University of Southern California Dissertations and Theses

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

(USC Thesis Other)

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

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content 1

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

By

Zhiyuan Yao

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree,
MASTER OF SCIENCE (Pharmaceutical Sciences)

May 2019

2

Acknowledgement
To Dr. Andrew Mackay for guiding me through research study in at the University of Southern California.

To Dr. Alan Epstein for thoughtful insights for anti-CD3 single-chain-ELP fusion research and co-mentor
the project “Development of the First Monoclonal Antibody for Detection of Elastin-Like Polypeptides”.

To Changrim Lee for his endeavor in ELP fusion protein projects and the help with fusion protein
refolding as well as SEC-MALS characterization.

To Aida Kouhi for her help as student mentor as well as research study in “Development of the First
Monoclonal Antibody for Detection of Elastin-Like Polypeptides” for immunizing BALB/c female mice,
generating monoclonal hybridoma line and conducting ELISA assay.

To Santosh Peddi and the research group of Dr. Honggang Cui for taking these cryo-TEM images.

Great help and inspirations with these projects have been received from Hao Guo, Zhe Li, Jordan
Despanie, Anh Truong, Yue Wang, Hugo Avila, Mincheol Park, David Tyrpak.

To my family members and Ms. Pepper who provide unfailing support to my study.

3

Table of Contents

Project 1: Generation and Characterization of Elastin-like Polypeptide and Anti-CD3 scFv Fusion Protein
Nanoworm ........................................................................................................................................ 5
Abstract ......................................................................................................................................... 5
Introduction .................................................................................................................................. 6
Result .......................................................................................................................................... 10
Generation of anti CD3-A192 fusion protein .......................................................................... 10
Characterization of Anti CD3-A192 Fusion Protein Nanostructure ......................................... 12
The thermal responsiveness property of anti CD3-A192 fusion protein ................................ 15
Anti CD3-A192 nanoworms target the CD3 Receptor in two CD3+ cell lines .......................... 17
Anti CD3-A192 Nanoworms inhibit the T Cell Activation by OKT3 antibodies on jurkat cells based
on interleukin-2. ...................................................................................................................... 19
Experimental Procedures ............................................................................................................ 21
Expression and Purification of Anti CD3-A1092 Fusion Protein .............................................. 21
Determination of Purity of Anti CD3-A192 Fusions Protein .................................................... 22
Refolding of Anti CD3-A192 Fusion Protein ............................................................................ 23
Light Scattering Analysis Electron Microscopy of Nanoparticles ............................................ 23
Transition Temperature of scFv-A192 Nanoparticles .............................................................. 24
Cell Surface Staining Assay ...................................................................................................... 25
Jurkat Cell Activation and IL-2 Detection ................................................................................ 26
Discussion.................................................................................................................................... 27
Project 2: Use of the First Monoclonal Antibody to Detect Elastin-Like Polypeptides in Western blot,
ELISA, and Immunofluorescence Microscopy ................................................................................. 30
Abstract: ...................................................................................................................................... 30
Introduction ................................................................................................................................ 31
Result .......................................................................................................................................... 34
Immunoreactivity of purified anti-ELP AK1, in an indirect ELISA ............................................ 34
Western blot analysis of anti-ELP AK1 specificity .................................................................... 36
AK1 Immunofluorescence staining of ELP-fusion proteins expressed in mammalian cells .... 39
4

Phase separation of ELPs in presence of anti-ELP AK1 ............................................................ 41
Method ....................................................................................................................................... 44
Cell culture and transfection ................................................................................................... 44
Enzyme Linked Immunosorbent Assay (ELISA) ........................................................................ 44
Western Blotting ..................................................................................................................... 44
Immunofluorescence staining and confocal microscopy ........................................................ 46
Phase separation behavior of ELPs in presence of anti-ELP AK1............................................. 47
Discussion.................................................................................................................................... 49
Reference .................................................................................................................................... 52

5

Project 1: Generation and Characterization of Elastin-like Polypeptide and
Anti-CD3 scFv Fusion Protein Nanoworm
Abstract
The TCR/CD3 complex is found on T cells and responsible for antigen recognition. The binding of anti-
CD3 antibody such as OKT3 to CD3 complexes would induce T cell activation in-vitro. Compared to
whole immunoglobulin molecules, antibody-derived fragments retain the binding properties and can
be readily (and cheaply) produced in bacterial systems. The hydrophilic protein polymer Elastin-like
polypeptides (ELPs) could be expressed recombinantly with single-chain variable fragment (scFv)
antibody in E. coil expression system and be used as a purification tag without any need for
chromatography.
In this study, a recombinant fusion protein between an anti-CD3 scFv and a soluble protein polymer
ELP has been successfully generated. The fusion protein could be expressed in E. coil, self-assemble to
worm-like nanostructures, and specifically binds with CD3 receptor on the cell surface. These anti-CD3
hybrid protein polymer nanoparticles could not activate the CD3 receptor and could block the in-vitro
T cell activation effect of OKT3 monoclonal antibody.

6

Introduction
The CD3 complex serves as a T cell co-receptor (TCR) that associates noncovalently with the TCR (Smith-
Garvin et al., 2009). The TCR/CD3 complex is found on T cells and responsible for the antigen
recognition mediated by major histocompatibility complex (MHC) molecules. (Gao et al., 2002; Sewell,
2012) The CD3 protein complex consisting of two heterodimers: CD3εγ, CD3εδ and one homodimer
CD3ζζ. (Kuhns et al., 2006; Punt et al., 1994) Among them, the cytoplasmic segments of CD3 ε, CD3 γ
and CD3 δ contain a single ITAM, whereas the cytoplasmic domain of the CD3 ζ subunit contains three
ITAMs. That resulting in ten ITAMs in CD3 complex in total, making the complex exquisitely sensitive to
antigen binding. Followed by antigen stimulation conformational changes within the cytoplasmic tails
of the CD3 polypeptides occur, ITAMs are required for initiation of the signaling cascade as they recruit
protein tyrosine kinases (PTKs), signaling intermediates and adapter molecules. For instance, the
phosphorylation conserved tyrosine residues present within the ITAMs in the CD3 complex, creating a
docking site for proteins with Src homology 2 domains, e.g. ζ -associated protein of 70 kDa (ZAP-70 )
(Pitcher and van Oers, 2003; Smith-Garvin et al., 2009). That phosphorylation process of the TCR-CD3
complex leads to various regulation of T cell function including T cell activation (Viola and Lanzavecchia,
1996), lymphokine production, internalization of the whole CD3-TCR complex(Krangel, 1987), and
induce T cell hyperstimulation and apoptosis (Combadiere et al., 1998).
Based on the function of TCR/CD3 complex, there are various kinds of antibodies and antibody-
derivatives are developed to target CD3 receptor and they are used for both therapeutics and
biomedical research purposes. For therapeutic purpose, for instance, in 1980, Van Wauwe et al.
7

reported report the generation of the OKT3 mAb to human CD3 on T cell surface. (Van Wauwe et al.,
2016) OKT3 is a mouse IgG2a antibody that recognize CD3 epsilon chain of the CD3/TCR complex on T
lymphocytes (Clevers et al., 1988). This OKT3 was approved by the US FDA in 1986 and has been
successfully used to treat allograft rejection in kidney, liver and heart transplantation (Hooks et al.,
1991). The binding of OKT3 monoclonal antibody to CD3 receptor would initially lead to the T cell
stimulation and subsequently induce blockage of function of cytotoxic T cells and apoptosis of the T
cells (Ferran et al., 1990). Blinatumomab is a ‘‘bispecific T-cell engager’’ approved in 2014 for the
treatment of acute lymphoblastic leukemia. (FDA News Release.,2014) The Blinatumomab antibody
consists of an anti-CD3 arm to engage CD3-positive T-cells without regard to T-cell receptor (TCR)
specificity and an anti-CD19 arm to bind to lymphoblasts expressing the CD19 marker. (Brischwein et
al., 2007) (Loffler et al., 2000)
In the application of anti-CD3 antibodies for biomedical research, solid phase-bound anti-CD3 mAbs
are used in inducing T cell activation in vitro. (Schwartz, 1990) Additionally, to provide adequate
activation efficiency, anti-CD3 antibodies are often bound to a solid surface or beads. (Li and Kurlander,
2010) anti-CD3 antibody could be bound to magnetizable superparamagnetic polystyrene spherical
microbeads and be used for T cell purification and isolation. (Neurauter et al., 2007)
Recently, synthetic and biomimetic polymeric materials are having increasing application in medical
applications such as tissue engineering, drug delivery, and protein modification. (Hortiguela et al., 2015)
The high degree of selectivity of antibodies for their ligands lends them to the application in creating
such addition-of-function polymeric materials. Furthermore, smaller, antibody-derived fragments that
8

retain the binding pockets and properties of whole immunoglobulin molecules are readily (and cheaply)
produced in bacterial systems. These fragments allow relatively simple engineering to modify
properties such as affinity, or to introduce novel characteristics such as fusion to a cytotoxic MOIETY
or modification for attachment to payload-containing nanoparticles.(Ferrer-Miralles et al., 2009)
Besides their wide applications in biological research such as protein purification (Terpe, 2003) and
imaging (Chen et al., 2013), recombinant fusion proteins have also become an important category of
biopharmaceuticals. (Chen et al., 2012; Schmidt, 2009)
Elastin-like polypeptides (ELPs) are a class of unique protein polymers inspired from human
tropoelastin, which is the precursor to elastin. In last few decades, ELPS have been extensively studied
as an attractive versatile biopolymer. ELPs are composed of repeated pentameric motif [Val-Pro-Gly-
Xaa-Gly] n, where Xaa can be any guest amino acid and n indicate the repeat number of the pentameric
sequence. (Urry, 1997) One unique feature of ELPS are they undergo inversible phase transition above
a characteristic transition temperature (Tt) determined by Xaa and n. Below the Tt, ELPs are highly
water-soluble and while the temperature raise above that ELPs would display a sharp phase separation
called a coacervate and from a secondary liquid phase. ELPs are attractive for multiple applications in
vitro and in vivo for tissue engineering and delivery of therapeutics because they are monodisperse,
biodegradable, biocompatible and low immunogenic. The advantages of using ELPs in generating novel
therapeutic include : i) being able to modulate cellular signaling;(Aluri et al., 2014a) ii) it could
recombinantly fused to antibody fragments without bioconjugation chemistry;(Mackay et al., 2010) iii)
potently engage multiple receptors;(Dhandhukia et al., 2017) iv) it would undergo proteolytic
9

