interface of the I domain of lymphocyte function-
associated antigen-1 (LFA-1) required for specific interaction with intercellular adhesion
molecule 1 (ICAM-1). J Biol Chem. 1995. 270, 19008-19016. doi: 10.1074/jbc.270.32.19008

Isobe M., Yagita H., Okumura K. and Ihara A. Specific acceptance of cardiac allograft after
treatment with antibodies to ICAM-1 and LFA-1. Science 1992;255:1125–7.

Ju Y., Guo H., Yarber F., Edman M.C., Peddi S., Janga S.R., MacKay J.A. and Hamm-Alvarez
S.F. Molecular targeting of immunosuppressants using a bifunctional elastin-like polypeptide.
Bioconjug Chem. 2019, 30, 2358-2372.

Kallen J., Welzenback K., Ramage P., Geyl D., Kriwacki R., Legge G., Cottens S., Weitz-
Schmidt G. and Hommel U. Structural Basis for LFA-1 Inhibition upon Lovastatin Binding to
the CD11a I-Domain. J Mol Biol. 1999. 292, 1-9. doi: 10.1006/jmbi.1999.3047

Kandula S. and Abraham C. LFA-1 on CD4+ T cells is required for optimal antigen-dependent
activation in vivo. J Immunol. 2004; 173:4443-4451. doi: 10.4049/jimmunol.173.7.4443

Kavanaugh AF, Davis LS, Jain RI, Nichols LA, Norris SH, Lipsky PE. A phase I/II open label
study of the safety and efficacy of an anti-ICAM-1 (intercellular adhesion molecule-1; CD54)
monoclonal antibody in early rheumatoid arthritis. J Rheumatol 1996;23:1338– 44.

Lefort C.T. and Ley K. Neutrophil arrest by LFA-1 activation. Front. Immunol. 2012. Vol. 3
(157). doi: 10.3389/fimmu.2012.00157.

Long E.O. ICAM-1: Getting a grip on leukocyte adhesion. J Immunol. 2001; 186:5021-5023.
doi: 10.4049/jimmunol.1100646

33
Manikwat P., Kiptoo P., Badawi A. H., Büyüktimkin B. and Siahaan T.J. Antigen-Specific
Blocking of CD4-Specific Immunological Synapse Formation Using BPI and Current Therapies
for Autoimmune Diseases. Med Res Rev. 2012 July; 32(4): 727-764. doi: 10.1002/med.20243

Minguet S., Swamy M., Alarcón B., Luescher I.F. and Schamel W.W.A. Full activation of the T
cell receptor requires both clustering and conformational changes at CD3. Immunity. 2007. 26,
43-54. doi: 10.1016/j.immuni.2006.10.019

Murphy C.J., Bently E., Miller P.E., McIntyre K., Leatherberry G., Dubielzig R., Giuliano E.,
Moore C.P., Philips T.E., Smith P.B., Prescott E., Miller J.M., Thomas P., Scagliotti R., Esson
D., Gadel T. and O’Neill C.A. The pharmacologic assessment of a novel lymphocyte function-
associated antigen-1 antagonist (SAR 1118) for the treatment of keratoconjunctivitis sicca in
dogs. Corea: Investigative Ophthalmology and Visual Science. 2011. Vol. 52, No. 6.

Nourshargh S. and Alon R. Leukocyte migration into inflamed tissues. Immunity 41. 2014
November. doi: 10.1016/j.immuni.2014.10.008.

Rosenblum D., Joshi N., Tao W., Karp J.M. and Peer D. Progress and challenges towards
targeted delivery of cancer therapeutics. Nat Commun. 2018. 1410 (9).

Rosenblum M.D., Remedios K.A. and Abbas A.K. Mechanisms of human autoimmunity. The J
Clin Invest. 2015 June; 125(6):2228-2233. doi: 10.1172/JCI78088.

Sagerström C.G., Kerr E.M., Allison J.P. and Davis M.M. Activation and differentiation
requirements of primary T cells in vitro. Immunology. 1993 October. Vol. 90, pp.8987-8991.