biodegradation after endocytosis;(Despanie et al., 2016) and v) and some ELP sequences have the
potential for low antigenicity. (MacEwan and Chilkoti, 2010)
In the previous study in our lab, a fusion between an anti-CD20 single chain antibody and ELP soluble
protein polymer has been generated that competitively binds CD20 on two B-cell lymphoma cell
lines, exhibits concentration-dependent induction of apoptosis, and induce apoptosis better than
Rituximab alone. (Aluri et al., 2014b) Based on this design, single chain variable regions (scFv)
antibody fragment was expressed recombinantly as a fusion with a protein polymer in E. coil
expression system, which eliminates the need for chemical bioconjugation. ELP protein polymer
segments in the fusion protein were used as the purification tag, enabling quick and efficient
purification via inverse transition cycling of ELP . Inverse transition cycling purification involves cycling
the protein above and below the transition temperature of the ELP fusion followed by centrifugation
to obtain the desired protein, without any need for chromatography. (Yeboah et al., 2016)
In this study, we describe a novel recombinant fusion protein between anti-CD3 scFv a single chain
and a soluble protein polymer ELP that could be expressed recombinantly expressed in E. coil
expression system and purified without any need for chromatography. The recombinant fusion was
designed with an anti-CD3 scFv fragment fused to the N-terminus of a 73.6 kDa molecular weight ELP
A192. Anti-CD3 scFv of the nanoworm is derived from the anti-CD3 segment of blinatumomab, which
targets epsilon-delta domain of the CD3 complex. The structure of fusion protein is characterized and
the binding properties, as well as the effect on T cell activation of this fusion protein are studied in
this manuscript.
10

Result
Generation of anti CD3-A192 fusion protein
The scFv-protein polymer was constructed with two moieties and fused together through molecular
cloning: 26.0 kDa anti-CD3 single chain Fv segment derived from the anti-CD3 segment of
blinatumomab and 73.6 kDa ELP protein polymer A192 fused in the N-terminus. (Figure. 1 A) (Table.
1) The anti CD3-A192 fusion protein construct was transfected to SHuffle T7 E. coli cell line. After
expression, bacterial lysate was collected and anti CD3-A192 fusion protein was purified and
concentrated through triggering the phase separation of the ELP A192. The yield of anti CD3-A192
fusion protein ranged from 18.5 mg/L of E.coli culture. Copper Staining SDS-PAGE determined the
purity was 85.8 % and the band correlated with 99.4 kDa molecular weight. (Figure. 1 B) After
purification, the scFv-protein polymer was refolded into the active form. They were denatured with 6
M guanidine hydrochloride and containing 2-Mercaptoethanol (beta-ME) reducing agent that reduces
any disulfide bonds that were aberrantly formed, and the scFv-protein polymer were renatured during
the dialysis refolding process.
11

Table 1. Amino acid sequences of anti CD3-A192 fusion protein and ELP A192.

Figure 1. Purity and identity of an anti-CD3 single-chain antibody fused to the ELP known as A192.
(a) the anti-CD3 single chain Fv segment is fused to the N-terminus of ELP protein polymer. (b) Copper
Staining SDS-PAGE determined the molecular weight correlated with estimated molecular weight 99.4
KDa. Purity of the fusion protein was 85.8 % determined through image analysis using ImageJ.

12

Characterization of Anti CD3-A192 Fusion Protein Nanostructure
These studies revealed in multiple methods that anti CD3-A192 fusion protein forms nanoparticles.
Dynamic light scattering (DLS) shows that the radius of nanostructure of the refolded anti CD3-A192
fusion protein is 34.8±5.9 nm, while ELP A192 alone has the radius of 9.9±2.1 nm. As the ELP A192
does not phase separate below its transition temperature, this suggests that the anti-CD3 scFv domain
mediates nanoparticle assembly. Additionally, the radius of anti CD3-A192 fusion protein before
refolding process is 74.0±15.1 nm, which is significantly higher than the radius of the particle after
refolding (Figure. 2 A). Therefore, a likely interpretation is that during the expression of fusion protein,
there exists unexpected assembly of anti CD3-A192 fusion protein into larger nanoparticles. Cryogenic
transmission electron microscopy (cryo-TEM) is also utilized in confirming the anti CD3-A192 fusion
protein nanoparticle size and morphology structure. (Cryo-TEM images were taken by Santosh Peddi
and the research group of Dr. Honggang Cui.) Through cryo-TEM, the renatured anti CD3-A192 fusion
protein nanoparticles were not spherical, in instead assemble worm-like nanostructures with a
constant width and variable length. (Figure. 2 B)
To reveal the structure of anti CD3-A192 fusion protein nanostructure and confirm the finding in cryo-
TEM imaging, the combination of Size Exclusion Chromatography with multiangle light scattering (SEC-
MALS) was used in the study. The size exclusion chromatography (SEC) was used to separate fusion
protein based on the difference of molecular weight. The renatured anti CD3-A192 fusion protein
nanostructures showed a mono peak on SEC chromatography, and the multiangle light scattering
confirmed the absolute molecular weight of 4403kDa (Figure.2 C), which suggested that the anti CD3-
13

A192 fusion protein nanostructures consisted ~44 anti CD3-A192 monomers in average. Shape factor
ρ, equals to the hydrodynamic radius (Rg)/radius of gyration (Rh) ratio, was used to characterize the
shape information about the fusion protein nanostructures. (Moffitt et al., 1998; Schmidt and
Stockmayer, 1984) The Rg/ Rh ratio of renatured anti CD3-A192 fusion protein nanostructures was 1.02.
High Rg/ Rh ratio (> 0.775) implied that the shape of the fusion protein nanostructure had a coil to rod-
like elongated structures. Thus, the light scattering experiments data correlate with cryoTEM
observations of these anti CD3-A192 fusion protein nanostructures. According to the characterization
of their size, composition and morphology, these renatured anti CD3-A192 fusion protein are defined
as hybrid protein polymer nanoworms.

14

Figure 2. Characterization of anti CD3-A192 fusion protein nanostructure (a) DLS intensity
data shows that the hydrodynamic radius of anti CD3-A192 fusion protein nanostructure is
significantly higher than A192. The refolded anti CD3-A192 fusion protein nanostructure had a
hydrodynamic radius of 34.8±5.9 nm and the native fusion protein nanostructure had a
hydrodynamic radius of 74.0±15.1 nm (b) Through cryoTEM, the renatured anti CD3-A192 fusion
protein nanoparticles are not spherical assembles to worm-like nanostructures. (Cryo-TEM images
were taken by Santosh Peddi and the research group of Dr. Honggang Cui.) (c) SEC-MALS reveals
the absolute molecular weight of refolded anti CD3-A192 fusion protein nanostructure is 4403
kDa, which implies that the fusion protein nanostructures consists of ~44 anti CD3-A192
monomers on average. The ratio of Rg / Rh is 1.02, which is consistent with a fusion protein
nanostructure with coil to rod-like elongated structures.
15

The thermal responsiveness property of anti CD3-A192 fusion protein
As previously mentioned, ELPs are temperature-sensitive polypeptides that undergo a reversible phase
separation above an inverse transition temperature. It has previously been shown that the transition
temperature of many ELP fusion proteins decreases compared to the pure ELP . (MacEwan et al., 2014)
To determine the phase separation behavior of anti CD3-A192 fusion protein, optical density (OD) at
350 nm is monitored while temperature increases. A192 is used to compare with anti CD3-A192 fusion
protein. The transition temperature of ELP A192 at 20μM alone is 60.1 °C, while the transition
temperature of ELP fusion protein is lower around 44.8°C. (Figure. 3 A, B) As the other ELPs alone, a
negative log–linear relationship could fit the relationship between the Tt and concentration of anti
CD3-A192 fusion protein. Thus, when ELP fusion protein binds with the receptor on cell surface and
gets locally concentrated, the transition temperature will be lower on cell surface than in circulation.

16

Figure 3. Anti CD3-A192 fusion protein is thermally-responsive. Optical density (350
nm) was plotted as a function of concentration and temperature for anti CD3-A192 fusion
protein and A192 (a) The transition temperature of CD3-A192 fusion protein at 20 μM is
around 44.8°C. (b) Concentration–temperature phase diagrams for the bulk phase transition
temperature (Tt) of anti CD3-A192 fusion protein and ELP A192. When compared to an
ELP that lacks the scFv domain, A192, the anti CD3-A192 fusion reduced the transition
temperature by ~15 °C. The dashed line indicates the 95% confidence interval around the
log-linear fit line.

17

Anti CD3-A192 nanoworms target the CD3 Receptor in two CD3+ cell lines
Cell surface staining experiments were performed based on flow cytometry and confocal microscopy
to test the binding activity of anti CD3-A192 nanoworm. Anti-ELP monoclonal antibody AK1 (Kouhi, A.,
Yao, Z.Y., Zheng, L., Hu,P ., Epstein, A., MacKay, J.A. (2019) Development of the First Monoclonal
Antibody for Detection of Elastin-Like Polypeptides, Unpublished manuscript) staining showed that anti
CD3-A192 nanoworm successfully binds on Jurkat and HUT 78 CD3+ Cell Line. (Figure. 4 A, D, G; B, E,
H) The same concentration of ELP A192 was used as a negative control to account for the non-specific
staining effect of anti-ELP monoclonal antibody to the ELP A192 region among the fusion protein. In
contrast, anti CD3-A192 nanoworms showed the minimal binding effect and in similar level with ELP
A192 negative control to CD3- cell line SU-DHL-7. These results confirmed that anti CD3-A192
nanoworms specifically recognize the CD3 receptor on the cell surface.

18

Figure 4. Anti CD3-A192 nanoworms cell surface CD3 receptor. Flow cytometry and
confocal microscopy was used to estimate the binding of anti CD3-A192 nanoworm. An anti-
ELP monoclonal antibody AK1 staining showed that anti CD3-A192 nanoworm successfully
binds on CD3+ cell line Hut 78 and Jurkat cell surface while the same concentration ELP A192
control shows minimal staining effect. In contrast, anti CD3-A192 nanoworms showed minimal
binding and in similar level with ELP A192 negative control to a CD3 negative cell line SU-
DHL-7. (a)(b)(c) Flow cytometry staining showed anti CD3-A192 nanoworm has 99.3%,
47.2%, 4.48% positive when gated by ELP A192 control on Hut 78, Jurkat, SU-DHL-7 cell lines
respectively. The data were analyzed using Flowjo software. (d)(e)(f), (g)(h)(i) confocal
microscope staining in fluorescence field and bright field of anti CD3-A192 nanoworm on Hut
78, Jurkat, SU-DHL-7 cell line. The data were analyzed using ImageJ.
19

Anti CD3-A192 Nanoworms inhibit the T Cell Activation by OKT3 antibodies on Jurkat
cells based on interleukin-2.
Solid phase-bound anti-CD3 monoclonal antibody OKT3 has been reported to induce TCR–CD3-
mediated activation in-vitro. (Lamers et al., 1992) In this study, Anti CD3-A192 nanoworms has been
observed to competitively bind with OKT3 to the CD3 receptor on Jurkat cell line and block the T cell
activation effect of OKT3 monoclonal antibodies. IL-2 secretion was tested by ELISA and used to
indicate the activation of Jurkat cells. To determine the effect of anti CD3-A192 nanoworms, 96-well
plates were precoated with different concentrations of OKT3 antibody (1-100ug/ml). For comparison
of IL-2 level after OKT3 activation, Jurkat cells were incubated with PBS and added to 96 well plate to
start culture. For the wells used to test the anti CD3-A192 nanoworm, Jurkat cell was pre-incubated
with anti CD3-A192 nanoworm for 45min at 4°C before adding. As the result, the pre-incubation of
anti CD3-A192 nanoworm could significantly lower the IL-2 secretion level compared to the cells
activated by the same concentration of OKT3 and the T-cell activation effect of pre-incubated cells
was minimal. 1-way ANOVA and Tukey HSD test showed a significant difference of the IL-2 secretion
level between OKT3 antibody stimulated group and preincubated group. (n=3 α= 0.05 P=0.0130, P
<0.0001). This result demonstrated that the effect of the OKT3 antibody was blocked when
pretreated with anti CD3-A192 nanoworms. And that suggests the anti CD3-A192 nanoworm
competitively bind to the same molecular target as OKT3 monoclonal antibody.

20

1
2
3
4
5
6
- 5 0
0
5 0
1 0 0
I L - 2 c o n c e n t r a t i o n p g / m l
1 . u n t r e a t e d
5 . p r e c o a t e d O K T 3 1 0 u g / m l
1 7 7 u g / m l n a n o w o r m t r e a t e d
6 . p r e c o a t e d O K T 3 1 0 0 u g / m l
1 7 7 0 u g / m l n a n o w o r m t r e a t e d
2 . O K T 3 1 u g / m l p r e c o a t e d
3 . O K T 3 1 0 u g / m l p r e c o a t e d
4 . O K T 3 1 0 0 u g / m l p r e c o a t e d
*
* * * *

Figure 5. Anti-CD3-A192 nanoworms suppress induction of IL-2 secretion from Jurkat
cells treated with OKT3. IL-2 secretion tested by ELISA was used to indicate the activation
of Jurkat cell. The group 2,3,4 showed the T cell activation effect of precoated OKT3 antibody
in concentration of 1, 10, 100ug/ml. Group 5 and 6 showed anti CD3-A192 nanoworm
preincubation significantly decrease the IL-2 level and T cell activation effect of OKT3
compared to group 3 and 4. Indicated errors are the mean ± SD. (n=3). (Tukey HSD, α=0.05
for OKT3 10ug/ml preincubated group (group 3 to group 5) P=0.0130 for OKT3 10ug/ml
preincubated group(group 4 to group 6) P <0.0001) Images and statistics were analyzed using
Prism Graphpad.

21

Experimental Procedures
Expression and Purification of Anti CD3-A1092 Fusion Protein
Through restriction enzyme digestion followed by sticky end ligation, anti-CD3 scFv was fused to ELPs.
The recombinant plasmid was verified as correct through DNA sequencing, and then the first amplified
in TOP10 competent cells and then transfected into chemically competent Origami B (DE3) Escherichia
coli using heat shock at 42 °C for 5 min. The transformed bacteria colonies were selected on ampicillin
(100 μg/L) agar plate for further fusion protein expression. Inoculate the selected colonies to 50 mL
terrific broth culture media with 100 μg/L of ampicillin overnight at 37 °C and 250rpm shaking.
Centrifuge the culture at 3000rpm, 4°C for 10 minutes to sediment the bacteria and Re-solubilized the
pellet with 5ml TB culture media. The bacteria suspension was inoculated into 5 L terrific broth media
with 100 μg/L of ampicillin overnight at 30 °C and 250rpm shaking. Bacterial cultures were centrifuged,
resuspended in cold phosphate buffered saline and lysed on ice using a periotic probe-tip sonicator
(Misonix, Farmingdales, NY). In order to remove insoluble debris, the cell lysate was centrifuged at
12000 rpm at 4 °C. Polyethyleneimine (0.5% w/v) was added to the crude cell lysate and incubated for
20min on ice to precipitate nucleic acids, and the insoluble cellular debris and DNA precipitates were
removed by centrifuging at 12,000rpm for 15 min at 4 °C. Soluble ELP proteins were purified from the
supernatant using an established technique called inverse transition cycling. (MacEwan et al., 2014)
These hot and cold centrifugation cycles were repeated twice to attain a sufficient purity. The final
purified protein batches were filtered using a 0.2 μm-micron sterile filter (Pall Corporation, Port
Washington, NY), and the protein concentrations were determined by measuring their absorbance at
22

280 nm and 350 nm using UV-Vis spectroscopy and protein concentration was determined using the
Beer-Lambert law:

concentration (M)=
𝐴 280
−A
350
MEC×l

Among the formula, A280 and A350 are the absorbances at 280 and 350 nm, the estimated molar
extinction coefficient (MEC) is 60,435 M
-1
cm
-1
and l is the path length (cm).
Determination of Purity of Anti CD3-A192 Fusions Protein
SDS-PAGE was used to estimate the molecular weight and the purity of fusion protein after purification.
3ug of fusion protein was added to SDS-PAGE loading buffer (final volume 20 μl) with reducing dye and
boiled at 95°C for 5 minutes. The samples were electrophoresed on 4-20% precast SDS-PAGE gel with
8 µL of Precision plus protein Kaleidoscope ladder at constant voltage 110V. After electrophoresis, the
gel was stained by a 10% copper chloride solution and imaged on a Biorad Versadoc imager (Hercules,
CA) using white light. The purity of the fusion protein was determined using ImageJ. In the software,
individual lanes were selected and the purity was determined by intensity profile of peak areas on each
lane:

Purity (%) =
𝐴 (𝑝𝑒𝑎𝑘 )
𝐴 (𝑡𝑜𝑡 )
× 100
Among the formula, A peak refers to the area of the peak of the target fusion protein, and A tot refers to
the area under all of the peaks.
23

Refolding of Anti CD3-A192 Fusion Protein
To refold the anti CD3-A192 scFv and ELP fusion protein, 6 M urea and 10mM 2-Mercaptoethanol (beta-
ME) were used in denaturing protein and reducing disulfide bonds. After added the denaturants, vortex
to completely solubilize the native fusion protein and incubate for 30 minutes at room temperature.
To facilitate gradual renaturation of the anti CD3-A192 scFv and ELP fusion protein, the denaturants
were gradually removed by step-wise dialysis using a dialysis cassette with a 20kDa molecular weight
cut off with a 100:1 sink condition at 4°C. Urea concentration was gradually reduced and beta-ME was
removed in the buffer, Arginine, GSH and GSSG were added in order to suppress protein aggregation
and facilitate disulfide-bond formation (Tsumoto et al., 1998; Tsumoto et al., 2004). denatured protein
was refolded by dialysis against 20 mM Tris base 150 mM NaCI (pH 8.0) with stepwise reduction of urea
(concentration 3,1,0.5,0 M) and arginine (concentration 500,250,125,62.5 mM). GSH/GSSG
(2mM/0.4mM) was added at first 2 steps of dialysis. Each dialysis was performed for about 24 hours.
After the aforementioned 4 changes of dialysis butter, the refolded fusion protein was dialysis against
PBS two times and the final stock was filtered through a sterile 0.2 μm filter.

Light Scattering Analysis Electron Microscopy of Nanoparticles
Dynamic light scattering (DLS) assay was performed to determine the hydrodynamic radius of anti CD3-
A192 fusions protein nanoworm. ELP A192 and anti CD3-A192 fusions protein nanoworm were diluted
with PBS to increasing concentration. All samples were sterile filtered using 0.45 μm filter. Prepared
24

samples were added to 384-well clear bottom plate and measured by Wyatt DynaPro Plate Reader.
CryoTEM imaging was used to determine the morphology of fusions protein nanoworm. Samples were
pipetted onto a TEM grid coated with a lacey carbon film (LC325-Cu, Electron Microscopy Sciences)
and imagines were acquired with accelerating voltage of 100 kV using FEI Tecnai 12 TWIN TEM
equipped with 16 bit 2Kx2K FEI eagle bottom mount camera (Hillsboro, OR).
To detect the absolute molecular weight and determine the R g/R h shape factor, the tandem size
exclusion chromatography and multiangle light scattering (SEC-MAL) are used in the study. The fusion
protein was sterile filtered using 0.45 μm filter before injected onto a Shodex size exclusion column
using PBS at 0.5 mL/min. The column eluents were analyzed on a Wyatt Helios system (Santa
Barbara,CA).

Transition Temperature of scFv-A192 Nanoparticles
ELP A192 and anti CD3-A192 fusion protein were diluted to varying concentrations (5 µM-35 µM) and
added to Tm microcell cuvettes (Beckman Coulter, Brea, CA). Then temperature was increased 1° C per
minute, and optical density (OD) at 350 nm was measured and recorded three times per minute (every
0.3 °C). The maximum first derivative of the OD with respect to temperature was defined to be the
transition temperature, indicating that the ELPs phase separated.

25

Cell Surface Staining Assay
The binding of anti CD3-A192 fusion protein nanoworm was estimated in CD3+ and CD3- cell line. For
CD3 cell line, Hut-78 cell and Jurkat cell line were used, and SU-DHL-7 was selected for CD3- cell line.
The cells were incubated for 1 h at 37 °C with humidified 5% carbon dioxide. Jurkat cell was cultured
in RPMI 1640 supplemented with 10% fetal bovine serum. Hut-78 cell was cultured in RPMI 1640
medium supplemented with 4.5g/L glucose, 0.2mM L-glutamine, and 10% fetal bovine serum. SU-DHL-
7 cell line was cultured in RPMI 1640 medium supplemented with HEPES and 10% fetal bovine serum.
10 µM fusion protein nanoworm and ELP A192 were added to 0.2 million cells resuspended in 1%
bovine serum albumin in Dulbecco's PBS. Cells were incubated at 4°C for 45min and washed 2 times
with 1% bovine serum albumin in Dulbecco's PBS. 0.5µg/ml anti-ELP AK1 (generated by Dr. Andrew
Mackay and Dr. Alan Epstein’s group) was used as primary antibody in staining and 1µg/ml Alexa Fluor
546 goat anti-mouse IgG(H+L) antibody was used as secondary antibody. The stained cells were
analyzed using flow cytometry and recorded data were analyzed using Flowjo (Ashland, OR).
After the flow cytometry analyses, the left samples were fixed by 2% paraformaldehyde on ice and
washed by Dulbecco's PBS. then cells were mounted in 20 ul Fluoromount™ Aqueous Mounting
Medium and sealed in slides. Confocal images were captured on a Zeiss LSM 800 confocal microscope
(Carl Zeiss Microscopy, Thornwood, NY). Images were analyzed using ImageJ.

26

Jurkat Cell Activation and IL-2 Detection
Jurkat cell was collected and diluted to 0.2million/ml in RPMI 1640 supplemented with 10% fetal
bovine serum. 96-well plates were used in cell culture and the plates were either untreated as
negative control or coated with OKT3 in the concentration 1ug/ml, 10ug/ml and 100ug/ml 70ul. For
the experiment group and OKT3 activation group, 60ul 35 uM anti CD3-A192 and 60ul PBS was added
to 60ul Jurkat cell suspension respectively and preincubated for 45min at 4 °C. Supernatant was
collected after 18 hours incubation at 37 °C with humidified 5% carbon dioxide. IL-2 was detected by
the ELISA assay and the microwell absorbances are read at 450 nm in proportion to the amount of IL-
2 present in the samples (Biorad benchmark plus plate reader (Hercules, CA). The IL-2 concentration
was determined by comparing to the standard curve build through the detection of stander IL-2
sample. Images and statistics were analyzed using Prism Graphpad.

27

Discussion
Utilizing molecular cloning and recombinant expression approaches, we here report a new anti CD3
scFv-based fusion protein nanoparticle. An anti-CD3 scFv and ELP A192 fusion protein may be useful
for low-cost expression and purification. The fusion protein is purified from the bacterial lysate of E-
coil expression system and ELPs are used as a purification tag based on inverse transition cycling
purification without any need for chromatography. This efficient and fast purification method and
expression system are economically favorable in comparison with mammalian-cell expression system.
The recombination expression of fusion protein polymer could eliminate the need for chemical
bioconjugation. The refolded fusion protein is observed to form a 34.8±5.9 nm worm-like
nanostructures. According to other studies of scFv-ELP fusion protein, the fusion protein
nanostructure before refolding process is spherical. And the formation of nanostructure could be due
to recombinant scFv multimerization. (Aluri et al., 2014b) (Dolezal et al., 2000) Base on that
hypothesis, the refolding process could control scFv multimerization and thus reduce nanoparticle
size and increase the binding efficiency of fusion protein nanostructure. That correlated with
phenomenon observed in this study and the previous study (Aluri et al., 2014b).
For the anti CD3-A192 nanoworms, in vitro binding experiment confirmed that it could specifically
recognize cell surface CD3 receptor on positive cell line. However, it requires a relatively high anti
CD3-A192 nanoworms concentration (10 µM). According to further study of scFv-ELP nanoparticles
and ELP fusion protein nanoparticles in other studies, the scFv remains in the core of the
nanoparticle and hydrophilic polypeptide is the corona. (Topcic et al., 2011) (MacKay et al., 2009) And
28

the detailed study about steric hindrance due to temperature-dependent β-turn maturation of the
ELP demonstrated scFv core remains accessible in the nanoparticle. (Topcic et al., 2011)
The in vitro T-cell activation study demonstrates that anti CD3-A192 nanoworm competitively binds
with OKT3 to CD3 receptor on Jurkat cell line and blocks the T cell activation effect of OKT3
monoclonal antibody. OKT3 recognize CD3 epsilon chain of the CD3 complex on T cells, while the
anti-CD3 scFv of the nanoworm is derived from anti-CD3 segment of Blinatumomab, which targets
the epsilon delta domain of the CD3 complex. (Clevers et al., 1988) Even though the OKT3 and anti
CD3-A192 nanoworm are targeting different region of CD3 complex, anti CD3-A192 nanoworm could
still competitively block the efficient of OKT3 antibody and turns the T-cell activation effect into
minimal. One possible explanation is that anti CD3-A192 nanoworm has a bulky structure and thus
blocks the binding of OKT3 antibody.
It is surprising that anti CD3-A192 nanoworm binding on CD3 receptor do not have the effect of T cell
activation. Based on this feature of anti CD3-A192 nanoworm, it could have application on T cell in-
vitro isolation and purification. For instance, currently, in the manufacture of CAR T cell therapy, anti-
CD3 antibody labeled magnetizable Dynabeads separation methods are used in order to isolate T cell
from mononuclear cells (MNCs) collected from patients. (Fesnak et al., 2016) However, this method
has some limitations, their binding can result in an artificial activation and modifications to cellular
functionality and morphology of the labeled cells. That could have a detrimental effect on isolated T
cell for further culture and downstream use (Abts et al., 1989). Consequently, an advanced protein
that could specifically bind to CD3 receptor but not activate T cell is desired for in-vitro T cell
29

purification and anti CD3-A192 nanoworm would be a good candidate for that application. Further
studies in estimating the efficiency of anti CD3-A192 nanoworm are thus warranted in improving the
widely used Dynabeads purification method and efficiently isolate T cell populations with a high
degree of purity and viability.
In conclusion, a recombinant fusion protein between anti-CD3 scFv a single chain and a soluble
protein polymer ELP has been successfully generated in this study. That fusion protein could self-
assemble worm-like nanostructures and specifically bind with CD3 receptor on the cell surface. And
these anti-CD3 hybrid protein polymer nanoparticles would not activate the CD3 receptor and could
block the in-vitro T cell activation effect of OKT3 monoclonal antibody.

30

Project 2: Use of the First Monoclonal Antibody to Detect Elastin-Like
Polypeptides in Western blot, ELISA, and Immunofluorescence Microscopy
Abstract:
Elastin-like polypeptides (ELPs) are protein-polymers derived from human tropoelastin that phase
separate above a tunable ‘transition temperature,’ which depends on their hydrophobicity, molecular
weight, and various solution properties. ELPs have been proposed for diverse applications, ranging
from drug delivery, protein purification, to biomaterials. Due to their overlap with mammalian self-
antigens found in the tropoelastin gene, some ELPs be regarded as ‘humanized.’ A major limitation to
their advancement has been a lack of specific antibodies, which could be used to characterize their
identity during purification, disposition upon cellular uptake, and biodistribution in the tissues and
plasma.
Consistent with the immune-tolerant properties of some ELPs, no anti-ELP monoclonal antibodies
(mAbs) have yet been reported. Therefore, to facilitate ELP studies, we successfully produced an anti-
ELP-mAb. The resulting anti-ELP mAbs are highly reactive and specific towards ELPs in ELISA assays,
western blotting, immunofluorescence assay, and through protein purification. In addition, anti-ELP
mAbs block the ELP-mediated phase separations under certain ratios.
These studies provide direct evidence that novel murine monoclonal antibodies can be raised against
purified ELPs. This new reagent empowers future studies to purify, detect, and characterize the cellular
and physiological disposition of ELPs and their resulting nanostructures.
31

Introduction
Elastin-like polypeptides (ELPs) are a class of unique protein polymers inspired from human
tropoelastin, which is the precursor to elastin. In last few decades, ELPS have been extensively studied
as an attractive versatile biopolymer. ELPs are composed of repeated pentameric motif [Val-Pro-Gly-
Xaa-Gly] n, where Xaa can be any guest amino acid and n indicate the repeat number of the pentameric
sequence. One unique feature of ELPS are they undergo inversible phase transition above a
characteristic transition temperature (Tt) determined by Xaa and n. Below the Tt, ELPs are highly
water-soluble and while the temperature raise above that ELPs would display a sharp phase separation
called a coacervate and from a secondary liquid phase. ELPs are attractive for multiple applications in
vitro and in vivo for tissue engineering and delivery of therapeutics because they are monodisperse,
biodegradable, biocompatible and low immunogenic. The advantages of using ELPs in generating novel
therapeutic include : i) being able to modulate cellular signaling;(Aluri et al., 2014a) ii) it could
recombinantly fused to antibody fragments without bioconjugation chemistry;(Mackay et al., 2010) iii)
potently engage multiple receptors;(Dhandhukia et al., 2017) iv) it would undergo proteolytic
biodegradation after endocytosis;(Despanie et al., 2016) and v) and some ELP sequences have the
potential for low antigenicity. (MacEwan and Chilkoti, 2010)
The antibodies based identification tools have become essential in biologic, clinical diagnostic, and
therapeutic protein analytical experiments. (Goding, 1996; Siddiqui, 2010) In particular, antibodies
based detection tools can quantitate the amount and location of a biologic target. antibody-based
assays are widely used both in early biologics development phases as well as mid- and late stage
32

discovery. (Clough and Hauer, 2005) For instance, from a regulatory standpoint, these reagents are
often used in batch release testing, and they have readily become a routine part of testing
procedures.(Clough and Hauer, 2005) Their critical role in product development has made it difficult
for biologics that lack a specific antibody against them to complete the development phase.
However, currently, due to the biocompatible and non-immunogenic feature of ELPs no anti-ELP mAbs
are not commercially available yet and no other groups have yet reported its successful production,
despite these potential applications. In the translation study of ELP and ELP biologics, a lack of mAbs
to characterize their identity during expression, and disposition upon cellular uptake has become a
major limitation. Aimed at facilitating ELP studies, we herein report successful production of an anti-
ELP mAb, designated AK1.
Therefore, to facilitate ELP studies, previous studies in our lab successfully produced an monoclonal
antibody AK1 towards ELP. (Kouhi, A., Yao, Z.Y., Zheng, L., Hu,P., Epstein, A., MacKay, J.A. (2019)
Development of the First Monoclonal Antibody for Detection of Elastin-Like Polypeptides, Unpublished
manuscript) A panel of three ELPs were used to hyperimmunize BALB/c female mice, and a monoclonal
hybridoma line was generated. This study provided direct evidence that novel murine monoclonal
antibodies can be raised against purified ELPs. The reactivity of these new reagents are showed in
ELISA assays, Western Blotting, and protein purification studies. Furthermore, this antibody can be
used in immunofluorescence assays to determine subcellular localization. Immunofluorescence based
assays that were employed in this study elucidated that AK1 has the potential to not only detect ELPs
in antibody-based assay, but also more importantly reveal specific behavior of these protein polymers
33

such as their ability to coacervate and form punctate like structures in vivo. This new reagent
empowers future studies to purify, detect, and characterize the cellular and physiological disposition
of ELPs and their resulting nanostructures.

34

Result
Immunoreactivity of purified anti-ELP AK1, in an indirect ELISA
The reactivity of purified anti-ELP AK1 against ELPs against ELPs are analyzed in an ELISA assay. ELPs
used in the detection including the three ELPs used in the immunization step and three other ELPs with
different sequence haven’t been used in the immunization step. Briefly, ELPs are coated 200ng/well in
multi-well plates and apparent antibody binding constant (Kd) was estimated through ELISA assay. The
result showed that 5 ELP antigens ranging from 0.4 to 0.6 nM. ELP S96 was the only notable exception
and the Kd was approximately one order of magnitude weaker, at 5 nM. (Figure 6.) Different ELPs in
the library consist of a repeated pentapeptide with 1 amino acids of the pentapeptide being different
and the number of times the pentapeptide is repeated. From the result it showed that the antibody-
antigen interaction is relatively independent of the guest amino acid residue (Xaa) and not only 3 ELPs
used in the immunization could be detected.

35

Figure 6. purified anti-ELP AK1 detects a panel of purified ELPs using indirect ELISA.
(a) Immuno-reactivity of AK1 was first tested against the ELPs used in immunization of the
mice. Based fitting to Eq. 1, the apparent K d for A96I96 is 0.6 nM ± 0.06 nM, for A192 is 0.8
nM ±0.1 nM, and for S48I48 is 0.4 nM ±0.07 nM. (b) Immuno-reactivity of AK1 was then
confirmed against three purified monoblocks ELPs, which were not included in the
immunization protocol. The K d for V96 is 0.6 nM ±0.07 nM, for S96 is 5 nM ±0.4 nM, and for
I48 is 0.6 nM ±0.07 nM. Data and estimates of K d represent the mean ± SD (n=3). This assay
was conducted in collaboration with Aida Kouhi.
36

Western blot analysis of anti-ELP AK1 specificity
To confirm the result of the indirect ELISA, western blotting was assessed to demonstrate anti-ELP AK1
was able to detect different ELPs including ELP fusion proteins regardless of their guest amino acid
residue. (Figure 2) Western blot result showed that anti-ELP AK1 can efficiently detect 6 different ELPs
and that corelated with result of indirect ELISA analysis. The only unexpected observation is I48 had to
be loaded in higher amount than other 5 ELPs in order to be detected. (Figure 7, A) For ELP A96I96,
A192, V96, S96 and S48I48, 0.1 ug loading amount was sufficient for anti-ELP mAb detection, while for
I48 anti-ELP mAb did not react with the antibody unless 2 μg of the protein was loaded onto the gel.
Since the antibody binding constant for I48 was similar to other 5 ELPs according to ELISA assay, one
possible explanation could be the difference between Western Blot and indirect ELISA procedures.
It is also demonstrated in cell lysate western blot assay that anti-ELP AK1 could also specifically detect
ELP without the interference of other unrelated proteins. Briefly, A96-CLC and V96-CLC
fusin protein plasmid was transfected into 293T cells and run on the gel. The western blot result shows
a these ELP fusions protein bind on 75 kD and no other band is detected in the lane which means the
antibody-antigen interaction is specific. (Figure 7, B)
Anti-ELP AK1 could be used in tracking ELP in the bacteria lysate during the inverse transition cycling
purification. As mentioned earlier, the inverse transition cycling purification involves cycling the protein
above and below the transition temperature of the ELP fusion followed by centrifugation to obtain the
desired protein, without any need for chromatography. However due to the lack of Anti-ELP mAb, there
was no qualitative study about the efficiency of this purification method. Based on the inverse
37

transition cycling purification, during the cooling cycle, the insoluble impurities when be removed in
pellets and ELP fusions are expected to become soluble and remain in the supernatant when
centrifuged. During the heating cycle, ELPs are expected to coacervate and collect in the pellet, and
the soluble impurities while be removed in supernatant. According to the result, the amount of ELP
remained in the pellet in cooling cycle and ELP remained in the supernatant in the heating cycle was
minimal, which indicated that the inverse transition cycling purification had good efficiency and ELP
lost during the purification step was minimal. (Figure 7, C)

38

Figure 7. AK1 efficiently detects ELPs during western immunoblotting from cellular
lysates. (a) 0.1 µg of purified ELPs (Table 1) were electrophoresed on lanes 2-7, while 2 µg was
loaded onto lane 8. To achieve equivalent detection, I48 required a higher loading amount than
the other ELPs, which possibly correlates with its less efficient electro-transfer to the
nitrocellulose membrane. (b) Immunoblotting was also used to verify the expression and purity
of two ELP clathrin-light chain fusions from cell lysates (20 µg) of HEK293T cells. Slightly
higher than their MW, these ELP fusions run near 75 kD. A 37 kD band was identified using an
anti-GAPDH mAb control. The control is untransfected 293T cell lysate. (c) AK1 was used to
track A192 during two rounds of ELP-mediated purification, which suggest that AK1 can be
useful optimize ELP purification procedures even in complex bacterial lysates. ‘Clarified lysate’
was separated from the ‘insoluble pellet’ and A192 was purified through two rounds of ELP-
mediated phase separation. The pellet (pel) and supernatant (sup) for hot centrifugation spins
(HS) and cold centrifugation spins (CS) are indicated.

-37-
kD
1 2 3 4 5 6 7 8 9 10 11 12
50
100
-75-
kDa
kDa
-75-
-50-
-20-
-75-
kDa
-37-
-100-
39

AK1 Immunofluorescence staining of ELP-fusion proteins expressed in mammalian cells
The ability of anti-ELP AK1 to stain ELP fusion proteins is also demonstrated in secondary
immunofluorescence imaging. In previous study of our team, genes encoding ELPs (A96 and V96) and
clathrin-light chain (CLC) fusion with myc tag was transiently transfected to 293T cell line. (Pastuszka
et al., 2014) In the original study, ELP fusion protein was expressed in 293 T cell line 2 days after
transfection. And cells were then incubated at either 4 °C or 37 °C to allow ELP fused CLC to solubilize
or coacervate and then stained with anti-myc antibody to detect the staining pattern. In this study, the
cell was stained with both anti-myc antibody and anti-ELP AK1 for comparison of staining ability and
staining pattern. The result showed that Anti-ELP AK1 successfully stained ELP fusion expressing cells
without any apparent cross-reactivity with untransfected cells (Figure 8). Furthermore, for anti-ELP
AK1 at 4 °C, which is below the intracellular Tt of V96.myc.CLC, the ELP fusion protein remains a
cytosolic staining diffuse pattern; while at 37 °C, which is above the intracellular Tt of V96.myc.CLC, the
fusions assemble punctate microdomains staining pattern. As a control, the A96.myc.CLC fusion
protein, whose apparent intracellular T t was higher than 37 °C, was not observed assembling
microdomains at 37 °C. That result correlates with anti-myc antibody staining pattern at 4 °C and 37 °C.
These data clearly show that it is possible to directly stain intracellular expressed ELP and
microdomains they formed without the need for additional epitope tags, such as myc.
40

Figure 8. Immunofluorescence microscopy using anti-ELP AK1 detects temperature-
dependent assembly of ELP fusions in mammalian cells. Genes encoding ELPs (A96 and
V96) and clathrin-light chain (CLC) fusion with myc tag was transiently transfected to 293T cell
line. After fusion protein was expressed, the samples was incubation at 4 or 37 °C and stained
by labeled anti-myc antibody (red), followed by anti-ELP AK1 (green). Correlating with anti-
myc antibody, A96-CLC remained soluble even when cells were incubated at 37 °C, which is
below the intracellular T t of A96-CLC. for anti-ELP AK1 at 4 °C, which is below the intracellular
Tt of V96.myc.CLC, the ELP fusion protein remains a cytosolic staining diffuse pattern; while
at 37 °C, which is above the intracellular Tt of V96-CLC, the fusions assemble punctate
microdomains staining pattern. This assay was conducted in collaboration with Aida Kouhi.
anti-myc
A96-CLC
37°C
Below T
t
A96-CLC
4°C
Below T
t
V96-CLC
37°C
Above T
t
V96-CLC
4°C
Below T
t

merged anti-ELP
41

Phase separation of ELPs in presence of anti-ELP AK1
As previously mentioned, ELPs are temperature sensitive polypeptides that undergo a reversible phase
separation above an inverse transition temperature. It has previously been shown that association of
ELPs with other molecules in the microenvironment can change their T t.(Dhandhukia et al., 2017) To
further examine this concept, the phase separation behavior of V96 ELPs in presence of varying AK1
concentrations was observed by measuring OD at 350 nm as a function of temperature. At the molar
ratio of ELP V96 to pentamer AK1 IgM monoclonal antibodies [50:1], which represents a great excess
of ELP to IgM, V96’s phase separation behavior was unchanged (Figure 7A). Interestingly, when the
anti-ELP antibody concentration increased in the solution to a molar ratio of V96:AK1 [5:1] or lower,
no temperature-dependent change in OD was observed. This loss of temperature-dependent phase
separation was also observed at a molar ratio of V96 to AK1 [10:1]; furthermore, this ratio is close to a
100:1 ratio between the concentration of an ELP pentameric motif (96 VPGVG repeats x 5 μM = 480
μM) and the Fv region on the AK1 IgM (10 x 0.5 μM= 5 μM). One possible explanation could be that
when AK1’s concentration in the mixture was high enough, all ELPs would be in association with
antibodies and no longer able to phase separate similar to free ELPs. This would also explain the high
baseline OD observed for the higher AK1 concentrations. As seen in Figure 7B the OD below T t shows
that from ELP V96 to antibody ratio [50:1], increasing amount of antibody added could result in higher
OD. However, one exception was the mixture at the V96 to antibody [3:1] ratio, which could be due to
the ELP-antibody complex, and may favor precipitation of crosslinked ELPs. 1-way ANOVA reveals a
significant difference between OD of V96 and AK1 mixtures in 4 different ratios (α= 0.05, P< 0.0001).
42

Post hoc analysis revealed a statistically significant difference between the OD of V96 and the OD of
groups with the ELP V96 to antibody ratio [3:1] (Tukey HSD, P =0.03*), [5:1] (Tukey HSD, P<0.0001****),
[10:1] (Tukey HSD, 0.008**).

43

Figure 7. An optimal ratio of anti-ELP AK1 suppresses the ELP temperature-dependent shift
in optical density (OD). (a) OD–temperature relationship was collected at 350 nm for V96 mixed
with AK1 IgM antibodies. The V96 concentration was fixed at 1 μM and the AK1 concentration
was mixed at the molar ratios of V96:IgM of 50:1, 10:1, 5:1, 3:1 and incubated for 30 mins prior
to heating. V96 alone phase separates above 37 °C, which is visible by an increase in OD.
Incubation with a 3:1 or 5:1 ratio of V96:IgM eliminated the temperature-dependent increase
in OD. (b) The OD at a fixed temperature (30 °C), which is below the phase transition
temperature for V96, was dependent on the mixture ratio to AK1 IgM. Indicated errors are the
mean ± SD. (n=3). (Tukey HSD,α=0.05 P =0.03*, <0.0001****, 0.008**, 0.997
ns
) (c)The increase
of OD may be consistent with the formation of complexes or networks with increased optical
density, albeit without temperature-dependent phase separation.

44

Method
Cell culture and transfection
293T cell line was used in this study for Immunofluorescence staining and cell lysate western blot. 293T
cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) that containing 10% FBS. For
plasmid transient transfection, Lipofectamine 3000 was used for 293T cells according to
manufacturer’s instructions. Cells were grown in a humidified, 5% CO 2 incubator at 37 °C and
hybridomas were frozen in liquid nitrogen for long-term storage using cryostor (Biolife Solutions Inc,
Bothel, WA).

Enzyme Linked Immunosorbent Assay (ELISA)
ELISA assay was used in detecting the reactivity of purified anti-ELP AK1 against ELPs against ELPs.
Three ELP antigens used in immunization and Three ELPs didn’t used in immunization were separately
diluted in PBS and coated in protein binding 96-well plates (100 μL/well, 2 μg/mL). Coated plate
was incubated overnight at 4 °C. After coated the plate was washed three times with PBS containing
0.5 % Tween-20 solution (PBST) for three times. 1X casein blocking buffer (MilliporeSigma, Burlington,
MA) blocking for one hour at 37 °C was used for preventing free protein binding. Serially diluted anti
ELP AK1 was incubated at 1 hour at 37 °C following three PBST washes. Horseradish peroxidase (HRP)-
conjugated goat anti-mouse IgM (Jackson Immunoresearch Labs, West Grove, Pennsylvania) was
diluted to 1:3000 with blocking buffer for one hour at 37 °C. 3,3´,5,5´-tetramentylbenzidine (TMB)
substrate (MilliporeSigma, Burlington, MA) was added to each well for 100 μL as a detection substrate
45

and were incubated at room temperature for 15 minutes. 100 μL of 2N H2SO4 was added to each well
to stop the reaction. The absorbance at 450 nm was detected using Synergy™ HT microplate reader
(BioTek, Winooski, VT). Following statistical analysis and calculation was conducted using GraphPad
Prism software. The apparent equilibrium disassociation of binding constant K d was determined
through
fitting the Abs 450 ELISA curve with nonlinear regression to the following saturation binding equation:
𝐴𝑏𝑠 450 𝑛𝑚
=
𝐵 𝑚𝑎𝑥 𝐶 𝑚𝐴𝑏 𝐾 𝑑 +𝐶 𝑚𝐴𝑏 + 𝐵 0
Eq. 1
Where C mAb is the concentration of the antibody, B max is the maximum shift in OD 450, B 0 is the baseline
background.
Western Blotting
To conduct Western Blotting assays for pure ELPs, a library of purified protein mixtures containing 4X
Laemmli sample buffer (Bio-Rad, Hercules, CA) with 2-Mercaptoethanol reducing reagent were
prepared and heated at 70 °C for 10 minutes. Pure ELP samples was Loaded in equal volume (20ul)
then separated by gel electrophoresis on a PAGEr EX 4-12% gradient gel along with molecular weight
markers. Gel electrophoresis was performed in constant voltage at 110V on ice in about 2hrs. Gel was
then transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA) using dry
blotting system iBlot2 (Thermo Fisher Scientific, Waltham, MA). The blot was blocked by TBST with 5%
Milk at room temperature for 1 hr. The blot was immunoblotted with anti-ELP AK1 (1:4000 dilution) as
primary antibody at 4 °C overnight in TBST with 5% Milk and then incubated with an HRP-conjugated
goat anti-mouse IgM as secondary antibody (Santa Cruz Biotechnology, Dallas, Texas).
46

Chemiluminescent substrate Super Signal™ West Pico Plus (Thermo Fisher Scientific, Waltham, MA)
was used to rinse the blot and visualize protein bands. Chemiluminescent signals were captured using
the ChemiDoc touch imager (Bio-Rad Laboratories, Hercules, CA).
To conduct Western Blotting assays for transfected ELP fusion protein in cell lysate, 293 T cell was
cultured and seeded in 35mm dishes. ELP fusion proteins plasmid were transfected to 293 T cell
according to method mentioned in cell culture method and incubated for 48 hours. The cell culture
dish in ice and wash the cells with ice-cold DPBS and whole cell lysates were prepared using RIPA buffer
(Cell Signaling Technology, Danvers, MA). The protein concentration for each cell lysate was
determined by BCA assay and total protein was loaded onto gels as described above and the same
protocol used for immunoblotting pure ELPs was followed.
To conduct Western Blotting assays to track ELP during purification in bacteria lysate, supernatant and
pellets was collected in purification method as described above in ELP production. 1.7 μL of bacteria
lysate from each sample was loaded onto the gel and samples are processed the same protocol as used
for Western Blotting of pure ELPs.
Immunofluorescence staining and confocal microscopy
To detect the reactivity of anti-ELP AK1 against ELPs fusion proteins expressed in cells,
Immunofluorescence assays were performed, and cells were imagined with confocal microscopy. 293T
cells was used in experiments and were cultured and plated on glass coverslips in 12-well plates. Cells
were transfected using lipofectamine 3000 with ELP-CLC fusion protein vectors that were previously
47

developed by our team (Thermo Fisher Scientist, Waltham, MA).(Kuhns et al., 2006; Pastuszka et al.,
2014) (Thermo Fisher Scientist, Waltham, MA).(Kuhns et al., 2006; Pastuszka et al., 2014) Cell was
incubated for 36 hours. Cells were first incubated on ice for 45 min to soluble ELP fusion protein,
followed by 45 min of incubation on ice or at 37 °C according to group design. After incubation, Cells
were then fixed with 4% paraformaldehyde (Alfa Aesar, Haverhill, MA) and washed with 50 mM
ammonium chloride, and permeabilized with 0.1% Triton X-100. Cells were blocked with 1% BSA for 1
hour at 37 °C and incubated with chicken anti-myc antibody (Abcam, Cambridge, MA) for 1 hour at
37 °C, washed, and incubated with Alexa 633 goat anti-chicken secondary antibody (Invitrogen,
Carlsbad, CA) for another hour at 37 °C. After the staining of anti-myc antibody, celled was incubated
with anti-ELP AK1 for 1 hour at 37 °C and followed by Alexa 430 goat anti-mouse secondary antibody
staining (Invitrogen, Carlsbad, CA) for 1 hour at 37 °. After the staining of anti-myc antibody and anti-
ELP AK1, cells were stained with DAPI and mounted with 20ul Fluoromount™ Aqueous mounting buffer
(Diagnostic Biosystems, Pleasanton, CA). Slides were imaged by Zeiss LSM 800 confocal microscope
(Carl Zeiss Microscopy, Thornwood, NY).
Phase separation behavior of ELPs in presence of anti-ELP AK1
To measure ELP’s T t in the presence of AK1, the following mixtures were prepared on ice. A sample
containing 1 µM V96 and no AK1; a sample containing 1 µM V96 and 0.33 µM AK1 ([3:1] ELP-AK1 molar
ratio); a sample containing 1 µM V96 and 0.2 µM AK1 ([5:1] ELP-AK1 molar ratio); a sample containing
1 µM V96 and 0.1 µM AK1 ([10:1] ELP-AK1 molar ratio); and a sample containing 1 µM V96 and 0.02
µM AK1 ([50:1] ELP-AK1 molar ratio). Samples were mixed on ice, and incubated at 4 °C for 30 min
48

while shaking to promote binding. Mixtures were then added to Tm microcell cuvettes (Beckman
Coulter, Brea, CA). Then temperature was increased 1° C per minute, and OD at 350 nm was measured
and recorded three times per minute (every 0.3 ° C). The maximum first derivative of the OD with
respect to temperature was defined to be the transition temperature, indicating that the ELPs have
phase separated.

49

Discussion
A novel monoclonal antibody that is highly reactive against Elastin-Like polypeptides (ELPs) has been
developed in previous study with augment immunogenicity of ELPs with Freund’s adjuvant and
Granulocyte-macrophage colony-stimulating factor (GM-CSF)(Leachman et al., 2000; Leenaars and
Hendriksen, 2005; Stills, 2005). A panel of three ELPs were used to hyperimmunize BALB/c female mice,
and a monoclonal hybridoma line was generated. Monoclonal antibody-producing cell lines were
further screened and two clones with high reactivity against the three ELPs were selected for further
characterization.
The anti-ELP antibody that was produced in this study was of an IgM isotype as revealed by an antibody
isotyping kit. Reactivity and affinity of the antibody was tested against ELPs in an ELISA assay and was
revealed anti-ELP antibody has high sensitivity to detect ELPs. Specificity of the antigen-antibody
interaction of ELP and ELP fusion proteins was tested in Western Blotting assays. Interestingly, the
purified AK1 was able to detect both ELP used in immunization and comprehensive library of ELP and
ELP fusion proteins. Furthermore, for other proteins that did not contain the pentapeptide of an ELP
in cell lysate, there was no cross-reactivity are observed. The finding that the antibody can detect a
broad range of ELPs may be due to a common epitope, Gly-Val-Pro-Gly, found in all of these constructs.
Furthermore, it is shown in this study that the anti-ELP antibody can be used in immunofluorescence
assays to determine subcellular localization. Immunofluorescence based assays that were employed in
this study elucidated that AK1 has the potential to reveal specific behavior of these protein polymers
50

such as their ability to coacervate and form punctate like structures in vivo.
As previously mentioned, ELPs are temperature sensitive polypeptides that undergo reversible phase
separation above an inverse transition temperature. When ELPs binds with anti-ELP antibody, with less
than 10 ELP chains per pentameric IgM complex, there may remain few unbound ELPs available to
phase separate. Loss of phase separation does not appear to require complete saturation of every ELP
binding motif. One explanation could be that when AK1’s concentration is high enough, ELPs interact
at multiple sites with the same pentameric IgM complex, which constrains ELPs into complex networks
unable to participate in temperature-dependent phase separation.
ELPs have been proposed for diverse applications, ranging from drug delivery, protein purification, to
biomaterials and have gained increased attention over the past decade.(Chilkoti et al., 2002; Dreher et
al., 2003; Kowalczyk et al., 2014; Lyons et al., 2014; Rodriguez-Cabello et al., 2016; Zhao et al., 2014;
Zhu et al., 2017) Because of lack of anti-ELP antibodies, other tools such as epitope tagging are used in
order to track ELPs.(Li et al., 2018; Ong et al., 2006). Even though the aforementioned techniques
successful is tracking ELPs, access to anti-ELP mAbs would be a superior approache to ELP detection
and tracking since antibody base identification methods are suitable to can quantitate the amount and
location of a biologic target and are widely used both in early biologics development phases as well as
mid- and late stage discovery. For instance, anti-ELP mAbs could be used to optimize bioprocessing,
quality control, cellular and tissue localization, toxicology, and ADME studies for ELPs and their fusions.
In conclusion, These studies provide direct evidence that novel murine monoclonal antibodies can be
raised against purified ELPs. This new reagent empowers future studies to purify, detect, and
51

characterize the cellular and physiological disposition of ELPs and their resulting nanostructures.

52

Reference
Abts, H., Emmerich, M., Miltenyi, S., Radbruch, A., and Tesch, H. (1989). CD20 POSITIVE HUMAN
LYMPHOCYTES-B SEPARATED WITH THE MAGNETIC CELL SORTER (MACS) CAN BE INDUCED TO
PROLIFERATION AND ANTIBODY SECRETION INVITRO. Journal of Immunological Methods 125, 19-28.
Aluri, S.R., Shi, P., Gustafson, J.A., Wang, W., Lin, Y.A., Cui, H., Liu, S., Conti, P.S., Li, Z., Hu, P., et al. (2014a).
A hybrid protein-polymer nanoworm potentiates apoptosis better than a monoclonal antibody. ACS Nano
8, 2064-2076.
Aluri, S.R., Shi, P., Gustafson, J.A., Wang, W., Lin, Y.A., Cui, H.G., Liu, S.L., Conti, P.S., Li, Z.B., Hu, P.S., et al.
(2014b). A Hybrid Protein - Polymer Nanoworm Potentiates Apoptosis Better than a Monoclonal Antibody.
Acs Nano 8, 2064-2076.
Brischwein, K., Parr, L., Pflanz, S., Volkland, J., Lumsden, J., Klinger, M., Locher, M., Hammond, S.A., Kiener,
P., Kufer, P., et al. (2007). Strictly target cell-dependent activation of T cells by bispecific single-chain
antibody constructs of the BiTE class. Journal of Immunotherapy 30, 798-807.
Chen, X.Y., Zaro, J.L., and Shen, W.C. (2012). Pharmacokinetics of recombinant bifunctional fusion proteins.
Expert Opinion on Drug Metabolism & Toxicology 8, 581-595.
Chen, X.Y., Zaro, J.L., and Shen, W.C. (2013). Fusion protein linkers: Property, design and functionality.
Advanced Drug Delivery Reviews 65, 1357-1369.
Chilkoti, A., Dreher, M.R., and Meyer, D.E. (2002). Design of thermally responsive, recombinant polypeptide
carriers for targeted drug delivery. Adv Drug Deliv Rev 54, 1093-1111.
Clevers, H., Alarcon, B., Wileman, T., and Terhorst, C. (1988). THE T-CELL RECEPTOR/CD3 COMPLEX - A
DYNAMIC PROTEIN ENSEMBLE. Annual Review of Immunology 6, 629-662.
Clough, N.E.C., and Hauer, P.J. (2005). Using Polyclonal and Monoclonal Antibodies in Regulatory Testing of
Biological Products. ILAR Journal 46, 300-306.
Combadiere, B., Sousa, C.R.E., Germain, R.N., and Lenardo, M.J. (1998). Selective induction of apoptosis in
mature T lymphocytes by variant T cell receptor ligands. Journal of Experimental Medicine 187, 349-355.
Despanie, J., Dhandhukia, J.P., Hamm-Alvarez, S.F., and MacKay, J.A. (2016). Elastin-like polypeptides:
Therapeutic applications for an emerging class of nanomedicines. J Control Release 240, 93-108.
Dhandhukia, J.P., Brill, D.A., Kouhi, A., Pastuszka, M.K., and MacKay, J.A. (2017). Elastin-like polypeptide
switches: A design strategy to detect multimeric proteins. Protein science : a publication of the Protein
Society 26, 1785-1795.
Dolezal, O., Pearce, L.A., Lawrence, L.J., McCoy, A.J., Hudson, P.J., and Kortt, A.A. (2000). ScFv multimers of
the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in V-
L to V-H orientation drives the formation of dimers, trimers, tetramers and higher molecular mass
multimers. Protein Engineering 13, 565-574.
Dreher, M.R., Raucher, D., Balu, N., Michael Colvin, O., Ludeman, S.M., and Chilkoti, A. (2003). Evaluation
of an elastin-like polypeptide-doxorubicin conjugate for cancer therapy. J Control Release 91, 31-43.
Ferran, C., Sheehan, K., Dy, M., Schreiber, R., Merite, S., Landais, P., Noel, L.H., Grau, G., Bluestone, J., Bach,
J.F., et al. (1990). CYTOKINE-RELATED SYNDROME FOLLOWING INJECTION OF ANTI-CD3 MONOCLONAL-
53

ANTIBODY - FURTHER EVIDENCE FOR TRANSIENT INVIVO T-CELL ACTIVATION. European Journal of
Immunology 20, 509-515.
Ferrer-Miralles, N., Domingo-Espin, J., Corchero, J.L., Vazquez, E., and Villaverde, A. (2009). Microbial
factories for recombinant pharmaceuticals. Microbial Cell Factories 8.
Fesnak, A., Lin, C.Y., Siegel, D.L., and Maus, M.V. (2016). CAR-T Cell Therapies From the Transfusion
Medicine Perspective. Transfusion Medicine Reviews 30, 139-145.
Gao, G.F., Rao, Z.H., and Bell, J.I. (2002). Molecular coordination of alpha beta T-cell receptors and
coreceptors CD8 and CD4 in their recognition of peptide-MHC ligands. Trends in Immunology 23, 408-413.
Goding, J.W. (1996). Monoclonal antibodies: principles and practice (Elsevier).
Hooks, M.A., Wade, C.S., and Millikan, W.J. (1991). MUROMONAB CD-3 - A REVIEW OF ITS
PHARMACOLOGY, PHARMACOKINETICS, AND CLINICAL USE IN TRANSPLANTATION. Pharmacotherapy 11,
26-37.
Hortiguela, M.J., Aumailley, L., Srivastava, A., Cunningham, C., Anandakumar, S., Robin, S., Pandit, A., Hu,
X.J., and Wall, J.G. (2015). Engineering recombinant antibodies for polymer biofunctionalization. Polymers
for Advanced Technologies 26, 1394-1401.
Kowalczyk, T., Hnatuszko-Konka, K., Gerszberg, A., and Kononowicz, A.K. (2014). Elastin-like polypeptides
as a promising family of genetically-engineered protein based polymers. World J Microbiol Biotechnol 30,
2141-2152.
Krangel, M.S. (1987). ENDOCYTOSIS AND RECYCLING OF THE T3-T CELL-RECEPTOR COMPLEX - THE ROLE OF
T3 PHOSPHORYLATION. Journal of Experimental Medicine 165, 1141-1159.
Kuhns, M.S., Davis, M.M., and Garcia, K.C. (2006). Deconstructing the form and function of the TCR/CD3
complex. Immunity 24, 133-139.
Lamers, C.H.J., Vandegriend, R.J., Braakman, E., Ronteltap, C.P.M., Benard, J., Stoter, G., Gratama, J.W., and
Bolhuis, R.L.H. (1992). OPTIMIZATION OF CULTURE CONDITIONS FOR ACTIVATION AND LARGE-SCALE
EXPANSION OF HUMAN LYMPHOCYTES-T FOR BISPECIFIC ANTIBODY-DIRECTED CELLULAR
IMMUNOTHERAPY. International Journal of Cancer 51, 973-979.
Leachman, S.A., Tigelaar, R.E., Shlyankevich, M., Slade, M.D., Irwin, M., Chang, E., Wu, T.C., Xiao, W.,
Pazhani, S., Zelterman, D., et al. (2000). Granulocyte-macrophage colony-stimulating factor priming plus
papillomavirus E6 DNA vaccination: effects on papilloma formation and regression in the cottontail rabbit
papillomavirus--rabbit model. Journal of virology 74, 8700-8708.
Leenaars, M., and Hendriksen, C.F. (2005). Critical steps in the production of polyclonal and monoclonal
antibodies: evaluation and recommendations. ILAR J 46, 269-279.
Li, Y.X., and Kurlander, R.J. (2010). Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-
CD3 for expanding human T cells: Differing impact on CD8 T cell phenotype and responsiveness to
restimulation. Journal of Translational Medicine 8.
Li, Z., Tyrpak, D.R., Park, M., Okamoto, C.T., and MacKay, J.A. (2018). A new temperature-dependent
strategy to modulate the epidermal growth factor receptor. Biomaterials 183, 319-330.
Loffler, A., Kufer, P., Lutterbuse, R., Zettl, F., Daniel, P.T., Schwenkenbecher, J.M., Riethmuller, G., Dorken,
B., and Bargou, R.C. (2000). A recombinant bispecific single-chain antibody, CD19 x CD3, induces rapid and
high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood 95, 2098-2103.
54

Lyons, D.F., Le, V., Kramer, W.H., Bidwell, G.L., 3rd, Lewis, E.A., Raucher, D., and Correia, J.J. (2014). Effect
of basic cell-penetrating peptides on the structural, thermodynamic, and hydrodynamic properties of a
novel drug delivery vector, ELP[V5G3A2-150]. Biochemistry 53, 1081-1091.
MacEwan, S.R., and Chilkoti, A. (2010). Elastin-like polypeptides: biomedical applications of tunable
biopolymers. Biopolymers 94, 60-77.
MacEwan, S.R., Hassouneh, W., and Chilkoti, A. (2014). Non-chromatographic Purification of Recombinant
Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli. Jove-Journal of
Visualized Experiments.
Mackay, J.A., Callahan, D.J., Fitzgerald, K.N., and Chilkoti, A. (2010). Quantitative model of the phase
behavior of recombinant pH-responsive elastin-like polypeptides. Biomacromolecules 11, 2873-2879.
MacKay, J.A., Chen, M.N., McDaniel, J.R., Liu, W.G., Simnick, A.J., and Chilkoti, A. (2009). Self-assembling
chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection.
Nature Materials 8, 993-999.
Moffitt, M., Yu, Y.S., Nguyen, D., Graziano, V., Schneider, D.K., and Eisenberg, A. (1998). Coronal structure
of star-like block ionomer micelles: An investigation by small-angle neutron scattering. Macromolecules 31,
2190-2197.
Neurauter, A.A., Bonyhadi, M., Lien, E., Nokleby, L., Ruud, E., Camacho, S., and Aarvak, T. (2007). Cell
isolation and expansion using Dynabeads((R)). In Cell Separation: Fundamentals, Analytical and Preparative
Methods, A. Kumar, I.Y. Galaev, and B. Mattiasson, eds., pp. 41-73.
Ong, S.R., Trabbic-Carlson, K.A., Nettles, D.L., Lim, D.W., Chilkoti, A., and Setton, L.A. (2006). Epitope tagging
for tracking elastin-like polypeptides. Biomaterials 27, 1930-1935.
Pastuszka, M.K., Okamoto, C.T., Hamm-Alvarez, S.F., and MacKay, J.A. (2014). Flipping the Switch on
Clathrin-Mediated Endocytosis using Thermally Responsive Protein Microdomains. Adv Funct Mater 24,
5340-5347.
Punt, J.A., Roberts, J.L., Kearse, K.P., and Singer, A. (1994). STOICHIOMETRY OF THE T-CELL ANTIGEN
RECEPTOR (TCR) COMPLEX - EACH TCR/CD3 COMPLEX CONTAINS ONE TCR-ALPHA, ONE TCR-BETA, AND 2
CD3-EPSILON CHAINS. Journal of Experimental Medicine 180, 587-593.
Rodriguez-Cabello, J.C., Arias, F.J., Rodrigo, M.A., and Girotti, A. (2016). Elastin-like polypeptides in drug
delivery. Adv Drug Deliv Rev 97, 85-100.
Schmidt, M., and Stockmayer, W.H. (1984). QUASI-ELASTIC LIGHT-SCATTERING BY SEMIFLEXIBLE CHAINS.
Macromolecules 17, 509-514.
Schmidt, S.R. (2009). Fusion proteins as biopharmaceuticals - Applications and challenges. Current Opinion
in Drug Discovery & Development 12, 284-295.
Schwartz, R.H. (1990). A CELL-CULTURE MODEL FOR LYMPHOCYTE-T CLONAL ANERGY. Science 248, 1349-
1356.
Sewell, A.K. (2012). Why must T cells be cross-reactive? Nature Reviews Immunology 12, 668-677.
Siddiqui, M.Z. (2010). Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci 72, 12-17.
Smith-Garvin, J.E., Koretzky, G.A., and Jordan, M.S. (2009). T Cell Activation. Annual Review of Immunology
27, 591-619.
Stills, H.F., Jr. (2005). Adjuvants and antibody production: dispelling the myths associated with Freund's
complete and other adjuvants. ILAR J 46, 280-293.
55

Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to
commercial systems. Applied Microbiology and Biotechnology 60, 523-533.
Topcic, D., Kim, W., Holien, J.K., Jia, F., Armstrong, P.C., Hohmann, J.D., Straub, A., Krippner, G., Haller, C.A.,
Domeij, H., et al. (2011). An Activation-Specific Platelet Inhibitor That Can Be Turned On/Off by Medically
Used Hypothermia. Arteriosclerosis Thrombosis and Vascular Biology 31, 2015-U2247.
Tsumoto, K., Shinoki, K., Kondo, H., Uchikawa, M., Juji, T., and Kumagai, I. (1998). Highly efficient recovery
of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled
introduction of oxidizing reagent - application to a human single-chain Fv fragment. Journal of
Immunological Methods 219, 119-129.
Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., Philo, J.S., and Arakawa, T. (2004). Role of arginine in
protein refolding, solubilization, and purification. Biotechnology Progress 20, 1301-1308.
Urry, D.W. (1997). Physical chemistry of biological free energy transduction as demonstrated by elastic
protein-based polymers. Journal of Physical Chemistry B 101, 11007-11028.
Van Wauwe, J.P., De Mey, J.H., and Goossens, J.G. (2016). OKT3: A MONOCLONAL ANTI-HUMAN T
LYMPHOCYTE ANTIBODY WITH POTENT MITOGENIC PROPERTIES. Journal of Immunology 197, 3431-3436.
Vaughan, T.J., Williams, A.J., Pritchard, K., Osbourn, J.K., Pope, A.R., Earnshaw, J.C., McCafferty, J., Hodits,
R.A., Wilton, J., and Johnson, K.S. (1996). Human antibodies with sub-nanomolar affinities isolated from a
large non-immunized phage display library. Nature Biotechnology 14, 309-314.
Viola, A., and Lanzavecchia, A. (1996). T cell activation determined by T cell receptor number and tunable
thresholds. Science 273, 104-106.
Weber, M., Bujak, E., Putelli, A., Villa, A., Matasci, M., Gualandi, L., Hemmerle, T., Wulhfard, S., and Neri, D.
(2014). A Highly Functional Synthetic Phage Display Library Containing over 40 Billion Human Antibody
Clones. Plos One 9.
Yeboah, A., Cohen, R.I., Rabolli, C., Yarmush, M.L., and Berthiaume, F. (2016). Elastin-Like Polypeptides: A
Strategic Fusion Partner for Biologics. Biotechnology and Bioengineering 113, 1617-1627.
Zhao, P., Dong, S., Bhattacharyya, J., and Chen, M. (2014). iTEP nanoparticle-delivered salinomycin displays
an enhanced toxicity to cancer stem cells in orthotopic breast tumors. Mol Pharm 11, 2703-2712.
Zhu, D., Wang, H., Trinh, P., Heilshorn, S.C., and Yang, F. (2017). Elastin-like protein-hyaluronic acid (ELP-
HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration. Biomaterials
127, 132-140.

Abstract (if available)

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Application of elastin-like polypeptides to therapeutics in leukemia

PDF

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

PDF

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

PDF

Immunotherapy of cancer

PDF

The role of CD99 in T cells

PDF

Stable cell lines expressing elastin-like polypeptide fusions with epidermal growth factor receptor modulate gene expression in a heat dependent manner

PDF

Temperature-mediated induction of caveolin-mediated endocytosis via elastin-like polypeptides

PDF

Physical chemistry and polymeric modification 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

Characterization of actin based motility in mammalian cells through LIM and SH3 domain protein 1 (LASP1) and elastin like polypeptide (ELP) fusion protein

PDF

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

PDF

Investigating the effects of targeting CD99 on T cells to enhance their antileukemia activity

PDF

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

PDF

FLT3/CD99 bispecific antibody-based nanoparticles (BiAbs) for acute myeloid leukemia

PDF

Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications

PDF

Development of engineered antibodies as novel anti-cancer agents

PDF

Generation and characterization of fully human anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies

PDF

Effects of particle architecture on in-vivo pharmacokinetics and bio-distribution of therapeutic nanostructures

PDF

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

Asset Metadata

Creator Yao, Zhiyuan (author)

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

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/24/2020

Defense Date 04/24/2019

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

Tag anti-CD3,anti-ELP,CD3 receptor,elastin-like polypeptide,microdomain monoclonal,OAI-PMH Harvest,single-chain variable fragment antibody

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, Andrew (committee chair), Xie, Jianming (committee member), Zhang, Yong (committee member)

Creator Email zhiyuanyao2016@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-145308

Unique identifier UC11662655

Identifier etd-YaoZhiyuan-7254.pdf (filename),usctheses-c89-145308 (legacy record id)

Legacy Identifier etd-YaoZhiyuan-7254.pdf

Dmrecord 145308

Document Type Thesis

Format application/pdf (imt)

Rights Yao, Zhiyuan

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