Salomon B. and Bluestone J.A. Cutting edge: LFA-1 interaction with ICAM-1 and ICAM-2
regulated Th2 cytokine production. J Immunol. 1998; 161:5138-5142.

Schulze-Koops H, Lipsky PE, Kavanaugh AF, Davis LS. Elevated Th1- or Th0-like cytokine
mRNA in peripheral circulation of patients with rheumatoid arthritis. Modulation by treatment
with anti-ICAM-1 correlates with clinical benefit. J Immunol 1995;155:5029–37.

Schwartz R.H. T cell anergy. Annu. Rev. Immunol. 2003. 21:305-34. DOI:
10.1146/annurev.immunol.21.120601.141110

Shah M., Hsueh P.Y., Sun G., Chang H.Y., Janib S.M. and MacKay J.A. Biodegradation of
elastin-like polypeptide nanoparticles. Protein Sci. 2012. Vol. 21:743-750. doi: 10.1002/pro.2063

Shi G., Zhang J., Zhang Z. and Zhang X. Systemic Autoimmune Diseases. Clin Dev Immunol.
2013. Article ID 728574. DOI: 10.1155/2013/728574

Stanley P. and Hogg N. The I domain of integrin LFA-1 interacts with ICAM-1 domain 1 at
residue Glu-34 but not Gln-73. J Biol Chem. 1998. Vol. 273, No. 6, Issue of February 6, pp.
3358-3362.

34
Sun Y., Zhang R., Gadek T.R., O’Neill C.A. and Pearlman E. Corneal inflammation is inhibited
by the LFA-1 antagonist. lifitegrast (SAR 1118). J Ocul Pharmacol Th. 2013. Vol. 29, No.4. doi:
10.1089/jop.2012.0102

Tinoco R., Carrette F., Barraza M. L., Otero D.C., Magaña J., Bosenberg M.W., Swain S.L. and
Bradley L.M. PSGL-1 is an immune checkpoint regulator that promotes T cell exhaustion.
Immunity. 2016. 44(5): 1190-1203. DOI: 10.1016/j.immuni.2016.04.015

Varga G., Nippe N., Balkow S., Peters T., Wild M.K., Seeliger S., Beissert S., Krummen M.,
Roth J., Suderkötter C. and Grabbe S. LFA-1 Contributes to Signal I of T-Cell Activation and to
the Production of Th1 Cytokines. J Invest Dermatol. 2010 April. Vol. 130(4); 1005-1012. doi:
10.1038/jid.2009.398

Vitali C., Bombardieri S., Jonsson R., Moutsopoulos H.M., Alexander E.L., Carsons S.E.,
Daniels T.E., Fox P.C., Fox R.I., Kassan S.S., Pillemer S.R., Talal N., Weisman M.H. and the
European Study Group on Classification Criteria for Sjögren’s Syndrome. Classification criteria
for Sjögren’s syndrome: a revised version of the European criteria proposed by the American-
European Consensus Group. Ann Rheum Dis. 2002; 61:554-558.

Voulgarelis M. and Tzioufas A.G. Pathogenetic mechanisms in the initiation and perpetuation of
Sjögren’s syndrome. Nat Rev Rheumatol. 2010. 6, 529-537. doi: 10.1038/nrrheum.2010.118

Walling B.L. and Kim M. LFA-1 in T cell migration and differentiation. Font Immunol. 2018.
9:952. doi: 10.3389/fimmu.2018.00952

Wang Y., Li D., Nurieva R., Yang J., Sen M., Carreño R., Lu S., McIntyre B.W., Molldrem J.J.,
Legge G.B. and Ma Q. LFA-1 affinity regulation is necessary for the activation and proliferation
of native T cells. J Biol Chem. 2009. Vol. 284, No. 19. 12645-12653. doi:
10.1074/jbc.M807207200

Wee H., Oh H., Jo J. and Jun C. ICAM-1/LFA-1 interaction contributes to the induction of
endothelial cell-cell separation: implication for enhanced leukocyte diapedesis. Exp Mol Med.
2009. Vol. 41. No.5. 341-348.

Woska J.R., Shih D., Taqueti V.R., Hogg N., Kelly T.A. and Kishimoto T.K. A small-molecule
antagonist for LFA-1 blocks a conformational change important for LFA-1 function. J Leukoc
Biol. 2001. Vol. 70.

Zhong M., Gadek T.R., Bui M., Shen W., Burnier J., Barr K.J., Hana E.J., Oslob J.D., Yu C.H.
Zhu J., Arkin M.R., Evanchik M.J., Flanagan W. M., Hoch U., Hyde J., Prabhu S., Silverman
J.A. and Wright J. Discovery and Development of Potent LFA-1/ICAM-1 Antagonist SAR 1118
as an Ophthalmic Solution for Treating Dry Eye. ACS Med Chem Lett. 2012. 3; 203-206.
dx.doi.org/10.1021/ml2002482

Abstract (if available)

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Development of novel immunosuppressant-based therapies to treat dacryoadenitis in a Sjögren’s syndrome mouse model

PDF

Trafficking of targeted elastin‐like polypeptide nanoparticles in the lacrimal gland

PDF

Development of an elastin-like polypeptide-based cyclosporine A formulation that improves autoimmune-mediated dry eye characteristic of Sj鰃ren抯 syndrome

PDF

Exploring serum and tear micro-RNA as biomarkers for early diagnosis of Sjögren’s Syndrome

PDF

The modulation of dynamin and receptor endocytosis machinery using elastin-like polypeptides

PDF

Flipping the switch on protein activity activity: elastin-like polypeptides assemble into cell switches and vesicles

PDF

Methods and protocols for detecting the intracellular assembly of elastin-like polypeptides

PDF

Cellular uptake mechanism of elastin-like polypeptide fusion proteins

PDF

Sustained and targeted delivery of rapamycin using FKBP-elastin like polypeptide fusion proteins

PDF

Secretion of exosomes through the potential endolysosomal pathway in Rab3DKO and Non-Obese Diabetic murine models

PDF

Increased cathepsin S plays an important role in ocular surface manifestations in Sjögren’s syndrome

PDF

The trafficking and pathogenesis of cathepsin S in Sjögren’s syndrome

PDF

Multivalent smart elastin-like polypeptide therapeutics with drug delivery and biosensing applications.

PDF

Investigation of mechanisms underlying development of autoimmune dacryoadenitis in lacrimal gland

PDF

Development and therapeutic assessment of multivalent protein polymers for cancer and eye diseases

PDF

Polymeric immunoglobulin receptor mediated drug carrier based on the genetically engineered temperature sensitive polypeptides

PDF

Characterization of IL-1β secretion by fusing elastin-like polypeptides to pro-caspase-1

PDF

Antibodies and elastin-like polypeptides: cellular and biophysical characterization of an anti-ELP monoclonal and an anti-CD3 single-chain-ELP fusion

PDF

Secretion of exosomal miRNAs in Rab3DKO, a model of exocrine secretory deficit

PDF

Elastin like polypeptide tagged angiotensin (1-7) is a potential effective treatment for retinitis pigmentosa

Asset Metadata

Creator Vega, Adrianna Giselle (author)

Core Title Targeting LFA-1/ICAM-1 interaction using ICAM-1 binding elastin-like polypeptide

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/24/2020

Defense Date 04/23/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag autoimmune disease,elastin-like polypeptide,intracellular adhesion molecule-1,lymphocyte function-associated antigen-1,OAI-PMH Harvest,Sjögren's syndrome

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hamm-Alvarez, Sarah (committee chair), MacKay, John Andrew (committee member), Okamoto, Curtis (committee member)

Creator Email agisellev3@gmail.com,vega@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-289295

Unique identifier UC11673850

Identifier etd-VegaAdrian-8323.pdf (filename),usctheses-c89-289295 (legacy record id)

Legacy Identifier etd-VegaAdrian-8323.pdf

Dmrecord 289295

Document Type Thesis

Rights Vega, Adrianna Giselle

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA