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Application of elastin-like polypeptides to therapeutics in leukemia

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Application of elastin-like polypeptides to therapeutics in leukemia

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Content Application of Elastin-like Polypeptides to Therapeutics in Leukemia
by
Mincheol Park
A Dissertation Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
PHARMACEUTICAL SCIENCES

August 2020

Copyright 2020 Mincheol Park
ii

Acknowledgements
First, I would like to thank Dr. J. Andrew MacKay for his constant advice, support, and belief during
my Ph.D. program at USC School of Pharmacy. I would also like to thank Dr. Houda Alachkar for
her guidance of Chapter 1, 2, and 3 studies and her support for those studies. I want to thank Dr.
Epstein for his advice and support in Chapter 4 and Dr. Jamie Spangler at Johns Hopkins
University for her advice in Chapter 5. Also, I want to thank all of my committee members, Dr.
Curtis Okamoto, Dr. Jianming Xie, and Dr. Pin Wang. These works would have not been done
without my colleagues and lab members. I want to especially thank Dr. Vijaya Pooja Vaikari from
Dr. Alachkar’s lab for her biggest contribution to Chapters 1, 2, and 3. Without her essential works,
these chapters would have not been completed. I want to thank Albert Lam from Dr. Zhang’s lab
for his contribution to Chapter 1. I want to thank Long Zheng from Dr. Epstein’s lab for his help
with Chapter 4 study. Finally, I am grateful to all of the previous and current lab members: Dr.
Jugal Dhandhukia, Dr. Zhe Li, Dr. Jordan Despanie, Dr. Santosh Peddi, Anh Truong, Changrim
Lee, David Tyrpak, Hao Guo, Hugo Avila, Jingmei Yu, Minchang Choi, Shruti Kakan, Siqi Lei,
Xiaoli Pan, Yaocun Li, Yaping Ju, Yue Wang, Yvette Kolodji, and Zhiyuan Yao.

iii

Table of Contents
Acknowledgements .................................................................................................................. ii
List of Tables ........................................................................................................................... vii
List of Figures ........................................................................................................................ viii
Abstract ..................................................................................................................................... x
Chapter 1: Formulation of elastin-like polypeptide nanoparticles targeting FMS-like
tyrosine kinase 3 for acute myeloid leukemia treatment....................................................... 1
1.1 Abstract ........................................................................................................................... 1
1.2 Introduction .................................................................................................................... 1
1.3 Materials and methods ................................................................................................... 3
1.3.1 α-FLT3-A192 cloning, expression and purification ................................................ 3
1.3.2 α-FLT3 scFv cloning, expression and purification ................................................ 8
1.3.3. α-FLT3-A192 protein concentration measurements ............................................10
1.3.4 α-FLT3-A192 purity and transition temperature analysis .....................................11
1.3.5 α-FLT3-A192 protein refolding and endotoxin measurement ..............................12
1.3.6 Measurements of the hydrodynamic radius of α-FLT3-A192 and colloidal
stability .............................................................................................................................13
1.3.7 Measurement of molecular weight of α-FLT3-A192 nanoparticles ......................13
1.3.8 α-FLT3-A192 binding study ....................................................................................13
1.3.9 Pharmacokinetic (PK) study of α-FLT3-A192 and α-FLT3 scFv ...........................14
1.3.10 Relative accumulation of α-FLT3-A192 nanoparticles using whole-tissue
fluorescence imaging ......................................................................................................15
1.3.11 Cell Culture ............................................................................................................16
1.3.12 Viability assays .....................................................................................................16
1.3.13 Flow cytometry analysis .......................................................................................17
1.3.14 Immunoblotting .....................................................................................................17
1.3.15 In vivo efficacy studies .........................................................................................18
1.3.16 Statistical Analysis ...............................................................................................19
1.4 Results ...........................................................................................................................19
1.4.1 Characterization of α-FLT3-A192 nanoparticles ...................................................19
1.4.2 α-FLT3-A192 binds specifically to FLT3 surface receptor....................................23
1.4.3 Antileukemic activity of α-FLT3-A192 in AML cells ..............................................25
1.4.4 α-FLT3-A192 inhibits the FLT3 signaling pathway................................................26
1.4.5 PK profiles of α-FLT3-A192 nanoparticles outperform α-FLT3 scFv ...................28
1.4.6 Anti-leukemia activity of α-FLT3-A192 in FLT3-ITD xenograft murine model .....32
iv

1.5 Discussion .....................................................................................................................34
1.6 Conclusion .....................................................................................................................38
1.7 Acknowledgements .......................................................................................................38
Chapter 2: Formulation of elastin-like polypeptide nanoparticles targeting CD99, a novel
target for acute myeloid leukemia treatment ........................................................................39
2.1 Abstract ..........................................................................................................................39
2.2 Introduction ...................................................................................................................39
2.3 Materials and methods ..................................................................................................41
2.3.1 Cloning and purification of α-CD99-A192 ..............................................................41
2.3.2 α-CD99-A192 protein concentration measurements.............................................43
2.3.3 α-CD99-A192 purity and transition temperature analysis ....................................43
2.3.4 α-CD99-A192 protein refolding ...............................................................................44
2.3.5 Measurements of the hydrodynamic radius of α-CD99-A192 and colloidal
stability .............................................................................................................................44
2.3.6 Measurement of the molecular weight of α-CD99-A192 nanoparticles ...............45
2.3.7 Patient Samples ......................................................................................................45
2.3.8 Cell Culture ..............................................................................................................45
2.3.9 Viability assays .......................................................................................................45
2.3.10 α-CD99-A192 competitive binding study .............................................................46
2.3.11 Cell apoptosis analysis .........................................................................................47
2.3.12 Pharmacokinetic (PK) and biodistribution study of α-CD99-A192 .....................47
2.3.13 In vivo efficacy studies .........................................................................................48
2.3.14 Statistical Analysis ...............................................................................................48
2.4 Results ...........................................................................................................................49
2.4.1 Characterization of α-CD99-A192 nanoparticles ...................................................49
2.4.2 α-CD99-A192 binds specifically to CD99 surface protein in vitro ........................51
2.4.3 α-CD99-A192 induces cell apoptosis in AML cells ...............................................54
2.4.4 α-CD99-A192 exhibits anti-leukemia activity in primary AML cells .....................56
2.4.5 PK profile of α-CD99-A192 nanoparticles fits the biexponential decay model ...57
2.4.6 α-CD99-A192 reduces leukemia burden and extends survival of mice in AML
xenograft model ...............................................................................................................60
2.5 Discussion .....................................................................................................................61
2.6 Conclusion .....................................................................................................................64
2.7 Acknowledgements .......................................................................................................64
Chapter 3: Formulation of ELP nanoparticles stimulating human granulocyte-
macrophage colony-stimulating factor receptors for post leukemia treatment .................65
v

3.1 Abstract ..........................................................................................................................65
3.2 Introduction ...................................................................................................................65
3.3 Materials and methods ..................................................................................................68
3.3.1 hGMCSF-A192 cloning & purification ....................................................................68
3.3.2 hGMCSF-A192 protein concentration ....................................................................69
3.3.3 hGMCSF-A192 purity and transition temperature analysis ..................................69
3.3.4 Thermodynamics of hGMCSF-A192 phase separation .........................................70
3.3.5 Characterization Studies of hGMCSF-A192 ..........................................................71
3.3.6 WST-1 cell proliferation assay ...............................................................................72
3.3.7 TF-1 mouse engraftment study In vivo ..................................................................73
3.3.8 Statistical Analysis .................................................................................................74
3.4 Results ...........................................................................................................................74
3.4.1 hGMCSF-A192 is highly expressed in E. coli, and the fusion of A192 to hGMCSF
serves as a solubilizing enhancer and a purification tag ..............................................74
3.4.2 Enthalpy and entropy of hGMCSF-A192 coacervation are higher for unmodified
A192 ..................................................................................................................................78
3.4.3 hGMCSF-A192 forms disulfide bonds and stable nanoparticles .........................79
3.4.4 hGMCSF-A192 nanoparticles are biologically active in vitro. ..............................83
3.4.5 hGMCSF-A192 enhances engraftment of TF-1 cells in xenograft murine model 84
3.5 Discussion .....................................................................................................................85
3.6 Conclusion .....................................................................................................................89
3.7 Acknowledgements .......................................................................................................89
Chapter 4: Design of an activatable anti-CD19 chimeric antigen receptor using elastin-like
polypeptides for B-cell acute lymphoblastic leukemia treatment .......................................91
4.1 Abstract ..........................................................................................................................91
4.2 Introduction ...................................................................................................................92
4.3 Materials and methods ..................................................................................................93
4.3.1 α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP cloning ..........................................93
4.3.2 Transient expression with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP ...........95
4.3.3 Live cell imaging with a temperature ramp ...........................................................95
4.3.4 Production of lentivirus with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP gene
..........................................................................................................................................95
4.3.5 Lentiviral transduction with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP gene
to make stable cell lines ..................................................................................................96
4.3.6 Confocal microscope imaging ...............................................................................97
4.4 Results ...........................................................................................................................97
vi

4.4.1 α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP express in 293T and Jurkat cells 97
4.4.2 Lentivirus with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP is producible, and
293T and Jurkat cells are transducible with the lentivirus ............................................97
4.5 Discussion .....................................................................................................................99
4.6 Conclusion ................................................................................................................... 101
4.7 Acknowledgements ..................................................................................................... 101
Chapter 5: Formulation of elastin-like polypeptide nanoparticles targeting cytotoxic T-
lymphocyte antigen-4 to harness the immune system for acute myeloid leukemia
treatment ............................................................................................................................... 102
5.1 Abstract ........................................................................................................................ 102
5.2 Introduction ................................................................................................................. 102
5.3 Methods........................................................................................................................ 104
5.3.1 Cloning and purification of α-CTLA4-A192 ......................................................... 104
5.3.2 α-CTLA4-A192 protein concentration measurements ........................................ 106
5.3.3 α-CTLA4-A192 purity and transition temperature analysis ................................ 107
5.3.4 α-CTLA4-A192 protein refolding .......................................................................... 107
5.3.5 Measurements of the hydrodynamic radius of α-CTLA4-A192 and colloidal
stability ........................................................................................................................... 107
5.3.6 Measurement of the molecular weight of α-CTLA4-A192 nanoparticles ........... 108
5.4 Results ......................................................................................................................... 109
5.4.1 α-CTLA4-A192 is highly expressed in E. coli, and the fusion protein purified
using temperature-dependent phase separation is soluble and pure. ....................... 109
5.4.2 The fusion of α-CTLA4 scFv to A192 decreases the transition temperature of
A192 by increasing ΔH coacervation and ΔScoacervation ......................................................... 110
5.4.3 α-CTLA4-A192 forms stable nanoparticles ......................................................... 111
5.5 Discussion ................................................................................................................... 112
5.6 Conclusion ................................................................................................................... 115
5.7 Acknowledgements ..................................................................................................... 115
References ............................................................................................................................ 116

vii

List of Tables
Table 1: Biophysical characterization of elastin-like polypeptides evaluated in Chapter 1……..22
Table 2: Pharmacokinetic Parameters of α-FLT3-scFv and α-FLT3-A192 nanoparticles
following IV administration using a non-compartmental analysis………………...………………..31
Table 3: Pharmacokinetic Parameters of α-FLT3-scFv and α-FLT3-A192
nanoparticles following IV administration using a two-compartment model…………..……….…31
Table 4: Characterization of α-CD99-A192 nanoparticles……………………………………..…51
Table 5: PK parameters for α-CD99-A192………………………………………………………...…59
Table 6: Sequence and biophysical characterization of A192 and hGMCSF-A192…………..…79
Table 7: Biophysical characterization of elastin-like polypeptides evaluated in Chapter 5……110
Table 8: Thermodynamics of A192 phase separation in scFv-A192…………………………….112

viii

List of Figures
Figure 1: Characterization of an elastin-like polypeptide (ELP) fusion protein targeting
the FLT3 receptor tyrosine kinase...…………………………………………………………………..21
Figure 2: Recombinant α-FLT3-A192 fusions form stable nanoparticles………………………...22
Figure 3: Recombinant α-FLT3-A192 binds to FLT3 receptor tyrosine kinase…………………..24
Figure 4: α-FLT3-A192 has anti-leukemic activity in AML cells…………………………………...26
Figure 5: α-FLT3-A192 inhibits FLT3 downstream signaling………………………………………27
Figure 6: The pharmacokinetic terminal half-life of α-FLT3-A192 nanoparticles exceed that
of a control α-FLT3 scFv……………………………......................................................................29
Figure 7: α-FLT3-A192 remains intact in the plasma over longer durations than α-FLT3 scFv.30
Figure 8: Anti-leukemia activity of α-FLT3-A192 in FLT3-ITD murine model…………………….33
Figure 9: Construction, purification and characterization of a recombinant elastin-like
polypeptide (ELP) fusion protein targeting CD99 proteins……….………………………………...50
Figure 10: Recombinant α-CD99-A192 binds specifically to CD99….…………………………...53
Figure 11: α-CD99-A192 has anti-leukemic activity in AML cells…………………………………55
Figure 12: α-CD99-A192 induces apoptosis in AML cells………………………………………….56
Figure 13: α-CD99-A192 has anti-leukemic activity in primary AML cells………………………..57
Figure 14: PK profile of α-CD99-A192 nanoparticles……………………………………………….58
Figure 15: Anti-leukemia activity of α-CD99-A192 in MOLM-13 murine model………………….61
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Figure 16: ELP fusion enables temperature-dependent purification of hGMCSF fusions from the
soluble fraction of bacterial lysate……………………………………………………………………..75
Figure 17: Fusion to hGMCSF shifts the phase diagram and thermodynamics of A192 phase
separation………………………………………………………………………………………………..77
Figure 18: Fusion of A192 to hGMCSF results in nanoparticles…………………………………..81
Figure 19: hGMCSF-A192 nanoparticles are stable colloids at physiological temperature……82
Figure 20: hGMCSF-A192 nanoparticles consist of several hundred fusion proteins…………..83
Figure 21: hGMCSF-A192 nanoparticles are biologically active……………………………….…84
Figure 22: An in vivo experiment verifies activity of hGMCSF-A192 in a mouse model………..85
Figure 23: α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP are expressible in 293T and Jurkat
cells and transducible with lentivirus………………………………………………………………...98
Figure 24: Cloning and purification of α-CTLA4-A192…………………………………………….109
Figure 25: α-CTLA4-A192 has a lower transition temperature than A192, and it forms stable
colloids at 37 °C……………………………………………………………………………………..…111
Figure 26: Size exclusion chromatography with multi-angle light scattering reveals that 310 α-
CTLA4-A192 molecules form a nanoparticle……………………………………………………….112

x

Abstract
In this dissertation, applications of protein polymers for leukemia treatment will be discussed.
Elastin-like polypeptides (ELPs), derived from human tropoelastin, are protein polymers that
consist of the pentameric amino acid repeat, (VPGXG) n where “X” represents a guest amino acid
that can be any amino acid, and “n” indicates the number of the pentameric repeat. ELPs have a
characteristic that they undergo reversible temperature-dependent phase separation above the
transition temperature, which is determined by “X” and “n.” As ELP amino acid sequences are
similar to that of human tropoelastin, they are biocompatible and biodegradable. ELPs are
genetically encodable, so it is easy to modify transition temperature and molecular weight.
Previously, our lab showed that a high molecular weight ELP, A192, fused to scFvs assembled
worm-like nanoparticles. In this dissertation, it will be shown how these characteristics of ELPs
will be harnessed in order to formulate nanoparticles, facilitate the purification, and enhance the
pharmacokinetic properties of small protein drugs for leukemia treatment. In addition, it will be
explored how the temperature-dependent phase separation of ELPs can be used as a molecular
activation switch of chimeric antigen receptors (CARs). In chapter 1, a high molecular weight ELP
is fused to an anti-FLT3 single-chain variable fragment (scFv) to formulate nanoparticles targeting
FMS-like tyrosine kinase 3 (FLT3) receptor, which is a known target for acute myeloid leukemia
(AML) treatment. It will be shown how the fusion of the ELP to anti-FLT3 scFv solubilizes the scFv,
makes the purification easier, forms stable nanoparticles, and enhances the pharmacokinetic
properties while maintaining the biological activity in vitro and in vivo. In chapter 2, similarly to
chapter 1, a high molecular weight ELP will be employed to formulate nanoparticles targeting the
CD99, which is a novel target for AML. In this Chapter, it will be shown that a high molecular
weight ELP fused to anti-CD99 scFv generates the soluble fusion protein, facilitates the
purification of the fusion protein, forms stable nanoparticles, and enhances the pharmacokinetic
properties of anti-CD99 scFv. In Chapter 3, a high molecular weight ELP will be fused to a small
protein, human granulocyte-macrophage colony-stimulating factor (hGMCSF) for post-leukemia
xi

treatment. hGMCSF is indicated for post-leukemia treatments to shorten time to neutrophil
recovery and accelerate myeloid reconstitution following peripheral blood progenitor cell
transplantation, autologous bone marrow transplantation, and allogeneic bone marrow
transplantation. In this Chapter, it will be shown that the fusion of ELP to hGMCSF solubilizes
hGMCSF, which is known to form protein aggregates, inclusion bodies, when produced in E. coli,
and that the ELP fusion allows the purification of pure hGMCSF without chromatography and
purification tags. In addition, it will be seen that the ELP fusion protein forms biologically active
nanoparticles in vitro and in vivo. In Chapter 4, it will be discussed ELPs as intracellular molecular
switches of an anti-CD19 CAR for acute lymphoblastic leukemia (ALL). In this chapter, it will be
shown the preliminary data of ELP fusion to an anti-CD19 CAR expressed in human cell lines and
will be discussed the potentials of CAR ELP fusion technology and future studies. Finally, in
Chapter 5, a high molecular weight ELP will be employed to formulate nanoparticles targeting
cytotoxic T-lymphocyte antigen 4 (CTLA4) to harness the host immune system for AML treatment.
In this chapter, preliminary data that show the expression and purification of the ELP fused to
anti-CLTA4 scFv and formulation of stable nanoparticles will be shown, and potentials and future
works will be discussed. Through Chapter 1 to Chapter 5, it is demonstrated that ELPs can serve
as a drug carrier by forming stable nanoparticles and may have potential as a molecular switch
for CAR activation.

1

Chapter 1: Formulation of elastin-like polypeptide nanoparticles targeting FMS-like
tyrosine kinase 3 for acute myeloid leukemia treatment
1

1.1 Abstract
Overexpression and mutation of FMS-like tyrosine kinase 3 (FLT3) are associated with poor
clinical outcome of acute myeloid leukemia (AML). For that reason, FLT3 is recognized as an
important therapeutic target for AML treatment, and targeted therapies against the FLT3, such as
small-molecule tyrosine kinase inhibitors (TKIs) and anti-FLT3 antibodies, have shown promising
efficacy in both preclinical and clinical studies. Currently, two small-molecule FLT3 inhibitors have
been approved by the FDA, but these inhibitors have shown limits, such as the development of
drug resistance and off-target toxicity. Although FLT3 is a valid therapeutic target, new
approaches to target FLT3 are necessary to enhance the therapeutic efficacy for AML treatment.
In Chapter 1, as a new approach to target FLT3, we employed elastin-like polypeptides to
formulate nanoparticles targeting FLT3. A high molecular weight elastin-like polypeptide, A192,
was fused to an α-FLT3 single-chain fragment variable, and the fusion protein, α-FLT3-A192,
assembled stable nanoparticles with great anti-leukemic activities in vitro and in vivo and
pharmacokinetic properties. In conclusion, α-FLT3-A192 nanoparticles appear to be a viable new
therapeutic approach for targeting FLT3 in AML, and the nanoparticles might have benefits over
TKIs and anti-FLT3 monoclonal antibodies, such as low toxicity and better efficacy through the
enhanced accumulation in the bone marrow and spleen.
1.2 Introduction
Acute myeloid leukemia (AML) is a hematological malignancy that exhibits uncontrolled
proliferation of poorly differentiated and immature myeloid cells (Lowenberg, 2008), and its
prognosis has been poor, which shows 5-year survival rate less than 30% (Daver et al., 2019).

1
This chapter was referenced to a manuscript, titled “Anti-FLT3 nanoparticles for acute myeloid leukemia: Preclinical
pharmacology and pharmacokinetics” by Park et al.

2

Among AML patients with the poor prognosis, the mutation of FMS-like tyrosine kinase 3 (FLT3)
is frequently observed (Kiyoi et al., 1999). One of the mutations, FLT3-internal tandem duplication
(FLT3-ITD), occurs in the juxtamembrane domain of FLT3, and it is associated with constitutive
activation of the FLT3 kinase and downstream of JAK/STAT5, Raf/MEK/ERK, and PI3K/Akt,
which regulate growth and survival of leukemic cells (Ravandi et al., 2010; Takahashi, 2011). As
constitutive activation of FLT3 kinase is observed, several small-molecule tyrosine kinase
inhibitors (TKIs), such as midostaurin, gilteritinib, and sorafenib, have been evaluated for AML
treatment (Chappell et al., 2019; Research, 2019; Stone et al., 2017). Although these TKIs have
shown great efficacy in patients with relapsed or refractory AML as first-line therapy, the
development of drug resistance has been observed due to a secondary mutation in FLT3 kinase
domain (Daver et al., 2015; Williams et al., 2013). Also, they are TKIs, so they can have off-target
toxicity resulted from inhibiting non-target tyrosine kinases. Therefore, antibodies specifically
targeting FLT3 could solve the low specificity problem of the current FLT3 targeted therapy.
Single-chain antibody fragments (scFvs) are derived from antibodies and consist of
variable heavy chain (VH) and light chains (VL). As derived from antibodies, they have high binding
affinity and specificity. They exhibit lower immunogenicity and are easy to engineer with
recombinant DNA technology. Unlike monoclonal antibodies, scFvs have shown very short half-
lives in murine models due to the rapid clearance from the kidneys as their average molecular
weight is 30 kDa (Hutt et al., 2012). Usually, monoclonal antibodies are expressed and purified
from mammalian cells, but scFvs can be purified from Escherichia coli, which can be more
economical and easier (Skerra and Pluckthun, 1988). However, scFvs expressed in E. coli tend
to form protein aggregates, known as inclusion bodies in the periplasm (Hayhurst and Harris,
1999; Schein, 1989). To solubilize, the inclusion bodies need to be denatured and refolded
(Lavallie et al., 1993), and the process takes time and lowers the final yield of the protein

3

(Thomson et al., 2012). Therefore, it is important to enhance both pharmacokinetic profiles and
stability to use scFvs in the clinic.
To improve both pharmacokinetic profiles and stability, elastin-like polypeptides (ELPs)
was fused to an anti-FLT3 scFv. ELPs are genetically engineered protein polymers that consist
of the pentameric amino acid repeats (VPGXG)n, which is derived from human tropoelastin. “X”
is a guest residue that can be any amino acid, and “n” indicates the number of the pentameric
repeats. ELPs are biocompatible and biodegradable as they are derived from human tropoelastin
(Despanie et al., 2016). ELPs undergo reversible phase separation above the transition
temperature, which is determined by the hydrophobicity and molecular weight. The phase
separation can be induced with mild salts and heat, and by using the reversible phase separation,
proteins fused to ELPs can be purified with high purity without chromatography and purification
tags (Christensen et al., 2009). Previously, our lab showed the fusion of a high molecular weight
ELP, A192, to an scFv targeting CD20 generated biologically active nanoparticles (Aluri et al.,
2014). In this chapter, we will show the formulation of nanoparticles targeting FLT3 by fusing A192
to anti-FLT3 scFv and enhanced pharmacokinetic profiles and stability of anti-FLT3 scFv by the
formulation. Also, the potential benefits of the nanoparticles over TKIs and anti-FLT3 monoclonal
antibodies will be discussed.
1.3 Materials and methods
1.3.1 α-FLT3-A192 cloning, expression and purification
The anti-FLT3 (α-FLT3) scFv gene was inserted to the amino terminus of a high molecular weight
ELP, A192 (73 kDa), in the pET-25b(+) vector, encoding α-FLT3-A192. The DNA sequence of the
α-FLT-A192 is:
ATGGAAGTCCAACTGGTCCAGTCAGGCGCTGAGGTAAAGAAGCCGGGCGCAAGCGTGAA
AGTATCTTGTAAGGCTTCCGGGTACACTTTTACTTCCTACTATATGCACTGGGTGCGTCAAG
CCCCCGGACAGGGCCTGGAGTGGATGGGGATTATTAACCCCTCGGGCGGTAGTACATCG

4

TACGCCCAAAAGTTTCAAGGCCGCGTAACTATGACGCGCGATACCTCCACAAGTACAGTAT
ATATGGAACTTAGCAGTTTGCGTTCCGAGGATACCGCCGTTTATTACTGCGCTCGCGGCGT
AGGTGCACACGATGCTTTCGACATCTGGGGCCAAGGGACAACAGTTACAGTCAGTAGTGG
TGGAGGTGGCTCGGGTGGCGGCGGCAGCGGAGGCGGTGGAAGCGACGTCGTAATGACG
CAATCTCCCCTTTCTTTGCCTGTTACGCCCGGCGAGCCTGCTTCCATTTCCTGTCGCTCCA
GTCAATCTTTGTTACATTCAAACGGGAACAATTATCTGGATTGGTACTTGCAAAAACCAGGC
CAAAGTCCTCAGCTGTTGATTTACTTGGGGAGCAATCGTGCATCGGGGGTGCCTGACCGC
TTTTCAGGTAGCGGGTCAGATACCGATTTCACGCTGCAAATCTCCCGTGTTGAGGCCGAAG
ACGTTGGCGTGTATTACTGCATGCAGGGAACTCATCCAGCAATCTCATTTGGTCAGGGGAC
TCGCTTGGAAATCAAGCTTGTACCCCGCGGAAGTGGTGTTCCGGGCGCTGGTGTACCAGG
TGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGG
GTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCT
GGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACC
AGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTC
CGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTA
CCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGT
ACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGT
GTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGG
CGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTG
GTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCC
GGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGC
CGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGC
GCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGG
TGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGG
GTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCG
GGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCC

5

GGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTAC
CGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTT
CCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGT
CCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTG
TACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGT
GTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGG
TGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAG
GTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCT
GGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGC
AGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTG
CAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGT
GCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGG
CGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAG
GTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCG
GGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACC
TGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTAC
CAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTT
CCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGT
ACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTG
TACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGT
GTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGG
CGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTG
GTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCC
GGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGC
CGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGC
GCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGG

6

TGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGG
GTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTTCCG
GGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGTCCC
GGGTGCCGGTGTTCCGGGTGCTGGTGTTCCGGGCGCTGGTGTACCAGGTGCAGGTGTAC
CGGGTGCCGGCGTACCTGGCGCAGGTGTCCCGGGTGCCGGTGTTCCGGGTGCTGGTGTT
CCGGGCGCTGGTGTACCAGGTGCAGGTGTACCGGGTGCCGGCGTACCTGGCGCAGGTGT
CCCGGGTGCCGGTGTTCCGGGTGCTGGTTAC
The encoded amino acid sequence of α-FLT3-A192 is:
MEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGAHDAFDIWGQGTTVTVSSGGG
GSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGNNYLDWYLQKPGQSP
QLLIYLGSNRASGVPDRFSGSGSDTDFTLQISRVEAEDVGVYYCMQGTHPAISFGQGTRLEIKL
VPRGSGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPG
AGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVP
GAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGV
PGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGA
GVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPG
AGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVP
GAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGV
PGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGA
GVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPG
AGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVP
GAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGV

7

PGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG
VPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGA
GVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPG
AGVPGAGVPGAGVPGAGVPGAGY
The molecular weight of α-FLT3-A192 is expected to be 100.1 kDa. For the cloning, α-FLT3 scFv
gene was purchased (Integrated DNA Technology, IA, USA), and the purchased gene was
inserted into an empty pET-25b(+) vector using NdeI and BamHI restriction enzymes (New
England Biolabs, MA, USA). After constructing pET-25b(+)-α-FLT3 scFv, BseRI and BssHII
restriction enzymes (New England Biolabs, MA, USA) were used to digest pET-25b(+)-α-FLT3
scFv and pET-25b(+)-A192 to construct pET-25b(+)-α-FLT3-A192. A diagnostic DNA digestion
was run using NdeI and BamHI restriction enzymes in order to confirm whether the cloned
construct has the right insert. To do so, the digested DNA was run on a 1% agarose gel for
electrophoresis, and the gel was imaged with the UV light. Further, the plasmid was sent for DNA
sequencing with the T7 promoter (TAATACGACTCACTATAGGG) and T7 terminator
(GCTAGTTATTGCTCAGCGG) to confirm in-frame insertion of α-FLT3-A192 into the pET-25b(+)
vector. To express and purity the fusion protein, Clearcoli® BL21 (DE) Electrocompetent Cells
(60810, Lucigen, WI, USA) were transformed with the α-FLT3-A192 plasmid using electroporation.
After the transformation, colonies were picked and culture in 60 mL of autoclaved Terrific Broth
with Glycerol (TB) (C8153, CulGeneX, CA, USA) with 100 µg/mL of carbenicillin at 37°C for
overnight. Next day, 10 mL of the bacteria culture was added to a fresh autoclaved 1 L of TB with
100 µg/mL carbenicillin to culture a total of 6 L of bacteria. The bacteria were cultured at 37 °C
until the optical density (OD) at 600 nm reached between 0.6 and 0.8. Then, 400 µL of 1 M
isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to each 1 L flask to bring the final
concentration to 400 µM IPTG. The IPTG induction was done for overnight at the room
temperature. Next day, bacteria were pelleted at 4,000 rpm for 15 minutes, and the supernatant

8

was discarded. Each pellet was resuspended with 30 mL of cold PBS, vortexed, and disrupted
using a probe-tip Misonix sonicator S-4000 (Misonix, NY, USA). After the sonication, 0.5 % of
polyethyleneimine (PEI) was added to each cell lysate, and the cell lysate was incubated on ice
for 5 minutes. The cell lysate was centrifuged at 13,000 rpm for 15 minutes at 4 °C. The
supernatant was collected, and solid NaCl was added to the supernatant to reach a final
concentration of 2 M. Each supernatant was placed in the water bath at 37 °C until phase
separation was observed. The supernatant was recovered by centrifugation at 4,000 rpm for 15
minutes at 37 °C. After centrifugation, the supernatant was discarded, and the pellet was
resolubilized with cold PBS on ice. The resolubilized pellet was centrifuged at 13,000 rpm for 15
minutes at 4 °C, and the supernatant was collected. This process, hot and cold centrifugation,
was repeated three times with a decreasing volume of PBS and NaCl concentration to obtain pure
α-FLT3-A192.
1.3.2 α-FLT3 scFv cloning, expression and purification
To purify α-FLT3 scFv, the gene was inserted into pFUSE vector with a C-terminal His6 tag, and
the gene was expressed in Expi293F cells (Thermo Fisher, MA, USA). The DNA sequence of α-
FLT3 scFv is:
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGTA
TCCATACGACGTACCGGATTATGCAGAAGTCCAACTGGTCCAGTCAGGCGCTGAGGTAAA
GAAGCCGGGCGCAAGCGTGAAAGTATCTTGTAAGGCTTCCGGGTACACTTTTACTTCCTAC
TATATGCACTGGGTGCGTCAAGCCCCCGGACAGGGCCTGGAGTGGATGGGGATTATTAAC
CCCTCGGGCGGTAGTACATCGTACGCCCAAAAGTTTCAAGGCCGCGTAACTATGACGCGC
GATACCTCCACAAGTACAGTATATATGGAACTTAGCAGTTTGCGTTCCGAGGATACCGCCG
TTTATTACTGCGCTCGCGGCGTAGGTGCACACGATGCTTTCGACATCTGGGGCCAAGGGA
CAACAGTTACAGTCAGTAGTGGTGGAGGTGGCTCGGGTGGCGGCGGCAGCGGAGGCGGT
GGAAGCGACGTCGTAATGACGCAATCTCCCCTTTCTTTGCCTGTTACGCCCGGCGAGCCT

9

GCTTCCATTTCCTGTCGCTCCAGTCAATCTTTGTTACATTCAAACGGGAACAATTATCTGGA
TTGGTACTTGCAAAAACCAGGCCAAAGTCCTCAGCTGTTGATTTACTTGGGGAGCAATCGT
GCATCGGGGGTGCCTGACCGCTTTTCAGGTAGCGGGTCAGATACCGATTTCACGCTGCAA
ATCTCCCGTGTTGAGGCCGAAGACGTTGGCGTGTATTACTGCATGCAGGGAACTCATCCA
GCAATCTCATTTGGTCAGGGGACTCGCTTGGAAATCAAGCACCATCACCACCACCAT
The encoded amino acid sequence of α-FLT3 scFv is:
MYRMQLLSCIALSLALVTNSYPYDVPDYAEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYM
HWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC
ARGVGAHDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISC
RSSQSLLHSNGNNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSDTDFTLQISRVEAE
DVGVYYCMQGTHPAISFGQGTRLEIKHHHHHH
The estimated molecular weight of α-FLT3 scFv is 27.8 kDa. To insert an N-terminal
hemagglutinin (HA) tag and a C-terminal 6×His tag, two primers were used in cloning α-FLT3
scFv into the pFUSE vector (InvivoGen, CA). The sequence of two primers is:
ATL255-
TGTCACGAATTCGTATCCATACGACGTACCGGATTATGCAGAAGTCCAACTGGTCCAGTCA
GGC
and ATL256-
TGGCCAGCTAGCTTATTAATGGTGGTGGTGATGGTGCTTGATTTCCAAGCGAGTCCCCTG
Purified PCR products and pFUSE vectors were digested with EcoRI and NheI restriction
enzymes before ligation with a T4 ligase (New England Biolabs, MA, USA). TOP10F’ E. coli
(Thermo Fisher, MA, USA) was transformed with the cloned plasmid and plated onto LB agar
plates with 25 µg/mL Zeocin (InvivoGen, CA, USA). To express and purify α-FLT3 scFv, Expi293F

10

cells (Thermo Fisher, MA, USA) were transfected with the purified plasmid using the Transport 5
Transfection Reagent (PolySciences, Inc., PA, USA). The cells were incubated for 5 days and
then pelleted, and the cell culture media was collected. The collected media was dialyzed to PBS
for 1000-fold dilution over 2 days. After the dialysis, the media was run through a gravity flow
column that is loaded with 1 mL of Ni-NTA beads (Thermo Fisher, MA, USA) and equilibrated with
a running buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5). After running the media
through the column, the column was washed with 15 mL of the running buffer, followed by 15 mL
of wash buffer (20 mM Tris, 500 mM NaCl, 30 mM imidazole, pH 7.5). The scFv bound to the
column was eluted with an elution buffer (20mM Tris, 500mM NaCl, 400 mM imidazole, pH 7.5)
and then concentrated using Amicon Ultra-15 Centrifugal Filter Units (MiliporeSigma, MA, USA).
Samples were loaded onto an Ä KTA pure chromatography system (GE Healthcare Life Sciences,
MA, USA) with a Superdex 75 10/300 GL column (GE Healthcare Life Sciences, MA, USA)
equilibrated with PBS for gel filtration and buffer exchange into PBS. Fractions containing α-FLT3
scFv were collected and concentrated. The yield of α-FLT3 scFv was measured to be 15 mg/L.
1.3.3. α-FLT3-A192 protein concentration measurements
As the fusion protein assembles nanoparticles, 6 M guanidine hydrochloride was used to disrupt
the nanoparticle assembly of the fusion protein to measure the concentration of α-FLT3-A192
more accurately. The protein concentration was measured using the following equation:
𝐶 𝐸𝐿𝑃 =
𝐴 280
−𝐴 350
𝜀𝑙
Eq. 1
Where A 280 is absorbance at 280 nm, A 350 is absorbance at 350 nm, ε is the molecular extinction
coefficient at 280 nm, and l is the path length (cm). The following equation was employed to define
ε of a protein (Pace et al., 1995):
𝜀 = 125𝑛 𝐶𝑦𝑠𝑡𝑖𝑛𝑒 + 5500 𝑛 𝑇𝑟𝑦𝑝𝑡𝑜𝑝 ℎ𝑎𝑛
+ 1490𝑛 𝑇𝑦𝑟𝑜𝑠𝑖𝑛𝑒 Eq. 2

11

Using the above equation, ε of α-FLT3 scFv was estimated to be 40,130 L mol
-1
cm
-1
assuming
all pairs of cysteine residues are oxidized to form disulfide bonds. ε of A192 was determined to
be 1,490 L mol
-1
cm
-1
. Therefore, ε of α-FLT3-A192 was calculated to be 41,620 L mol
-1
cm
-1
. The
ε of α-FLT3 scFv purified from Expi293F cells was estimated to be 44,600 L mol
-1
cm
-1
assuming
all pairs of cysteine residues are oxidized to form disulfide bonds. The optical absorbance at 280
and 350 nm was measured with a NanoDrop 2000 (ThermoFisher Scientific Inc., MA, USA), which
has a path length of 0.1 cm.
1.3.4 α-FLT3-A192 purity and transition temperature analysis
The purity of α-FLT3-A192 was measured based on SDS-PAGE gel images using ImageJ (NIH,
MD, USA). To run SDS-PAGE, 10 µg and 20 µg of α-FLT3-A192 were loaded to a 4-20% precast
SDS-PAGE gel (4561095, Bio-Rad Laboratories, CA, USA). The SDS-PAGE gel was stained with
GelCode
TM
Blue Safe Protein Stain (24596, Thermo Fisher, MA, USA) and then imaged with a
ChemiDoc Touch Image System (Bio-Rad Laboratories, CA, USA). To analyze the gel, each lane
was plotted to obtain area under each peak using ImageJ. The following equation was employed
to measure the purity of the protein:
% 𝑝𝑢𝑟𝑖𝑡𝑦 = (
𝐴 𝑝𝑒𝑎𝑘 𝐴 𝑡𝑜𝑡𝑎𝑙 𝑝𝑒𝑎𝑘 ) × 100% Eq. 3
Apeak is the area under the peak of the protein of interest, and Atotal peak is the area under all peaks
observed on the SDS-PAGE gel. The transition temperature (Tt) of α-FLT3-A192 was measured
using Beckman Coulter DU 800 UV/Vis spectrometer (Beckman Coulter, CA, USA). To measure
the Tt, four different concentrations of α-FLT3-A192, 3.125, 6.25, 12.5, and 25 µM, were prepared.
The samples were transferred to micro cuvettes and heated at a rate of 1 °C/min starting from
20 °C to 85 °C. To detect the phase separation, OD 350 was measured every 18 seconds. Tt was
estimated where the maximum first derivative of the OD 350 with respect to temperature occurred.
This data was fit to the following equation:

12

𝑇 𝑡 = 𝑏 − 𝑚 𝑙𝑜𝑔
10
[𝐶 𝐸𝐿𝑃 ] Eq. 4
Where b is the y-intercept representing the extrapolated Tt at a concentration of 1 µM, m is a
slope representing the change in °C per 10-fold change in ELP concentration, CELP.
1.3.5 α-FLT3-A192 protein refolding and endotoxin measurement
As it is observed that protein refolding enhances the biological activity, the fusion protein was
refolded after purification. The fusion protein in PBS was mixed with the same volume of of 8 M
urea buffer containing 10 mM β-mercaptoethanol (BME). The denatured fusion protein was
dialyzed to 3 M urea buffer with 2 mM glutathione (GSH) and 0.4 mM oxidized glutathione (GSSH)
for 24 hours at 4 °C. Then, the protein was dialyzed to 1 M urea buffer with 2 mM GSH and 0.4
mM GSSH, 0.5 M urea buffer, 0 M urea buffer, and then 2 times of PBS for 24 hours for each
step at 4 °C.
After the refolding, the endotoxin level of α-FLT3-A192 was measured. To quantify the
endotoxin in the fusion protein, a chromogenic endotoxin testing kit, Pyrochrome® (Associates of
Cape Cod, Inc., MA, USA) was used. To plot a standard curve, a control standard endotoxin
(CSE) of 50 EU/mL in 2.6 mL of LAL Reagent water was prepared, and then five different
concentrations of CSE were prepared. The fusion protein was diluted into 1 to 1000, 1 to 10000,
and 1 to 100000 ratios. After the dilution, 50 µL of CSE and diluted protein samples were added
to a 96-well plate. Then, 50 µL of limulus amebocyte lysate was added to the CSE and protein
samples. The plate was placed in a Biotek Synergy H1 Hybrid Multi-Mode Reader (Biotek, VT,
USA), and the absorbance at 405 nm was measured. The runtime was 2 hours, and the
measurement interval was 35 seconds. By using the time at maximum velocity, the standard curve
was plotted, and then the endotoxin burden of the fusion protein was calculated based on the
curve.

13

1.3.6 Measurements of the hydrodynamic radius of α-FLT3-A192 and colloidal stability
To measure the hydrodynamic radius and the stability of α-FLT3-A192, dynamic light scattering
(DLS) was used. To run DLS, 25 µM of α-FLT3-A192 was prepared, filtered with a 0.22 µm filter,
and 60 µL of the filtered protein was pipetted to each well of a 384-well plate. To prevent
evaporation of the solvent, 15 µL of mineral oil was pipetted to each sample. The 384-well plate
was placed to a DynaPro Plate Reader II (Wyatt Technology, CA, USA), and the hydrodynamic
radius was measured at 37 °C. As a control, the hydrodynamic radius of A192 was also measured.
To measure the stability of α-FLT3-A192 nanoparticles in PBS, the plate was incubated at 37 °C,
and every 24 hours, the hydrodynamic radius was measured over 3 days.
1.3.7 Measurement of molecular weight of α-FLT3-A192 nanoparticles
To estimate the average molecular weight of α-FLT3-A192 nanoparticles, size exclusion
chromatography-multi-angle light scattering (SEC-MALS) was employed. To run SEC-MALS, 10
µM of α-FLT3-A192 was prepared and filtered with a 0.22 µm filter. A Shodex protein KW-804
(8.0mmI.D.×300mm) (Showa Denko America, NY, USA) was equilibrated with PBS before
running. The fusion protein elusion was observed with three detectors, UV 210 mm detector
(SYC-LC-1200) (Agilent Technologies, CA, USA), multi-angle static light scattering detector
(DAWN HELEOS) (Wyatt Technology, CA), USA, and differential refractometer (Optilab rEX)
(Wyatt Technology, CA, USA). The data were analyzed by ASTRA 6 software.
1.3.8 α-FLT3-A192 binding study
To confirm the binding and specificity of α-FLT3-A192 to FLT3 receptors, the fusion protein was
labeled with 5(6)-Carboxytetramethylrhodamine succinimidyl ester (NHS-rhodamine) (46406,
Thermo Fisher, MA, USA), and unlabeled rhodamine was removed by Zeba
TM
Spin Desalting
Columns (Thermo Fisher, MA, USA). Absorbance at 555 nm was used to quantify the rhodamine
concentration for labeled ELPs using the following equation:

14

𝐶 𝐸𝐿𝑃 =
𝐴 555
𝜀 𝑅 ℎ𝑜𝑑𝑎𝑚𝑖𝑛𝑒 ×𝑙 Eq. 5
Where CELP is the concentration of rhodamine-labeled proteins, A 555 is absorbance at 555 nm,
εRhodamine is molecular extinction coefficient of NHS-rhodamine, which is 80,000 L mol
-1
cm
-1
. As a
control, A192 was also labeled with NHS-rhodamine, and the degree of labeling was 1.2 and 1.6
rhodamine per A192 and α-FLT3-A192, respectively. To analyze the binding of the proteins to
FLT3 receptors, MOLM-13, MV4-1, and U937 cells were treated with 1 and 10 µM of the
rhodamine-labeled α-FLT3-A192 and incubated for 30 minutes at 4 °C. As a negative control, 25
µM of rhodamine-labeled A192 was used. After the binding, cells were washed with PBS twice
and transferred to glass-bottom MatTek plates (P35G-0.170-14-C, MatTek Corporation, MA,
USA) for live cell imaging. Live cell images were captured using ZEISS LSM 880 Confocal Laser
Scanning Microscope (Zeiss, Germany) with a 63x objective. To quantify the binding of the
proteins, images were analyzed with imageJ to compare the intensity levels among the different
cell lines and treatment doses. To run competitive binding study, 0.5×10
5
MOLM-13 and MV4-11
cells were pre-incubated with 0.4 µg IgG (Santa Cruz, Cat # 2025) or anti-FLT3 mAb (Thermo
Fisher, Cat # 17135182) at 0.1, 0.2, and 0.4 µg in 100 µL PBS for 30 mins on ice. After the
pre-incubation, the cells were washed twice with PBS and then treated with 10 µM of
rhodamine-labeled α-FLT3-A192 for 30 mins on ice. Unbound α-FLT3-A192 was washed twice
with PBS, and the binding of the fusion protein was analyzed by measuring the shift in the
mean fluorescent intensity (MFI) by flow cytometry.
1.3.9 Pharmacokinetic (PK) study of α-FLT3-A192 and α-FLT3 scFv
To analyze the PK parameters of α-FLT3-A192 nanoparticles and free α-FLT3 scFv, both
formulations were labeled with NHS-rhodamine, filtered with a 0.45 µm filter, and then injected
intravenously into tail vein of female 4 to 6-week-old NOD-scid /Il2rg
-/-
(NSG) mice (n=5).
Rhodamine-labeled α-FLT3-A192 was dosed at 150 µL/20g BW of 360 µM rhodamine (220 µM
of labeled and unlabeled protein) and α-FLT3 scFv was dosed at 150 µL/20g BW of 30 µM

15

rhodamine (110 µM of labeled and unlabeled protein). Blood samples (10-30 µL) were collected
by venipuncture and mixed instantaneously with 80 µL of heparinized cold PBS (1,000 U/mL).
Blood samples of mice injected with rhodamine-labeled α-FLT3-A192 were collected at 3 min, 30
min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 72 h post injection. Blood samples of mice
injected with rhodamine-labeled α-FLT3 scFv were obtained at 3 min, 15 min, 30 min, 45 min, 1
h, 2 h, 4 h, 6 h, 10 h, 24 h, 48 h, and 72 h post injection. Samples were pelleted at 2,000 g for 10
minutes at 4 °C to collect diluted plasma. The diluted plasma was pipetted to a 96-well plate, and
the plate was placed to a Biotek Synergy H1 Hybrid Multi-Mode Reader (Biotek, VT, USA) in order
to measure the fluorescence (Excitation/Emission: 540/580 nm). To plot a standard curve for α-
FLT3-A192, the rhodamine concentrations of 2, 5, 14, 41, 123, 370, 1111, 3333, and 10000 nM
were prepared with rhodamine-labeled α-FLT3-A192. To plot a standard curve for α-FLT3-scFv,
the rhodamine concentrations of 12, 37, 111, 333, and 1000 nM were prepared with rhodamine-
labeled with α-FLT3 scFv. After measuring the rhodamine-labeled protein concentration of α-
FLT3-A192 and α-FLT3 scFv in the plasma over time, the collected plasma was run on SDS-
PAGE gels, and the gels were excited with a ChemiDoc Touch Image System to visualize all
fluorescent proteins on the gels. The gel images were analyzed with ImageJ to calculate the
fluorescent intensity fraction of the protein of interest at each time point. The fraction was applied
to the measured concentration of rhodamine-labeled α-FLT3-A192 and α-FLT3 scFv in the
plasma, and the adjusted concentrations over time were plotted to obtain PK curves.
1.3.10 Relative accumulation of α-FLT3-A192 nanoparticles using whole-tissue
fluorescence imaging
To analyze the relative accumulation of rhodamine-labeled α-FLT3-A192, the liver, kidneys, and
spleen were collected from NSG mice (n=5) after 96 hours post injection, and the organs were
imaged with In Vivo Imaging System (IVIS) Lumina Series III (PerkinElmer, MA, USA).The
exposure time for the imaging was set to 1 second. To analyze the relative accumulation of

16

rhodamine-labeled α-FLT3 scFv, the liver, kidneys, and spleen were collected from NSG mice
(n=5) after 96 hour post injection, and the collected organs were imaged with iBright FL1000
Imaging System (Thermo Fisher, MA, USA). The exposure time for this imaging was set to 1
second. To compare the relative accumulation of rhodamine-labeled α-FLT3-A192 and α-FLT3
scFv in the liver, kidneys, and spleen, organ images obtained from IVIS and iBright FL 1000
Imaging System were analyzed with ImageJ, and % fluorescence was measured in those organs
by using the following equation:
% Mean fluorescence =
𝑀𝑒𝑎𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑇𝑜𝑡𝑎𝑙 𝑚𝑒𝑎𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 Eq. 6
where total mean fluorescence is the sum of mean fluorescence of the liver, kidneys, and spleen.
Mean fluorescence from each kidney was averaged to measure the accumulation to the kidneys.

1.3.11 Cell Culture
MV4-11 cells were purchased from ATCC. MOLM-13 and U937 cells, were kindly provided by Dr.
Wendy Stocks’s lab. All the cell lines were authenticated at the University of Arizona Cell
Authentication Core. All cell lines were cultured in Roswell Park Memorial Institute 1640 (RPMI
1640) medium (Thermo Fisher, MA, USA) supplemented with 10% fetal bovine serum (FBS) and
100 U/mL penicillin (Thermo Fisher, MA, USA).
1.3.12 Viability assays
Cell viability assays were performed by incubating 0.5×10
5
cells with either A192 or α-FLT3-A192
(1,10, 25 and 50 µM) for 30 minutes on ice and then seeding the cells at a concentration of
5×10
5
/mL in a 12 well plate. After 72 hours of incubation at 37 °C, the number of live cells was
counted using trypan blue (cat.no:15250-061, Life Technologies, Carlsbad, USA), and cell viability
was determined by the ratio of the number of live cells in treated samples to that in untreated cells.
The assays were performed three times. To determine the IC 50 for MOLM-13, MV4-11 and U937

17

cells, 5×10
4
cells treated with α-FLT3-A192 (0.01, 0.1, 0.5, 1, 10, 25, 50, 75 and 100 µM) were
seeded in triplicate wells in a 96 well plate. Viability was then measured using alamar blue assay
(cat no: DAL 1100, Invitrogen, Carlsbad, USA). Briefly, after 72 hours of incubation at 37°C, 10
µL of alamar blue was added per well, and incubated for another four hours at 37 °C. The
fluorescence was measured on a synergy H1 microplate reader after the incubation. Change in
viability was calculated by normalizing the fluorescence of treated cells to that of untreated cells.
IC 50 was calculated by fitting non-linear regression.
1.3.13 Flow cytometry analysis
The binding of A192 or α-FLT3-A192 to FLT3 receptors was assessed by flow cytometry. Briefly,
0.5×10
5
FLT3-ITD
+
MOLM-13, MV4-11 cells or the FLT3
-
U937 cells were treated with either
rhodamine-labeled A192 or α-FLT3-A192 (1, 10, and 25 µM) for 30 minutes on ice. Cells were
then washed to remove unbound proteins. Bound rhodamine-labeled A192 and α-FLT3-A192
were analyzed by assessing the shift in the mean fluorescence intensity (MFI) by flow cytometry.
Data were analyzed by normalizing MFI to unstained cells. For in vitro apoptosis assays, cells
were stained using the Annexin V and PI APC kit according to the manufactures protocol
(Invitrogen, Cat no:88-8007-72). The percentage of APC
+
cells were compared among groups.
For in vivo experiments, engraftments were analyzed using cell-surface expression of human
CD45 (huCD45: cat.no:25-0459-41, eBioscience, CA, USA). Cells were stained with PE-Cy7-A
conjugated anti-huCD45. Mean florescence intensity (MFI) of PE-Cy7-A was used to quantify data.
Data were analyzed using the LSRII BD Fortessa X20 flow cytometer and processed using FloJo
software (BD, Franklin Lakes, NJ, USA).
1.3.14 Immunoblotting
Immunoblotting was performed by lysing cells in Pierce-Protease lysis buffer (cat.no:8788,
Thermo Fisher, MA, USA), supplemented with a protease inhibitor mix (cat.no: A32959, Thermo
Fisher, MA, USA). Protein concentrations in cell lysates were measured using the Bicinchoninic

18

acid (BCA) protein assay reagent (Pierce, Thermo Scientific, MA, USA). 30 µg of cell lysates were
added to each lane of SDS-PAGE gels. Lysates were run on the gel by electrophoresis and
transferred overnight to a polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were
blocked with 5% non-fat milk or bovine serum albumin and probed with indicated antibodies: ERK
(Cell Signaling, cat.no.9102), P-ERK (Cell Signaling, cat.no.9101), STAT5 (Cell Signaling,
cat.no.9351), STAT5 (Santa Cruz, cat.no.sc-835), GAPDH (Santa Cruz, cat.no. sc-32233.
Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, TX,
USA) were used for detection. Immunodetection was achieved with the SuperSignal
Chemiluminescent Substrate (Thermo Fisher) and detected by a Bio-Rad Chemidoc Gel Imaging
machine.
1.3.15 In vivo efficacy studies
All animal protocols were approved by the Institution for Animal Care and Use Committee of the
University of Southern California. For xenograft experiments, male and female 4- to 6-week-old
NOD-scid /Il2rg
-/-
(NSG) were purchased from Jackson Laboratory (Bar Harbor, ME).
Approximately 2.5×10
6
MOLM-13 cells were administered via tail vein injection. Two independent
experiments were done to obtain the data. For the first experiment, male mice were engrafted
and treated with A192 (n=5) or α-FLT3-A192 (n=5). For the second experiment female mice were
engrafted with MOLM-13 cells were treated with A192 (n=3) or α-FLT3-A192 (n=3). In both
experiments, mice were treated with 200 µL of 220 µM A192 or α-FLT3-A192 via tail vein on Day
7, 10, 13 and 16 post engraftments. Mice were euthanized on Day 17 and organs (spleen, bone
marrow and blood) were collected for further analyses. Isolated cells from bone marrow and
peripheral blood, tissues were stained for huCD45, and huCD45 engraftments were measured
using flow cytometry. For survival analysis, female mice engrafted with MOLM-13 cells were
treated with 200 µL of 220 µM of A192 (n=7) or α-FLT3-A192 (n=7) on Days 7, 10, 13 and 16 post

19

leukemia engraftments. Mice were euthanized when they displayed signs of distress including:
lethargy, severe weight loss, hair loss, and/or hunched appearance.
1.3.16 Statistical Analysis
A global one-way ANOVA followed by the Tukey post-hoc test was used to determine whether
mean values are significantly different between groups for confocal and IVIS image analysis. All
the data is presented as mean ± standard deviation (SD). Student t test was used to determine if
the difference in means between two groups was statistically significant for viability, apoptosis,
and engraftment analysis. For the mice survival analysis, Kaplan Meier survival analysis was
performed based on Log-rank (Mantel Cox). p < 0.05 was considered significant.
1.4 Results
1.4.1 Characterization of α-FLT3-A192 nanoparticles
The fusion protein, α-FLT3-A192, was expressed in Clearcoli® BL21, and the produced fusion
protein was soluble in PBS. Both A192 and α-FLT3-A192 (Table 1) undergo reversible phase
separation above a transition temperature (T t) (Despanie et al., 2016). Through induction of this
phase separation, α-FLT3-A192 was purified at a level of 77 mg/L culture with an estimated purity
of ~82 % (Fig. 1B). The purity measured on fluorescent imaging of an SDS-PAGE gel with NHS-
rhodamine labelled α-FLT3-A192 was ~92 %. The fusion of α-FLT3 scFv to A192 substantially
lowered Tt compared to A192. As shown in Fig. 1C, α-FLT3-A192 undergoes phase separation
above 42.3 °C while A192 phase separates above 59.9 °C (25 µM in PBS). The endotoxin level
of A192 and α-FLT3-A192 was measured to be 0.06 EU/nmol and 23.9 EU/nmol respectively.
Since the α-FLT3-A192 was produced in ClearColi
®
expression system, these nanoparticles are
expected not to activate human TLR-4 (Mamat et al., 2015). DLS analysis shows that α-FLT3-
A192 forms nanoparticles (Fig. 2A). To measure the stability, the hydrodynamic radius of α-FLT3-
A192 was monitored for 72 hours, and it demonstrated that α-FLT3-A192 nanoparticles are stable
colloids at 37 °C in PBS (Fig. 2B). The average of molecular weight of assembled nanoparticles

20

was analyzed with SEC-MALS, and it indicated that approximately 60 α-FLT3-A192 molecules
form a nanoparticle. SEC resolved a minor peak and major peak with a molecular weight of
5.6×10
6
Da and 6.4×10
6
Da, respectively (Fig. 2C). The Rg/Rh ratio = 1.1 for the major peak, which
is consistent with an extended rod-like nanostructure (Table 1).

21

Figure 1. Characterization of an elastin-like polypeptide (ELP) fusion protein targeting the FLT3
receptor tyrosine kinase. A) A single-chain variable fragment (scFv) targeting the FLT3 receptor
tyrosine kinase was genetically fused to the amino-terminus of a high molecular weight ELP, A192 and
expressed in E. coli. It is conjectured that the highly soluble A192 blocks the formation of protein
aggregates by α-FLT3 scFvs, resulting in the formation of stable nanoparticles. B) The fusion protein
was purified using ELP-mediated phase separation, which was induced by increasing temperature and
sodium chloride concentration. Using cycles of ‘cold’ and ‘hot’ centrifugation, recombinant fusion protein
was obtained at high purity as demonstrated by SDS-PAGE stained by Coomassie. The red arrow
indicates the major band for α-FLT3-A192. Lanes 2, 3 show the sample after 2, 3 purification cycles
respectively. The purity of the major band in Lane 3 was estimated at 81.8%. On the right side, the
fluorescent imaging of an SDS-PAGE gel shows the results of rhodamine labeling of α-FLT3-A192 used
for PK study. The purity of the major band observed in the fluorescent imaging was estimated at 92.3 %.
C) Optical density (350 nm) was measured to evaluate ELP phase behavior over a range of
temperatures and concentrations. While attachment of the scFv reduces the phase diagram curve with
respect to free A192, at physiological salt concentrations (PBS) these ELP nanoparticles are expected
to remain soluble during circulation at 37 C.

22

Table 1. Biophysical characterization of elastin-like polypeptides evaluated in Chapter 1
ELP Amino acid
sequence
M.W.
(kDa)
b

T t
(°C)
c

R h
(nm)
d

R g
(nm)
d

R g/R h Shape
e

A192

MG(VPGAG)192Y 73.6 59.9 7.5
(0.2)
N/A N/A N/A
α-FLT3-A192 α-FLT3
a
-
G(VPGAG)192Y
100.1 42.3 38.6
(0.7)
42.6 (0.7) 1.1 extended
a
the amino acid sequence is:
MEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDT
STSTVYMELSSLRSEDTAVYYCARGVGAHDAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTP
GEPASISCRSSQSLLHSNGNNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSDTDFTLQISRVEAEDVGVYY
CMQGTHPAISFGQGTRLEIK-LVPRGS
b
expected molecular weight based on the amino acid sequence and confirmed by SDS-PAGE (Fig. 1B)
c
transition temperature defined as the maximum first derivative of the optical density at 350 nm for 25 µM ELP in
PBS (Fig. 1C)
d
values indicate the mean (standard deviation) of n=5
e
the Rg/Rh ratio of α-FLT3-A192 is consistent with a ‘rod-like’ nanoparticle of an extended aspect ratio as estimated
using DLS and SEC-MALS

Figure 2. Recombinant α-FLT3-A192 fusions form stable nanoparticles. A) The hydrodynamic
radius (Rh) of recombinant α-FLT3-A192 was measured using dynamic light scattering (DLS) at 37 °C
in PBS. While A192 has a size consistent with the molecular weight of a free polymer, α-FLT3-A192
formed a larger and more polydisperse population of nanoparticles. B) Nanoparticle stability at 37 °C
was measured with DLS over three days. The Rh did not change substantially, which suggests the
nanoparticles remain stable colloids. C) Size exclusion chromatography multi-angle light scattering
(SEC-MALS) was employed to measure the molecular weight and the radius of gyration of the
nanoparticles. Two peaks were observed, and their average molecular weights were 6.4×10
6
Da and
5.6×10
6
Da for major peak 1, minor peak 2 respectively. Based on the expected molecular weight of
the expressed fusion protein, these nanoparticles consist of sixty α-FLT3-A192 molecules. These
nanoparticles have Rg/Rh ratio = 1.1, which is consistent with an extended nanoworm shape.

23

1.4.2 α-FLT3-A192 binds specifically to FLT3 surface receptor
To assess the binding affinity and specificity of α-FLT3-A192 to the FLT3 receptor, MOLM-13 and
MV4-11 cells (FLT3-ITD
+
AML cell lines) or U937 cells (FLT3
-
AML cell line) were incubated with
1 µM or 10 µM of rhodamine-labeled α-FLT3-A192 or 25 µM of rhodamine labeled A192 for 30
minutes. Confocal microscopy images showed dose-dependent increase in the fluorescence
signals obtained from FLT3-ITD
+
AML cell lines treated with α-FLT3-A192 compared with the
fluorescent signal obtained from A192 treated cells. In addition, FLT3 negative U937 cells did not
show any binding of α-FLT3-A192, which suggests that the fusion protein binds to FLT3-ITD
+
AML
cell lines specifically. A192 did not result in fluorescence signals in any of the cell lines, suggesting
lack of binding. (Fig. 3A and B).
The binding of rhodamine labeled α-FLT3-A192 to the FLT3 receptor was also assessed
using flow cytometry. In MOLM-13 cells, a significant increase in binding was observed in cells
treated with α-FLT3-A192 10 µM compared to A192 (normalized MFI 1.0 vs. 2.0; p<0.0001). In
MV4-11, a significant increase in binding was also observed between cells treated with A192 vs
1 µM of α-FLT3-A192 (normalized MFI 1.0 vs. 1.36; p=0.03) and in α-FLT3-A192 10 µM
(normalized MFI 1.0 vs. 1.7; p=0.006). On the contrary, α-FLT3-A192 showed no significant
binding in FLT3 negative U937 cells (Fig. 3C).
To confirm the specificity of α-FLT3-A192 binding to FLT3 receptors, a competitive
binding assay was performed with anti-FLT3 mAb. MOLM-13 and MV4-11 cells were pre-
treated with anti-FLT3 mAb and were then washed to remove unbound mAbs. Then, the cells
were treated with rhodamine-labeled α-FLT3-A192. After normalizing to untreated cells, MFI of
samples pre-treated with anti-FLT3 mAb were normalized to cells treated with α-FLT3-A192
alone. In both MOLM-13 and MV4-11 cells pre-treated with anti-FLT3 mAb (0.1, 0.2 and 0.4
µg) showed a significant decrease in binding of α-FLT3-A192. Pre-treatment with an irrelevant

24

IgG did not affect the binding of α-FLT3-A192 to FLT3 receptors. No significant binding was
observed in cells treated with rhodamine-labeled A192 (Fig 3D).

Figure 3. Recombinant α-FLT3-A192 binds to FLT3 receptor tyrosine kinase. FLT3 ITD
+
AML cell
lines MOLM-13 and MV4-11 and FLT3 negative U937 cells (0.5 x 10
6
cells each) were treated with 1
µM or 10 µM of rhodamine labeled α-FLT3-A192 or 25 µM of rhodamine labeled A192 for 30 minutes
on ice. Binding was only observed in MOLM-13 and MV4-11 via A) Laser scanning confocal microscopy.
B) Cell fluorescence was quantified with ImageJ by analyzing images obtained from laser scanning
confocal microscopy. Higher fluorescence signal was observed in cells treated with 10 µM α-FLT3-A192
compared with 1 µM. (the number of analyzed cells: U-937, n=35, 56, 20; MOLM-13, n=203, 70, 84;
MV4-11, n=76, 62, 24) C) Binding of rhodamine labeled α-FLT3-A192 to MV4-11 and MOLM-13 cells
was confirmed via flow cytometry by measuring peak shift in rhodamine to bounds cells and quantifying
based on mean fluorescence imaging. No binding was observed in U937 cells (FLT3 negative). A
rhodamine-labeled A192 control failed to bind to any of the cell lines. D) Competitive binding assay was
performed in MOLM-13 and MV4-11 cells by pre-treating cells with either anti-FLT3mAb (1, 2, 4 µg/mL)
or IgG and then observing binding with rhodamine α-FLT3-A192 (10 µM). Binding was measured via
Flow cytometry by measuring the peak shift in rhodamine to bound cell and quantifying based on mean
fluorescence intensity. MFI was normalized to cells treated with α-FLT3-A192 alone. Data represented
as mean ± SD, n=3. * p≤0.05 ** p≤0.01*** p≤0.001**** p≤0.0001
A
B
MOLM-13 MV4-11 U-937
α-FLT3-A192 10 µM A192 25 µM
C
D
0.0
0.5
1.0
1.5
MFI:PE
(a-FLT3-A192)
*
**
***
MOLM-13
A192
α-FLT3-A192
IgG + α-FLT3-A192
mAb FLT3 1µg/mL+ α-FLT3-A192
mAb FLT3 2µg/mL+ α-FLT3-A192
mAb FLT3 4µg/mL+ α-FLT3-A192

25

1.4.3 Antileukemic activity of α-FLT3-A192 in AML cells
Next, to determine the anti-leukemia effects of α-FLT3-A192 in vitro, FLT3-ITD
+
cells (MOLM-13
and MV4-11 cells) were treated with α-FLT3-A192 at 1, 10, 25 and 50 µM, or the same
concentrations of A192. Live cells were counted using the trypan blue assay. The percentage of
viable cells was significantly reduced when MOLM-13 cells were treated with α-FLT3-A192
compared with A192 at 1 µM (p=0.0009, 17% decrease), 10 µM (p=0.0002, 23% decrease), 25
µM (p<0.0001, 34% decrease) and 50 µM (p<0.0001, 36% decrease) (Fig. 4A). Similarly, the
percentage of viable MV4-11 cells was significantly reduced at 1 µM (p<0.0001, 67% decrease),
10 µM (p<0.0001, 71% decrease), 25 µM (p<0.0001, 80% decrease) and 50 µM of α-FLT3-A192
(p<0.0001, 83% decrease) (Fig. 4B). No effect was seen on FLT3 negative U937 cells (Fig. 4C).
IC 50 values of α-FLT3-A192 for MOLM-13 and MV4-11 cells were determined to be 46.87 µM and
5.65 µM, respectively (Fig. 4D and E respectively). We also assessed whether the decrease in
cell viability was due to an enhanced apoptosis in cells treated with α-FLT3-A192. Apoptosis
measured by Annexin V staining was significantly increased at 72 hours for MOLM-13 cells
treated with α-FLT3-A192 (25 µM: p=0.007, 5.27% vs. 42.76%; and 50 µM: p=0.05, 5.58% vs.
53.83%; Fig. 4F). Similarly, MV4-11 cells treated with α-FLT3-A192 showed significantly higher
levels of apoptosis compared with A192 treated cells (25 µM: p=0.003, 25.36% vs 62.77%; and
50 µM: p=0.0004, 24.43% vs 66.36%; Fig. 4G). No effect was observed in FLT3 negative U937
cells (Fig. 4H).

26

1.4.4 α-FLT3-A192 inhibits the FLT3 signaling pathway
FLT3-ITD mutations cause a constitutive activation of FLT3 kinase and subsequent activation of
its downstream proliferative signaling pathways, including the Ras/MAPK kinase
(MEK)/extracellular signal-regulated kinase (ERK) pathway and PI3K/Akt pathway (Gilliland and
Griffin, 2002). In contrast to FLT3-WT signaling, FLT3-ITD also activates the STAT5 pathway
(Choudhary et al., 2005; Grundler et al., 2005; Mizuki et al., 2003). To determine the effect of α-
FLT3-A192 nanoparticles on FLT3-ITD downstream signaling, FLT3-ITD
+
MOLM-13 and MV4-11

Figure 4: α-FLT3-A192 has anti-leukemic activity in AML cells. A-C) A trypan blue viability assay
was performed in MOLM-13, MV4-11 (FLT3 ITD
+
) and U937 (FLT3 negative) cells treated with α-FLT3-
A192 or an A192 control for 72 hours. The number of live cells was normalized to untreated cells. Data
represented as mean ± SD, n=6. D-E) An IC50 of α-FLT3-A192 was measured using alamar blue
staining in MOLM-13 and MV4-11 cells at 72 hours post treatment with the increasing concentration of
α-FLT3-A192 and plotted based on non-linear regression. F-H) Apoptosis was measured by flow
cytometry in MOLM-13, MV4-11 (FLT3 ITD
+
) and U937 (FLT3 negative) cells at 72 hours post treatment
with α-FLT3-A192 or control A192 using APC conjugated Annexin V stain and normalized to untreated
cells. Data represented as mean ± SD, n=3.

27

cells were treated with α-FLT3-A192 (25 µM) for 1, 6 and 24 hours. FLT3 targets, phospho-STAT5
and phospho-ERK, were assessed by western blot. There was a significant decrease in both
phospho-STAT5 and phospho-ERK at 6- and 24-hours post treatment with α-FLT3-A192
compared with cells treated with A192 (25 µM) (MOLM-13 cells, Fig. 5A; MV4-11 cells, Fig. 5B).

Figure 5: α-FLT3-A192 inhibits FLT3 downstream signaling A) MOLM-13 and B) MV4-11 cells were
treated with A192 or α-FLT3-A192 (25 µM). Cell lysates were collected at 0.5, 6 and 24 hours and
prepared for western blot analysis; immunoblots were probed for STAT5 and ERK pathway. C)
Quantified analysis for pERK/ERK and pSTAT5/STAT4 in MOLM-13 and MV4-11 cells. Experiment was
performed in duplicates.
Figure 5
A B
MOLM-13 MV4-11
0.0
0.5
1.0
1.5
pERK/ERK
24 hours
A192
α-FLT3-A192
* *
MOLM-13 MV4-11
0.0
0.5
1.0
1.5
pSTAT5 /STAT5
6 hours
A192
α-FLT3-A192 *
*
C

28

1.4.5 PK profiles of α-FLT3-A192 nanoparticles outperform α-FLT3 scFv
PK study of α-FLT3-A192 showed that α-FLT3-A192 nanoparticles fit a biexponential decay
model using the following equation:
𝐂𝐩 = 𝐀 𝒆 −𝜶𝒕
+ 𝑩 𝒆 −𝜷𝒕
Eq. 7
Where A, B, α, and β are ‘macroconstants’ that can be fit with nonlinear regression. Using a non-
compartmental and two-compartmental analysis, the PK parameters of α-FLT3-A192 and α-FLT3
scFv were obtained. The terminal half-life of α-FLT3 scFv was estimated to be 2.34 hours and
mean residence time (MRT) was measured to be 1.74 hours. The clearance of the protein was
calculated to be 0.03 mL/hr· g, and the steady-state volume of distribution 0.06 mL/g. On the other
hand, the terminal half-life of the fusion protein was estimated to be 14.7 hours, and the MRT was
measured to be 18.7 hours. The clearance of the fusion protein was determined to be 0.07
mL/hr· g, and the steady-state volume of distribution was calculated to be 1.50 mL/g (Fig. 6A,
Table 2, and Table 3). Images of spleen, kidneys, and liver showed that both α-FLT3 scFv and
α-FLT3-A192 nanoparticles still remained in those organs after 96 hours from the injection (Fig.
6B and 6C). The whole-tissue fluorescence imaging analysis indicated that the relative
accumulation of α-FLT3-A192 in the kidneys was significantly lower than that of α-FLT3 scFv
(p≤0.05). On the other hand, the relative liver accumulation of α-FLT3-A192 was not statistically
different from that of α-FLT3 scFv (p>0.05). The relative spleen accumulation of α-FLT3-A192
was significantly higher than that of α-FLT3 scFv (p≤0.01).

29

Figure 6. The pharmacokinetic terminal half-life of α-FLT3-A192 nanoparticles exceed that of a
control α-FLT3 scFv. 220 µM of rhodamine-labeled α-FLT3-A192 and 110 µM of α-FLT3 scFv in PBS
was injected via the tail vein to mice (n=5) in 150 µL/25g B.W. Plasma was collected and measured for
fluorescence. Rhodamine-labeled α-FLT3-A192 and α-FLT3 scFv concentrations were calculated using
a standard curve. As quality control, plasma samples were all analyzed by SDS-PAGE to determine
what fraction of total fluorescence remained associated with the intact, labeled protein. A) The plasma
concentrations were fit to Eq. 7 to obtain the PK curve, and the generated curve shows rhodamine-
labeled α-FLT3-A192 and α-FLT3 scFv concentration in the plasma over time. After 96 hours from the
injection, spleen, kidneys, and liver were collected from mice treated with rhodamine-labeled α-FLT3-
A192. B-C) IVIS images (α-FLT3-A192) and iBright images (α-FLT scFv) of organs were obtained 96
hours post-injection and quantified based on the mean fluorescence measured from images using
ImageJ. The fraction of total mean fluorescence (the sum of mean fluorescence of the liver, kidneys,
and spleen) was calculated for each organ (Eq. 6), and the % mean fluorescence was plotted to
compare the relative accumulation in major clearance organs was plotted. The accumulation of α-FLT3-
A192 in the liver was not significantly different from that of α-FLT3 scFv (p>0.05). On the other hand,
the accumulation of α-FLT3-A192 in the kidneys was significantly lower than that of α-FLT3 scFv
(p≤0.05). In contrast, accumulation of α-FLT3-A192 in the spleen was significantly higher than that of
α-FLT3 scFv (p≤0.01).

30

Figure 7. α-FLT3-A192 remains intact in the plasma over longer durations than α-FLT3 scFv. A)
SDS-PAGE gels were run with the collected plasma at different time points from NOD/SCID mice
injected with rhodamine-labeled α-FLT3 scFv or α-FLT3-A192 and were excited to visualize the
rhodamine signal in order to verify α-FLT3 scFv and α-FLT3-A192 were intact in the blood plasma over
time. These gels were analyzed with ImageJ to measure the fraction of the α-FLT3 scFv or α-FLT3-
A192 band intensity in each lane. Each lane represents different time points: 1) protein ladder 2) 3 min
3) 30 min 4) 1 h 5) 2 h 6) 4 h 7) 6 h 8) 8 h 9) 10 h 10) 24 h 11) 48 h 12) rhodamine-labeled α-FLT3 scFv
control for the top panel. For the bottom panel, each lane represents: 1) protein ladder 2) rhodamine
labeled α-FLT3-A192 control 3) 3 min 4) 30 min 5) 1 h 6) 2 h 7) 4 h 8) 8 h 9) 12 h 10) 24 h 11) 36 h 12)
48 h B) The graph shows that the fraction of α-FLT3-A192 signal is over 0.7, indicating that the major
portion of fluorescent signals come from α-FLT3-A192. On the other hand, the fraction of intact α-FLT3
scFv rapidly decreases, supporting that α-FLT3 scFv has a poor PK profile.

31

Table 2. Pharmacokinetic Parameters of α-FLT3-scFv and α-FLT3-A192 nanoparticles following IV
administration using a non-compartmental analysis
Table 3. Pharmacokinetic Parameters of α-FLT3-scFv and α-FLT3-A192 nanoparticles following IV
administration using a two-compartment model

Parameter (Unit) α-FLT3 scFv
a
(n=5) α-FLT3-A192
a
(n=5) p value
CL/g BW (mL/h· g) 0.03 (0.01) 0.07 (0.02) 0.009
AUC (µM*h) 7.14 (2.14) 39.7 (14.9) 0.001
AUMC (µM*h
2
) 11.7 (1.70) 665 (238) 0.0003
MRT (h) 1.74 (0.46) 18.7 (7.8) 0.001
Vd/g BW (mL/g) 0.03 (0.01) 0.17 (0.09) 0.008
Vss/g (mL/g) 0.06 (0.04) 1.50 (0.76) 0.003
Parameter (Unit) α-FLT3 scFv
a
(n=5) α-FLT3-A192
a
(n=5) p value
dose of total protein (nmol) 20.6 (1.7) 31.1 (5.7) 0.004
dose of rhodamine (nmol) 5.52 (0.45) 50.9 (9.4) <0.0001
Body weight (g) 25.0 (2.0) 18.8 (3.5) 0.009
A (µM) 5.45 (1.80) 31.6 (43.7) 0.21
B (µM) 2.30 (1.37) 1.83 (1.27) 0.59
α (h
-1
) 12.1 (18.9) 4.56 (1.63) 0.40
β (h
-1
) 0.44 (0.19) 0.056 (0.030) 0.002
t1/2α (h) 0.26 (0.21) 0.17 (0.05) 0.36
t1/2β (h) 2.34 (2.20) 14.7 (5.6) 0.002
C0 (µM) 7.75 (2.31) 33.4 (44.9) 0.24
AUC (µmol*h) 6.73 (2.05) 38.8 (14.6) 0.001
CL/g BW (mL/h· g) 0.04 (0.01) 0.08 (0.02) 0.009
V1/g BW (mL/g) 0.03 (0.01) 0.17 (0.09) 0.008
kelimination (h
-1
) 1.17 (0.27) 0.69 (0.62) 0.15
kplasma tissue (h
-1
) 5.18 (8.19) 3.47 (1.52) 0.66
ktissue plasma (h
-1
) 6.18 (10.9) 0.46 (0.28) 0.27
a
values indicate the mean (standard deviation) of n=5 following a dose of 2.7 nanomoles of rhodamine-labeled α-
FLT3-A192 (1.7 nanomoles of the total dose of α-FLT3-A192) per gram of mouse body weight or a dose of 0.2
nanomoles of rhodamine-labeled α-FLT3 scFv (0.8 nanomoles of the total dose of α-FLT3 scFv) alone.
a
values indicate the mean (standard deviation) of n=5 following a dose of 2.7 nanomoles of rhodamine-labeled α-
FLT3-A192 (1.7 nanomoles of the total dose per gram BW of α-FLT3-A192) per gram of mouse body weight or a
dose of 0.2 nanomoles of rhodamine-labeled α-FLT3 scFv (0.8 nanomoles per gram BW of the total dose of α-
FLT3 scFv) alone. The average mouse body weight across these studies is 22 g.

32

1.4.6 Anti-leukemia activity of α-FLT3-A192 in FLT3-ITD xenograft murine model
To assess the in vivo therapeutic effects of α-FLT3-A192 nanoparticles, the MOLM-13 leukemia
murine model was used. NSG mice were engrafted with 2.5×10
6
MOLM-13 cells. Mice were then
randomized into two treatment groups. Mice were treated with 176 mg/kg/mouse with either A192
(n=8) or α-FLT3-A192 (n=8) on Day 7, 10, 13 and 16 post leukemia engraftments. Mice were
euthanized on day 17 and organs were evaluated for leukemia burden. Mice treated with α-FLT3-
A192 had smaller spleens that weighed significantly less compared with the A192 treated mice
(48 vs. 126 mg, p=0.03) (Fig. 7A). Flow cytometry analysis of huCD45 for leukemia engraftment
exhibited that α-FLT3-A192 mice had significantly less engraftment compared with the mice
treated with A192 in the peripheral blood (%huCD45: 17.5 vs. 43.44, p=0.007; Fig.7 C-D) and the
bone marrow (%huCD45: 10.2 vs. 26.1%, p<0.0001; Fig. 7 E-F).
Next, the effect of α-FLT3-A192 was observed on the survival of MOLM-13 engrafted mice.
For survival analysis, 2.5X10
6
MOLM-13 cells were engrafted in NSG mice. Mice were treated
with four doses 176 mg/kg/mouse of either A192 (n=7) or α-FLT3-A192 (n=7) on Day 7, 10, 13
and 16. Mice treated with α-FLT3-A192 survived significantly longer than mice treated with A192
(median survival: 36 vs. 30 Days, p=0.0015).

33

Figure 8: Anti-leukemia activity of α-FLT3-A192 in FLT3-ITD murine model. Fig A-F: 2.5 × 10
6

cells MOLM-13 cells/mouse were engrafted in NSG mice. Mice were treated with 200 µL of 220 µM of
A192 (n=8) or α-FLT3-A192 (n=8) on day 7, 10, 13 and 16 post engraftment and euthanized on day 17.
100mg/kg Midostaurin (N=3) on day 7-11 post leukemia engraftments. A) Images of mice spleens show
decreased in spleen size in mice treated with α-FLT3-A192 compared withA192 treated mice.
Enlargement of spleen is consistent with progression of this AML model in A192 control mice. B)
Spleens weights were measured, and spleens of mice treated with α-FLT3-A192 weighed significantly
less compared with the A192 treated mice (0.48 vs. 126 mg, p=0.03). C-D) Leukemia engraftment was
measured using human CD45 (huCD45) antibody by flow cytometry in the peripheral blood (%huCD45-
α-FLT3-A192: 17.5 vs. A192: 43.44, p=0.007) and (%huCD45- midostaurin: 13.73 vs. A192: 43.44,
p=0.03) E-F) Similarly, CD45 engraftment was reduced by α-FLT3-A192 treatment in the bone marrow
(%huCD45- α-FLT3-A192: 10.2 vs. A192: 26.1, p<0.0001) and (%huCD45- midostaurin: 11.59 vs.
A192: 26.1, p=0.0003). There was no difference in the engraftment of mice treated with midostaurin or
α-FLT3-A192. G) 2.5 × 10
6
MOLM-13 cells/mouse were engrafted in NSG mice and treated with 200
µL of 220 µM A192 (n=7) or α-FLT3-A192 on day 7, 10, 13 and 16 post engraftment and survival was
recorded. Kaplan Meier survival analysis showed that mice in the α-FLT3-A192 treatment group
survived significantly longer than mice in the A192 group (median survival: 36 vs. 30 days, p=0.0015).
A B
C E
G F
A192 a- FLT3-A192
A192 a- FLT3-A192
D
Log-rank p=0.0015
(n=7)
(n=7)
Blank
A192
α-FLT3-A192
Midostaurin
0
100
200
300
400
Weight in mg
Spleen weight
A192
α-FLT3-A192
Midostaurin
0
20
40
60
80
CD45 Engraftment %
Peripheral Blood
*
*
ns
A192
α-FLT3-A192
Midostaurin
0
10
20
30
40
CD45 Engraftment %
Bone Marrow
***
***
ns
SSC
CD45
SSC
CD45
Blank
A192
α-FLT3-A192
Midostaurin

34

1.5 Discussion
The FLT3 receptor is a key therapeutic target for AML because it is frequently overexpressed or
mutated in leukemic blasts. Also, the presence of the FLT3-ITD mutation is associated with a poor
prognosis (Abu-Duhier et al., 2000; Drexler, 1996; Hawley et al., 2019; Zheng et al., 2019).
Targeted therapies against FLT3-ITD have long been investigated, and small-molecule FLT3
tyrosine kinase inhibitors (TKIs) and anti-FLT3 antibodies have shown promising results in clinical
trials. Recently two FLT3 inhibitors were approved by the FDA for treating FLT3-ITD
+
patients.
Midostaurin is approved for the treatment of pre-transplant patients in combination with standard
therapy. Despite its clinical effects, half of these patients die of their disease within 4 years.
Gilteritinib is approved for relapsed and refractory AML in patients with FLT3-ITD. Yet, the Phase
III ADMIRAL Trial showed that only 37% of treated patients survived longer than one year (Daver
et al., 2019; Stone et al., 2017). In addition, the potential drug resistance resulted from the
selective oncogene inhibition by a potent and highly specific kinase inhibitor like gilteritinib is a
major clinical challenge. The presence of FLT3-WT, which is sensitive to FLT3 ligand and
relatively resistant to FLT3 inhibitors, may also contribute to the drug resistance. In addition, the
high level of FLT3 ligand found in the bone marrow microenvironment results in persistent activity
of the FLT3/MAPK pathway and provides survival signals to leukemic blasts. Activation of
downstream pathways of FLT3 and FLT3-ITD, such as MAPK and STAT5, also contribute to the
drug resistance to FLT3 inhibitors (Heidel et al., 2006; von Bubnoff et al., 2009; Williams et al.,
2013). Furthermore, due to the limited specificity of first-generation TKIs, there is increased
toxicity associated with their high dose for clinical use (Fischer et al., 2010; Levis et al., 2011;
Serve et al., 2013). Altogether, these data challenge us to design and develop better therapeutic
approaches for patients with FLT3-ITD. Antibodies against FLT3 were developed in an attempt to
address the limitations associated with TKIs. Previously, two human anti-FLT3 mAbs, IMC-EB10
and IMC-NC7 were shown to have promising anti-leukemia activity both in vitro and in FLT3-ITD
murine models (Piloto et al., 2005). However, phase 1 clinical trial (NCT00887926) to determine

35

the safety of IMC-EB10 (LY3012218) in patients with relapsed or refractory AML was terminated
due to lack of efficacy, even though the drug was safe (Sanford et al., 2015). It remains unclear
why this monoclonal antibody failed in the clinical trial. However, limitations associated with
monoclonal antibody development may have contributed to this outcome. Factors related to the
lack of therapeutic concentrations of the antibody on the target cells and limited clustering on the
target receptors are critical pharmacological challenges of antibody therapies. Our study presents
the development and validation of a new antibody-based FLT3 nanoparticle, which is composed
of α-FLT3 single-chain variable fragment (scFv) linked to a high molecular weight elastin-like
polypeptide (ELP), A192, derived from human tropoelastin.
Many studies regarding scFvs produced from E. coli report the scFvs tend to forms protein
aggregates, known as inclusion bodies; furthermore, the inclusion bodies require specialized
strategies to recover scFvs from the inclusion bodies (Heo et al., 2006; Kipriyanov et al., 1997;
Sanchez et al., 1999). As expected, the fusion of a highly soluble A192 to α-FLT3 scFv generated
a high yield of fusion protein (77 mg/L) in the soluble fraction after bacterial lysis. ELPs phase
separate above tunable transition temperatures, and this characteristic facilitates the
chromatography-free purification of the fusion protein. By using three cycles of hot and cold
centrifugation, high purity of α-FLT3-A192 (82, 92 % purity by Coomassie, Rhodamine-labeling
respectively) was obtained without attaching any additional purification tags, which can
compromise biological activities of proteins (Goel et al., 2000; Sabaty et al., 2013). As derived
from human tropoelastin, ELPs are biocompatible and biodegradable (Despanie et al., 2016).
Conjugating A192 to α-FLT3 scFv increases its molecular weight and stabilizes these antibodies
into nanoparticles. Although A192 consists of repeats containing aliphatic amino acids, their
solubility is high because of the hydration of a predominant random-coil peptide backbone.
Conversely, most scFvs tend to form inclusion bodies when produced in E. coli as well as
precipitation during protein refolding, which represents their tendency to oligomerize into

36

aggregates. Therefore, the most likely mechanism of assembly is that fusion of A192 blocks
formation of inclusion bodies of α-FLT3 scFv, favoring the formation of stable colloids. Neither
A192 nor α-FLT3-A192 nanoparticles undergo ELP-mediated coacervation until temperatures
much higher than 37 °C. Meanwhile, the nanoparticles are observed even at 4 °C. Thus, the
tendency of scFv to precipitate and the high solubility of A192 suggests the nanoparticle core
forms through oligomerization of the scFv protein domains.
Based on DLS and SEC-MALS, Rg/Rh ratio suggests that α-FLT3-A192 forms worm-like
nanoparticles, but there is a limitation of using the ratio to determine the shape of nanoparticles
as the fusion protein consists of two completely different proteins. To verify the fusion protein
forms nanoworms, Cryo-TEM imaging can be employed in future studies. When produced in E.
coli, the stability of scFv fragments has always been a major challenge for developing scFvs as
therapeutic antibodies (Brockmann et al., 2005; Worn and Pluckthun, 1999, 2001). However, the
engineered α-FLT3-A192 nanoparticles proved to be stable for three days at 37 °C in PBS. This
supports the feasibility for moving forward with this approach into further clinical development.
The endotoxin levels of α-FLT3-A192 were significantly reduced by expressing the fusion protein
in ClearColi cells instead of the Shuffle T7 cells. Lipopolysaccharide is known to be the major
pyrogen of concern for recombinant protein therapeutics produced from E. coli; however,
traditional anion exchange ‘polymixin’ chromatography is ineffective at removing endotoxin signal
from these nanoparticles. As an alternative, this fusion protein was expressed in ClearColi, which
produce a LipidA1 variant that is unable to activate human TLR4 and lacks the extended
polysaccharide chain (Mamat et al., 2015). This strategy also reduced observed endotoxin levels
based on the chromogenic LAL test; however, levels remain higher than for free A192 or for FDA
recommended guidelines. Therefore, even though free endotoxin was unable to induce the cell
apoptosis while α-FLT3-A192 induced, it is hard to rule out TLR4-mediated recruitment of the
innate immune system that remains in NOD/SCID mice.

37

Western blot analysis indicated that α-FLT3-A192 nanoparticles inhibited STAT5 and ERK
phosphorylation whose pathways are known to drive cell survival and proliferation induced by
FLT3-ITD mutations in FLT3-ITD
+
cells (Fig.5C) (Benekli et al., 2009; Wingelhofer et al., 2018).
This western blot data supports that α-FLT3-A192 nanoparticles are directly associated with the
FLT3 signaling pathway. This result is consistent with a previous study showing that an anti-FLT3
antibody, IMC-EB10 that targets the same domain as α-FLT3-A192 (domain 4 of FLT3) inhibited
the FLT3-mediated activation of downstream signaling kinases(Li et al., 2004). It was observed
that IC 50 between MOLM-13 and MV4-11 were one magnitude different. MV4-11 cells have
homozygous FLT3-ITD mutation while MOLM-13 cells have heterozygous mutation, and it seems
this characteristic affects the sensitivity to α-FLT3-A192 nanoparticles.
The PK analysis and pharmacodynamic study of α-FLT3-A192 suggested the
nanoparticles had an excellent activity with enhanced PK profiles. In the NSG murine model, α-
FLT3-A192 nanoparticles reduced the leukemia burden and extended survival of MOLM-13
engrafted mice. Due to their small molecular weight, scFvs are rapidly cleared from the systemic
circulation by the kidneys. The PK profiles of multiple scFvs administered to mice were assessed
to get an idea of the average half-life and mean residence time (MRT). Datapoints were extracted
from the published papers and then analyzed. The average of half-lives was 2.2 ± 1.5 hours
(mean ± 95% Cl, n=7), and the average of MRT was estimated to be 2.4 ± 2.0 hours (mean ±
95% Cl, n=7) (Hutt et al., 2012; Li et al., 1998; Muller et al., 2007; Pavlinkova et al., 1999a;
Pavlinkova et al., 1999b; Rios et al., 2019; Yang et al., 2003). Similarly, α-FLT3 scFv exhibited
short MRT and half-life. In contrast, the terminal half-life of α-FLT3-A192 nanoparticles was
estimated to be 14.7 hours and the MRT was measured to be 18.7 hours. Both of these
parameters are significantly greater than the parameters of α-FLT3 scFv.
Formulation of nanoparticles targeting FLT3 receptors with a long half-life can have
several advantages over anti-FLT3 monoclonal antibodies. Leukemic cells are produced in the

38

bone marrow and circulate systemically. Thus, targeting leukemic cells in both the systemic
circulation and the bone marrow is crucial to enhance the efficacy of AML treatment. The size of
monoclonal antibodies is around 10 nm (Reth, 2013), which allows monoclonal antibodies to
extravasate to normal tissues from the blood vessel (Sarin, 2010; Stylianopoulos, 2013).
Therefore, monoclonal antibodies do not specifically target a leaky microenvironment, which is
frequently exhibited by tumors. On the other hand, the average diameter of α-FLT3-A192
nanoparticles is 77.2 nm, which will not allow them to extravasate to normal tissues in comparison
with monoclonal antibodies (Bergqvist et al., 1987). In addition, the vasculature of the bone
marrow is leaker in patients with AML than in healthy people (Mu et al., 2018; Passaro et al.,
2017), which may enhance the accumulation of α-FLT3-A192 in the bone marrow than for
monoclonal antibodies. Therefore, α-FLT3-A192 nanoparticles have the potential to be superior
to anti-FLT3 monoclonal antibodies by targeting both peripheral blood and bone marrow more
effectively.
1.6 Conclusion
In this chapter, we show the formulation of nanoparticles targeting FLT3 receptors. α-FLT3-A192
nanoparticles purified from Clearcoli show great stability at 37 °C and anti-leukemic activities both
in vitro and in vivo. We also demonstrate that α-FLT3-A192 nanoparticles have excellent PK
parameters in comparison with α-FLT3 scFv.
1.7 Acknowledgements
This work was made possible by University of Southern California (USC), the Gavin S. Herbert
Professorship, the United States of America National Institutes of Health R01 GM114839 to JM,
the USC Ming Hsieh Institute, P30 CA014089 to the USC Norris Comprehensive Cancer Center,
P30 EY029220 to the USC Ophthalmology Center Core Grant for Vision Research, Center of
Excellence in Nanobiophysics at USC, and the Translational Research Laboratory at USC School
of Pharmacy.

39

Chapter 2: Formulation of elastin-like polypeptide nanoparticles targeting CD99, a novel
target for acute myeloid leukemia treatment
2

2.1 Abstract
CD99 is a transmembrane glycoprotein, and its overexpression has been associated with various
malignancies. Previously, the Alachkar lab has reported CD99 is upregulated in acute myeloid
leukemia (AML) and has presented CD99 as a viable therapeutic target for AML. Currently, no
CD99 targeted therapies are in clinical trials, so preclinical studies of CD99 targeted therapy for
AML is urgently needed. In this chapter, similarly to Chapter 1, we will present the formulation of
elastin-like polypeptide (ELP) nanoparticles targeting CD99 for AML treatment. To formulate the
ELP nanoparticles, a high molecular weight ELP, A192, was fused to an anti-CD99 single-chain
variable fragment, forming a fusion protein, α-CD99-A192. The fusion protein spontaneously
assembles into stable nanoparticles with both excellent pharmacological and pharmacokinetic
properties. In this Chapter, we suggest α-CD99-A192 nanoparticles as a new modality of AML
treatment with the 16 hours long half-life, excellent anti-leukemic activity in vitro and in vivo, and
prolonged overall survival of mice engrafted with MOLM-13 cells.
2.2 Introduction
Despite the poor prognosis of acute myeloid leukemia (AML), therapeutics of AML has been
limited to chemotherapy and hematopoietic cell transplantation for decades (Liao et al., 2019). In
recent years, several drugs have been approved by the FDA, such as tyrosine kinase inhibitors
(TKIs), for AML treatment thanks to the understanding of the biology of acute myeloid leukemia
(AML), but still, the 5-year survival rate remains low (Döhner et al., 2017). As an effort to find a
novel target for AML, the Alachkar lab has recently shown CD99 is highly expressed in AML
compared with normal hematopoietic cells. In addition, the lab has established CD99 as a viable

2
This chapter was referenced to a manuscript, titled “Anti-CD99 scFv-ELP nanoworms for the treatment of acute myeloid
leukemia” by Vaikari et al.

40

therapeutic target for AML. The lab showed that silencing CD99 resulted in decreased cell
proliferation and increased cell apoptosis. Also, targeting CD99 with a monoclonal antibody (mAb)
induced anti-leukemic effects, such as reduced leukemic cell proliferation and migration and
increased cell apoptosis and differentiation (Vaikari et al., 2019). In addition, anti-CD99 mAb
significantly reduced the leukemic burden in AML xenograft models, demonstrating therapeutic
effects for AML in vivo (Chung et al., 2017).
Currently, mAbs targeting CD99 are available, but none of them are clinically approved
for AML treatment. In addition, except mAbs and single-chain variable fragments (scFvs) targeting
CD99, there are no other modalities available to target CD99. While the production of mAbs can
be costly and cumbersome, scFvs can be easy to produce while expecting a similar efficacy to
that of monoclonal antibodies. scFvs are small proteins whose molecular weight is around 30 kDa,
and they consist of a variable heavy chain (VH) and light chain (VL), which are connected by a
flexible peptide linker. These scFvs exhibit similar binding affinity and specificity to mAbs, and
they can be easily produced in Escherichia Coli using recombinant protein technology (Skerra
and Pluckthun, 1988), which is much cheaper compared to the expensive production of mAbs in
a mammalian expression system (Hu et al., 2007). However, their low molecular weight makes
scFvs susceptible to rapid glomerular filtration in the kidneys resulting in a short half-life (Hutt et
al., 2012). In addition, their low stability compromises their suitability for clinical use (Hayhurst
and Harris, 1999).
To overcome these limitations, we have employed a high molecular weight elastin-like
polypeptide (ELP) to fuse an anti-CD99 scFv. ELPs are genetically engineered protein polymers
derived from human tropoelastin. They consist of the pentameric amino acid repeats, (VPGXG) n,
where X can be any amino acid (Despanie et al., 2016). As derived from humans’ tropoelastin,
ELPs are biodegradable and biocompatible. ELPs undergo reversible phase separation above a
transition temperature, and this characteristic allows ELPs purified without chromatography

41

(Christensen et al., 2009). As ELPs are genetically encodable, their molecular weight can be
easily modified with DNA recombinant technology. To avoid the glomerular filtration, we have
fused a high molecular weight ELP, A192, to an anti-CD99 scFv. In this chapter, we report the
fusion protein, α-CD99-A192, assembles into stable nanoparticles that target CD99 with strong
anti-leukemic activity and a long half-life, suggesting α-CD99-A192 as a new modality of AML
therapy.
2.3 Materials and methods
2.3.1 Cloning and purification of α-CD99-A192
To construct an elastin-like polypeptide (ELP) targeting CD99, α-CD99 scFv gene was fused to
the amino terminus of a high molecular weight ELP, A192, in the the pET-25b(+) vector, encoding
α-CD99-A192. the α-CD99 scFv gene was purchased from Integrated DNA Technologies, IA,
USA, and the DNA sequence is:
ATGGCTGAAGTACAGTTAGTGGAATCAGGAGGTGGTTTAGTACGCCCAGGTGGTTCTTTAC
GCCTTAGCTGTGCAGCTTCTGGCTTCACATTTAGTTCCTACGCAATGAGTTGGGTCCGTCA
AGCGCCGGGCAAAGGATTAGAGTGGGTGAGCGCAATTAGCGGTTCAGGGGGTTCGACCT
ACTACGCAGACAGTGTCAAGGGTCGCTTTACAATTTCTCGCGATAATTCTAAGAATACCTTG
TATTTGCAAATGAACTCGCTTCGTGCGGAAGATACTGCAGTGTACTATTGTGCAAAAAGCC
ACAAACGCTTTGACTACTGGGGACAGGGAACCTTAGTGACGGTTTCACGCGGAGGCGGTG
GATCTGGCGGCGGAGGGTCGGGGGGAGGTGGCTCATCAGAATTGACACAGGACCCTGCC
GTGTCAGTTGCCCTGGGGCAAACCGTACGCATTACATGCCAGGGTGATTCACTTCGTTCTT
ACTATGCTTCCTGGTACCAGCAAAAGCCAGGCCAAGCCCCCGTGCTGGTTATCTATGGGA
AAAACAACCGCCCGTCGGGCATTCCAGACCGCTTTTCCGGTTCTTCTTCAGGTAATACGGC
AAGTCTGACCATTACTGGAGCACAAGCCGAGGACGAGGCAGACTACTATTGTAATTCGAGT
TTCCCTCGCACCAGTTCCGTAGTGTTTGGGGGCGGAACTAAATTGACAGTGCTTGGA.

42

To construct α-CD99-A192, the α-CD99 scFv was cloned into an empty pET-25b(+) vector using
NdeI and BamHI restriction enzymes (New England Biolabs, MA, USA). After the confirmation of
the ligation of α-CD99 scFv in pET-25b(+) vector, BseRI and BssHII restriction enzymes were
used to cut pET-25b(+)-α-CD99 scFv and pET-25b(+)-A192 to construct pET-25b(+)-α-CD99-
A192. After the ligation, a diagnostic DNA digestion was run using NdeI and BamHI restriction
enzymes followed by electrophoresis on a 1% agarose gel to confirm the insertion of α-CD99-
A192 into pET-25b(+) vector. After the diagnostic DNA digestion, the plasmid was sent for
sequencing using the T7 promoter (TAATACGACTCACTATAGGG) and T7 terminator
(GCTAGTTATTGCTCAGCGG), which confirmed in-frame insertion into the pET-25b(+). To
express and purify α-CD99-A192, Clearcoli BL21 (DE) Electrocompetent Cells (60810, Lucigen,
WI, USA) were transformed with the α-CD99-A192 plasmid using electroporation. After
transformation, colonies were picked and cultured in 60 mL of Terrific Broth with Glycerol (TB)
(C8153, CulGeneX, CA, USA) with 100 µg/mL of carbenicillin at 37 °C for 16-18 hours. For each
autoclaved 1 L of TB containing 100 µg/mL carbenicillin, 10 mL of cultured Clearcoli was added.
A total of 6 L Clearcoli was cultured at 37 °C until the optical density (OD) at 600 nm reached
between 0.6 and 0.8. Once the OD 600 reached above 0.6, 400 µL of 1 M isopropyl β-D-1-
thiogalactopyranoside (IPTG) was added to each 1 L Clearcoli culture to bring the final
concentration to 400 µM IPTG. IPTG induction was allowed to proceed overnight at room
temperature. The next day, bacteria were pelleted by centrifugation at 4,000 rpm for 15 minutes,
and the supernatant was discarded. Each pellet was resuspended with 30 mL of cold PBS,
vortexed, and sonicated using a probe-tip Misonix sonicator S-4000 (Misonix, NY, USA). After
sonication, 0.5 % of polyethyleneimine (PEI) was added to each cell lysate, and the cell lysate
was incubated on ice for 5 minutes. The cell lysates were pelleted at 13,000 rpm for 15 minutes
at 4 °C, and the supernatant was collected. To induce the temperature-dependent phase
separation, solid NaCl was added to the supernatant to bring a final concentration of 2 M. Each
supernatant mixed with 2 M of NaCl was placed in the water bath at 37 °C until phase separation

43

was induced. After the heat-induced phase separation, the supernatant was pelleted at 4,000 rpm
for 15 minutes at 37 °C, and the supernatant was discarded. Each pellet was resuspended with
cold PBS on ice. The resolubilized pellet was centrifuged at 13,000 rpm for 15 minutes at 4 °C to
remove any impurities. The supernatant was recovered, and this process, a cycle of hot and cold
centrifugation, was repeated three times by gradually decreasing the volume of PBS and NaCl
concentration to increase the purity of α-CD99-A192.
2.3.2 α-CD99-A192 protein concentration measurements
As it forms nanoparticles that scatter ultraviolet light, α-CD99-A192 was denatured with 6 M
guanidine hydrochloride to disrupt the assembly of nanoparticles. The protein concentration was
measured using the Eq. 1. To obtain ε of the fusion protein, the Eq. 2. was employed. By
employing the equation, ε of α-CD99 scFv was estimated to be 41,620 L mol
-1
cm
-1
assuming all
pairs of cysteine residues are oxidized to form disulfide bonds. ε of A192 was calculated to be
1,490 L mol
-1
cm
-1
as it has only one tyrosine at the end of A192 sequence. As a result, ε of α-
CD99-A192 was calculated to be 43,110 L mol
-1
cm
-1
. The absorbance at 280 and 350 nm was
measured using a NanoDrop 2000 (Thermo Fisher, Waltham, MA, USA), whose path length is
0.1 cm.
2.3.3 α-CD99-A192 purity and transition temperature analysis
To estimate the purity of α-CD99-A192, an SDS-PAGE gel image was analyzed using ImageJ.
To run SDS-PAGE, 10 and 20 µg of α-CD99-A192 was loaded to a 4-20% precast SDS-PAGE
gel (4561095, Bio-Rad Laboratories, CA, USA). The gel was stained with Gelcode
TM
Blue Safe
Protein Stain (24596, Thermo Fisher, MA, USA). After the staining at room temperature, the gel
image was captured with a ChemiDoc Touch Image System (Bio-Rad Laboratories, CA, USA).
The gel image was analyzed with ImageJ (NIH, MD, USA) to estimate the purity of the purified α-
CD99-A192. Each lane was plotted to obtain the area under each peak, and the Eq. 3 was used
to measure the purity of the protein. To measure the transition temperature (Tt) of α-CD99-A192,

44

Beckman Coulter DU 800 UV/VIS spectrometer (Beckman Coulter, CA, USA) was employed. To
analyze the Tt, four different concentrations of α-CD99-A192 were prepared: 3.125, 6.25, 12.5,
and 25 µM. The samples were transferred to micro cuvettes and then heated at a rate of 1 °C/min
starting from 20 °C to 85 °C. Optical density (OD) at 350 nm was measured every 18 seconds to
plot a phase diagram. The Tt was determined where the maximum first derivative of the OD at
350 nm with respect to temperature occurred. The transition temperature data were fit to the Eq.
4.
2.3.4 α-CD99-A192 protein refolding
To optimize the biological activity of α-CD99-A192, α-CD99-A192 was refolded. First, the fusion
protein in PBS was 2-fold diluted with 8 M urea buffer with 10 mM β-mercaptoethanol (BME) to
denature the fusion protein. After the denaturation, the mixture was dialyzed to 3 M urea buffer
with 2 mM glutathione (GSH) and 0.4 mM oxidized glutathione (GSSH) for 24 hours at 4 °C. Then,
the fusion protein was dialyzed to 1 M urea buffer with 2 mM GSH and 0.4 mM GSSH, 0.5 M urea
buffer, a buffer containing 20 mM Tris, 150 mM NaCl, and 50 mM arginine with pH 8.0, and then
PBS (twice) for 24 hours at 4 °C for each step.
2.3.5 Measurements of the hydrodynamic radius of α-CD99-A192 and colloidal stability
To measure the hydrodynamic radius of α-CD99-A192 in PBS, 25 µM of α-CD99-A192 samples
were prepared and then filtered with a 0.22 µm filter. After the filtration, 60 µL of the protein was
pipetted to each well of a 384-well plate. to prevent evaporation of the solvent, 15 µL of mineral
oil was added to 60 µL of the protein. The plate was placed into DynaPro Plate reader II (Wyatt
Technology, CA, USA), and the hydrodynamic radius was measured. As a control, the
hydrodynamic radius of A192 was also measured. To measure the stability of α-CD99-A192 in
PBS, the 384-well plate was incubated at 37 °C, and the hydrodynamic radius of the fusion protein
was measured every 24 hours over 3 days.

45

2.3.6 Measurement of the molecular weight of α-CD99-A192 nanoparticles
To estimate the average molecular weight of α-CD99-A192 nanoparticles, size exclusion
chromatography-multiangle light scattering (SEC-MALS) was employed. To run SEC-MALS, 10
µM of α-CD99-A192 was prepared and filtered with a 0.22 µm filter. A Shodex protein KW-804
(8.0mmI.D.x300mm) (Showa Denko America, NY,USA) was equilibrated with PBS before loading
the samples, and the prepared sample was run through the column. The fusion protein elusion
was observed with three detectors, UV 210 mm detector (SYC-LC-1200) (Agilent Technologies,
CA, USA), multi-angle static light scattering detector (DAWN HELEOS) (Wyatt Technology, CA),
USA, and differential refractometer (Optilab rEX) (Wyatt Technology, CA, USA). The data were
analyzed by ASTRA 6 software.
2.3.7 Patient Samples
Peripheral blood was obtained from patients with AML at the time of diagnosis at the Norris
Comprehensive Cancer Center at USC following written informed consent. The use of human
materials was approved by the Institutional Review Boards of USC in accordance with the Helsinki
Declaration.
2.3.8 Cell Culture
U937 and MOLM-13 cells, were kindly provided by Dr. Wendy Stocks’s lab at the University of
Chicago. MV4-11 cells were purchased from ATCC. All the cell lines were authenticated at the
University of Arizona Cell Authentication Core. Cells were cultured in Roswell Park Memorial
Institute 1640 (RPMI 1640) medium (Thermo Fisher, MA, USA) supplemented with 10% fetal
bovine serum (FBS) and 100 U/mL penicillin (Thermo Fisher, MA, USA). Primary cells (USC001)
were grown in RPMI plus FBS (20%) and cytokine cocktails CC100 (Flt3L, SCF, IL-3 and IL-6).
2.3.9 Viability assays
Cell viability was analyzed by treating 0.5×10
5
cells with either A192 or α-CD99-A192 at 1,10, 25
and 50 µM. Cells were incubated for 30 minutes on ice and then seeded in a 12-well plate at a

46

concentration of 5×10
5
/mL. The number of live cells was counted using trypan blue (cat.no:15250-
061, Life Technologies, Carlsbad, USA) after 72 hours of incubation, and cell viability was
estimated by normalizing the number of live cells in samples treated with A192 or α-CD99-A192
to that in untreated cells. To determine the IC 50 of α-CD99-A192 for U937, MOLM-13, and MV4-
11, cells were incubated in a 96-well plate at 5×10
5
cells/mL and treated with α-CD99-A192 at
various concentration: 0.01, 0.1, 0.25, 0.5, 1, 10, 25, 50, and 100 µM. To measure the cell viability,
after 72 hours of the incubation, 10 µL of alamar blue (cat no: DAL 1100, Invitrogen, Carlsbad,
USA) was added to each well, and the plate was incubated for four hours at 37 °C. Fluorescence
was measured with a synergy H1 microplate reader. The change in viability was measured by
normalizing the fluorescence of cells treated with A192 or α-CD99-A192 to that of untreated cells.
IC 50 was calculated by fitting the data to non-linear regression.
2.3.10 α-CD99-A192 competitive binding study
To verify the binding and specificity of α-CD99-A192 to CD99 proteins on the cell surface, α-
CD99-A192 was labeled with 5 molar excess NHS-rhodamine for 2 hours at the room temperature.
Any unbound NHS-rhodamine was removed by running the mixture through Zeba
TM
Spin
Desalting Columns (Thermo Fisher, MA, USA). Absorbance was measured to quantify the
rhodamine concentration of labeled ELPs using the Eq. 5. Based on these measurements, the
degree of rhodamine labeling was estimated to be ~1.2, ~1.8 rhodamine per A192, α-CD99-A192
respectively. To measure the binding of α-CD99-A192 and A192 to CD99 proteins, flow cytometry
was employed. To prepare samples to run, 0.5×10
5
MOLM-13, MV4-11, U937 or CD99
-
293T
cells were treated with either rhodamine-labeled A192 or α-CD99-A192 (1, 10, and 25 µM) and
incubated for 30 minutes on ice. Unbound proteins were washed away and the bound rhodamine-
labeled A192 and α-CD99-A192 were assessed by measuring the shift in the mean fluorescence
intensity (MFI) by flow cytometry. The flow data were analyzed with the normalization of MFI to
untreated cells. To assess the specificity of α-CD99-A192 to CD99 proteins, 0.5×10
5
MOLM-13

47

cells were pre-incubated with IgG, anti-CD99 mAb 2.5 µg/mL and 5 µg/mL for 30 mins on ice.
Cells were washed to remove unbound antibodies and then treated with rhodamine-labeled α-
CD99-A192 (10 µM) for 30 mins on ice. Unbound α-CD99-A192 was washed and the binding of
α-CD99-A192 was assessed by measuring the shift in the MFI by flow cytometry. Remaining cells
were transferred to glass-bottom MatTek plates (P35G-0.170-14-C, MatTek Corporation, MA,
USA) for live cell imaging. Cell images were captured using ZEISS LSM 880 Confocal Laser
Scanning Microscope (Carl Zeiss, Oberkochen, Germany) with a 63x objective lens. Images were
analyzed with ImageJ to quantify the fluorescent intensity levels in the cells.
2.3.11 Cell apoptosis analysis
Apoptosis assay in vitro was performed using the Annexin V and PI APC kit according to the
manufactures protocol (Invitrogen, Cat no:88-8007-72). The percentage of apoptotic cells (APC
+

cells) were compared in cells treated with α-CD99-A192 and normalized to untreated cells. For in
vivo experiments, cell-surface expression of human CD45 (hCD45: cat.no:25-0459-41,
eBioscience, CA, USA) were measured to confirm engraftment. Mouse peripheral blood, bone
marrow and spleen cells were stained with PE-Cy7-A conjugated anti-hCD45 for 30 mins on ice.
Unbound anti-hCD45 washed away, and the bound anti-hCD45 was measured via flow cytometry.
The MFI of PE-Cy7-A was used to quantify data. Flow cytometry was run on the LSRII BD
Fortessa X20 flow cytometer and processed using FloJo software (BD, Franklin Lakes, NJ, USA).
2.3.12 Pharmacokinetic (PK) and biodistribution study of α-CD99-A192
To analyze the PK parameters of α-CD99-A192 in mice, 150 µL/20g BW of 340 µM of rhodamine
labeled to α-CD99-A192 (190 µM of total protein concentration) was injected into tail vein of
females 4 to 6-week-old NOD-scid /Il2rg
-/-
(NSG) mice (n=4). Blood samples (10-20 µL of blood)
were collected by venipuncture and mixed with 80 µL of heparinized cold PBS instantaneously
(1,000 U/mL) at the following time points: 3 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36
h, 48 h, and 72 h post injection. The diluted blood samples were pelleted at 2,000 g for 10 minutes

48

at 4 °C to collect the plasma. The diluted plasma was transferred to a 96-well plate, and a Biotek
Synergy H1 Hybrid Multi-Mode Reader (Biotek, VT, USA) was employed to measure the
fluorescence (Excitation/Emission: 540/580 nm). To plot a standard curve, various concentrations
of α-CD99-A192 were prepared: 2, 5, 14, 41, 123, 370, 1111, 3333, and 10000 nM of rhodamine
labeled to α-CD99-A192. After the PK study is completed, mice were euthanized, and the kidneys,
liver, and spleen were collected to image for bioluminescence using the iBright FL1000 gel/cell
imager. To quantify the fluorescent signals, region of interests (ROIs) were selected for the
kidneys, liver, and spleen, and the fluorescent intensity was assessed with ImageJ.
2.3.13 In vivo efficacy studies
Animal protocols were approved by the Institution for Animal Care and Use Committee of the
University of Southern California. To perform xenograft experiments, 2.5×10
6
MOLM-13 cells
were administered via tail vein injection into 4- to 6-week-old female NOD-scid /Il2rg
-/-
(NSG) mice
purchased from Jackson Laboratory (Bar Harbor, ME). Engrafted mice were treated with 200 µL
of 220 µM of A192 (n=4) or α-CD99-A192 (n=4) via tail vein on Day 7, 10, 13 and 16 post
engraftments. Mice were euthanized on Day 21, and the spleen, bone marrow and blood were
collected. The collected organs and blood were stained for hCD45 and then analyzed using flow
cytometry. For survival analysis, male mice engrafted with MOLM-13 cells were treated with 200
µL of 220 µM of A192 (n=7) or α-CD99-A192 (n=7) on Days 7, 10, 13 and 16 post MOLM-13
engraftment. Once they showed signs of severe weight loss, lethargy, hair loss, and/or hunched
appearance, mice were euthanized.
2.3.14 Statistical Analysis
For confocal and biodistribution image analysis, a global one-way ANOVA followed by the Tukey
post-hoc test was employed to determine whether mean values are significantly different between
groups. Student t test was used to determine the significance in means between two groups for
viability, apoptosis, and engraftment analysis. Mice survival analysis was performed by the Kaplan

49

Meier survival analysis based on Log-rank (Mantel Cox). For all analysis, p < 0.05 was considered
significant. All the data is presented as mean ± standard deviation (SD).
2.4 Results
2.4.1 Characterization of α-CD99-A192 nanoparticles
The fusion protein was purified using three cycles of hot and cold centrifugation, and the yield of
α-CD99-A192, expressed in Clearcoli, was calculated to be 34 mg/L with an apparent purity of
~74% (Fig.8B). The purity estimated on fluorescent imaging of an SDS-PAGE gel with
rhodamine-labeled α-CD99-A192 was ~77%. The fusion of α-CD99 scFv to A192 apparently
lowered the Tt of A192 compared to A192 alone. As shown in Fig. 8C, α-CD99-A192 undergoes
phase separation above 45.3 °C while A192 undergoes phase separation above 59.9 °C at 25
µM in PBS. DLS analysis shows that α-CD99-A192 forms nanoparticles at 37 °C with the
hydrodynamic radius of 46.6 nm (Fig. 8D). In addition, the hydrodynamic radius of α-CD99-A192
was not changed much over 72 hours, which demonstrated that α-CD99-A192 nanoparticles are
stable colloids at 37 °C in PBS (Fig. 8E). The average of molecular weight of nanoparticles was
assessed with SEC-MALS and indicated that approximately 170 α-CD99-A192 molecules form a
nanoparticle (Fig. 1F). The Rg/Rh ratio was 1.0 for the major peak, which is consistent with a
nanoworm-like nanostructure (Table 1).

50

Figure 9. Construction, purification and characterization of a recombinant elastin-like
polypeptide (ELP) fusion protein targeting CD99 proteins. A) To construct an ELP fusion protein
targeting CD99 proteins, α-CD99 scFv was fused to the N-terminus of a high molecular weight ELP,
A192. It has believed that fusion of A192 to α-CD99 scFv assembles nanoparticles spontaneously in
PBS by blocking the formation of inclusion bodies. B) The ELP fusion protein was purified by using
ELP-mediated temperature-dependent phase separation. Three rounds of hot and cold centrifugation
were used to purify α-CD99-A192, and the purity of the fusion protein was calculated to be 73.5 %
based on the analysis of the SDS-PAGE gel on the left side: lane 1: ladder lane 2: 10 µg of α-CD99-
A192 lane 3: 20 µg of α-CD99-A192. The SDS-PAGE gel on the right side shows the purity of NHS-
rhodamine labeled α-CD99-A192, and the purity was calculated to be 77 %: lane 4: 8.5 µg of NHS-
rhodamine labeled α-CD99-A192 lane 5: 17 µg of NHS-rhodamine labeled α-CD99-A192 C) The
transition temperature of A192 and α-CD99-A192 was measured at 3.125, 6.25, 12.5, and 25 µM. The
fusion of α-CD99 scFv to A192 substantially lowers the transition temperature of A192, and the transition
temperature is less affected by the change of the protein’s concentration. Transition temperature
becomes less sensitive to the change of the protein’s concentration. D) Hydrodynamic radius (Rh) of
refolded α-CD99-A192 was measured with DLS at 37 °C. While A192 is monomeric, α-CD99-A192
formed nanoparticles. E) The stability of α-CD99-A192 in PBS was observed with DLS for 72 hours.
The Rh of the fusion protein did not change much over 72 hours, indicating that the fusion protein
remains stable colloids in PBS at 37 °C. F) To measure the molecular weight of α-CD99-A192
nanoparticles, SEC-MALS was employed. The average molecular weight of a nanoparticle was
measured to be 1.7×10
7
Da, which is equivalent to the molecular weight of 171 α-CD99-A192 molecules.

51

Table 4. Characterization of α-CD99-A192 nanoparticles
ELP Amino acid
sequence
M.W.
(kDa)
b

T t (°C)
c
R h (nm) R g/R h Shape
e

A192 G(VPGAG)192Y 73.6 59.9 7.5 ± 0.2 N/A monomer
α-CD99-A192 αCD99
a
-
G(VPGAG)192Y
99.2 45.3 46.6 ± 0.5 1.0
d
Extended rod-
like shape
a
αCD99 amino acid sequence:
MAEVQLVESGGGLVRPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCAKSHKRFDYWGQGTLVTVSRGGGGSGGGGSGGGGSSELTQDPAVSVALGQTV
RITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSFPRTS
SVVFGGGTKLTVLGLVPRGS
b
It is the expected molecular weight based on the amino acid sequence
c
Transition temperature was determined at the maximum first derivative of the optical density at 350 nm
d
This number represents Rg/Rh of peak 1 observed in SEC-MALS
e
The shape of α-CD99-A192 was determined by using the ratio Rg/Rh, which were obtained from DLS and SEC-
MALS

2.4.2 α-CD99-A192 binds specifically to CD99 surface protein in vitro
The binding of α-CD99-A192 to CD99 proteins was assessed in CD99
+
AML cell lines MOLM-13,
MV4-11, U937 and CD99
-
293T cells. Flow cytometry analysis revealed that rhodamine labeled
α-CD99-A192 showed significantly higher binding to MOLM-13 compared with A192 at both 1 µM
(2.4-fold; p=0.04) and 10 µM (3.1-fold; p=0.01) (Fig 9A). Significantly higher level of binding of α-
CD99-A192 was also detected in MV4-11 cells compared with A192 at both 1 µM (1.7-fold;
p=0.001) and 10 µM (2.8-fold; p=0.02) (Fig 9B). Similarly, in U937 cells, significantly higher
binding of α-CD99-A192 was observed at both 1 µM (1.5-fold; p=0.002) and 10 µM (1.9-fold;
p=0.0001) (Fig 9C). As expected, α-CD99-A192 did not bind to CD99
-
293T, suggesting that the
binding was specific to cell lines expressing CD99 proteins (Fig 9D). A192 at 1 µM or 10 µM
showed no binding to the cells as compared with untreated cells.
To verify whether the binding of α-CD99-A192 to CD99 proteins is specific, we performed
a competitive binding assay with an anti-CD99 monoclonal antibody. MOLM-13 cells were pre-
treated with either an anti-CD99 mAb or IgG, and unbound antibodies were washed away. Then,
the cells were treated with rhodamine-labeled α-CD99-A192. After normalizing to untreated cells,
it was found that cells pre-treated with 2.5 and 5 µg/mL anti-CD99 mAb had a significant decrease

52

in binding to α-CD99-A192 as compared to cells treated with α-CD99-A192 alone ( 35%, p=0.03
and ~60%, p=0.001 respectively). Pre-treatment with IgG did not affect the binding of α-CD99-
A192 to CD99 proteins. No binding was observed in MOLM-13 cells treated with rhodamine
labeled A192 (Fig 9E). Confocal microscope image analysis also showed the similar results to
that of flow cytometry. The treatment of anti-CD99 mAb significantly inhibited the binding of α-
CD99-A192 (10 µM) to CD99 proteins, while IgG did not affect the binding of α-CD99-A192,
suggesting that the binding of α-CD99-A192 is specific to CD99 surface protein (Fig 9F).

53

Figure 10. Recombinant α-CD99-A192 binds specifically to CD99. CD99
+
AML cell lines MOLM-13,
MV4-11, U937 cells, and CD99
-
293T cells (0.5 x 10
6
cellls) were treated with 1 µM or 10 µM of
rhodamine labeled α-CD99-A192 or rhodamine-labeled A192 for 30 minutes on ice. The binding was
measured via flow cytometry by analyzing the MFI of rhodamine and normalizing data to untreated cells.
The significant binding was observed in A) MOLM-13 B) MV4-11 C) U937 cells. D) No binding was
observed in 293T cells. The competitive binding assay was performed in MOLM-13 cells by pre-treating
cells with either CD99 Mab (2.5 µg/mL or 5 µg/mL) or IgG and then observing binding with rhodamine
α-CD99-A192 (10 µM). The binding was measured via E) Flow cytometry by measuring the peak shift
in rhodamine to bound cell and quantifying based on mean fluorescence intensity and F) laser scanning
confocal microscopy where cell fluorescence was quantified with ImageJ. Data represented as mean ±
SD. * p≤0.05 ** p≤0.01*** p≤0.001**** p≤0.0001

54

2.4.3 α-CD99-A192 induces cell apoptosis in AML cells
To verify the anti-leukemia activity of α-CD99-A192, MOLM-13, MV4-11 and U937 cells were
treated with α-CD99-A192 at 1, 10, 25 and 50 µM, or the same concentrations of A192. To
determine the number of live cells, trypan blue assay was performed. A significant decrease in
the percentage of viable cells was observed in MOLM-13 cells treated with α-CD99-A192
compared with cells treated with A192 at 1 µM (p<0.0001, 30% decrease), 10 µM (p<0.0001, 43%
decrease), 25 µM (p<0.0001, 78% decrease) and 50 µM (p<0.0001, 82% decrease) (Fig. 10A).
Similarly, α-CD99-A192 significantly decreased the cell viability in MV4-11 cells at 1 µM (p<0.0001,
30% decrease), 10 µM (p<0.0001, 46% decrease), 25 µM (p<0.0001, 80% decrease) and 50 µM
(p<0.0001, 84% decrease) (Fig. 10B). In U937 cells, the fusion protein significantly reduced the
number of live cells at 1 µM (p<0.0001, 30% decrease), 10 µM (p<0.0001, 41% decrease), 25 µM
(p<0.0001, 75% decrease) and 50 µM (p<0.0001, 83% decrease) (Fig. 10C). Based on these
results, IC 50 of α-CD99-A192 for MOLM-13, MV4-11 and U937 cells were determined as 8.1 µM,
4.9 µM and 6.05 µM, respectively (Fig. 10D-F). Cells treated with α-CD99-A192 exhibited
enhanced cell apoptosis. Using Annexin V staining, it was observed that MOLM-13 cells treated
with α-CD99-A192 showed a significant increase in cell apoptosis (1 µM: p=0.03,1.3-fold; 10 µM:
p=0.01, 1.5-fold; 25 µM: p=0.001, 1.6-fold and 50 µM: p=0.01, 1.6-fold; Fig. 11A). In MV4-11 cells
treated with α-CD99-A192, higher levels of cell apoptosis were observed compared with cells
treated with A192 (1 µM: p=0.04,1.4-fold; 10 µM: p=0.005, 1.7-fold; 25 µM: p=0.007, 2-fold and
50 µM: p=0.007, 2-fold; Fig. 11B). Similarly, U937 cells treated with α-CD99-A192 showed
significantly higher levels of cell apoptosis compared with cells treated with A192 (1 µM:
p=0.04,1.48-fold; 10 µM: p=0.007, 1.7-fold; 25 µM: p=0.01, 2.3-fold and 50 µM: p=0.009, 2.3-fold;
Fig. 11C).

55

Figure 11. α-CD99-A192 has anti-leukemic activity in AML cells. A-C) A trypan blue viability assay
was performed in MOLM-13, MV4-11, and U937 cells treated with α-CD99-A192 or an A192 control for
72 hours. The number of live cells was normalized to untreated cells. Data represented as mean ± SD,
n=6. D-E) Alamar blue staining was performed in MOLM-13, MV4-11 and U937 cells to determine the
IC50 of α-CD99-A192 at 48 hours post-treatment with the increasing concentration of α-CD99-A192 and
plotted based on non-linear regression.

56

2.4.4 α-CD99-A192 exhibits anti-leukemia activity in primary AML cells
To study the anti-leukemic activity of α-CD99-A192 further, primary AML cells (USC001) were
obtained from patients with AML. Primary AML cells treated with rhodamine-labeled α-CD99-
A192 (20 µM) showed significantly increased binding of α-CD99-A192 to CD99 proteins
compared to A192 (2.6-fold, p<0.001; Fig. 12A). In addition, the fusion protein significantly
reduced cell survival of AML primary cells (24 hours: p=0.002, 50% decrease; 48 hours µM:
p<0.0001, 50% decrease; Fig. 12B). AML primary cells treated with α-CD99-A192 also exhibited
a significant increase in cell apoptosis at 48 hours (p=0.002, 1.2-fold; Fig. 12C).

Figure 12. α-CD99-A192 induces apoptosis in AML cells. Annexin V-PI assay was performed in A)
MOLM-13, B) MV4-11 and C) U937 cells treated with α-CD99-A192 or control A192. Apoptosis was
measured by flow cytometry using APC conjugated Annexin V stain normalized to untreated cells.
Representative flow cytometry images and quantified Annexin V data are presented. Data represented
as mean ± SD, n=3.

57

2.4.5 PK profile of α-CD99-A192 nanoparticles fits the biexponential decay model
PK analysis of α-CD99-A192 indicated that α-CD99-A192 nanoparticles fit a biexponential decay
model using the Eq. 7. Using a non-compartmental and two-compartmental analysis, the PK
parameters of α-CD99-A192 were determined (Table 5). The terminal half-life of α-CD99-A192
nanoparticles was estimated to be 15.8 hours, and mean residence time was 21.3 hours (Fig.
13A). The clearance of the fusion protein was determined to be 0.28 mL/hr, and the volume of
distribution was estimated to be 1.92 mL. Bioluminescence images of spleen, kidneys, and liver
showed that α-CD99-A192 nanoparticles still resided in those organs 96 hours post injection and
accumulated at higher level in the liver as compared to the spleen and kidney (p ≤0.05) (Fig. 13B
and C).

Figure 13. α-CD99-A192 has anti-leukemic activity in primary AML cells. A) A trypan blue viability
assay was performed in MOLM-13, MV4-11 and U937 cells treated with α-CD99-A192 or an A192
control for 72 hours. The number of live cells was normalized to untreated cells. Data represented as
mean ± SD, n=6. B-C) Alamar blue staining was performed in MOLM-13, MV4-11, and U937 cells to
determine the IC50 of α-CD99-A192 at 48 hours post-treatment with the increasing concentration of α-
CD99-A192 and plotted based on non-linear regression.

58

Figure 14. PK profile of α-CD99-A192 nanoparticles. 190 µM of α-CD99-A192 labeled covalently
with rhodamine in 150 µL/25g B.W. of PBS was injected via tail vein in mice (n=4 mice). Blood was
collected and plasma was measured for fluorescence. Rhodamine-labeled α-CD99-A192 plasma
concentration was calculated using a standard curve. A) The plasma concentrations were fit to Eq. 7 to
obtain the PK curve, and the generated curve shows the rhodamine-labeled α-CD99-A192
concentration in the plasma over 72 hours. After 96 hours from the injection, spleen (S), kidneys (K),
and the liver (L) were collected from mice. B-C) Bioluminescence images of organs were obtained 96
hours post-injection and quantified based on the fluorescent intensity obtained from images.

59

Table 5: PK parameters for α-CD99-A192
a
determined using non-compartmental analysis
b
determined by fitting to Eq. 7 and fit to a two-compartment model of an intravenous bolus(Guo and MacKay, 2020)

Parameter (Unit) α-CD99-A192 IV (SD) (n=4) 95% Conf Interval
[Lower to Upper bound]
Dose (nmol)
a
66.8 (2.8) [62.4 to 71.2]
CL (mL/hr)
a
0.28 (0.02) [0.24 to 0.32]
AUC (µM*hr)
a
240 (17) [212 to 268]
AUMC (µM*hr
2
)
a
5130 (959) [3610 to 6660]
MRT (hr)
a
21.3 (2.6) [17.1 to 25.4]
Vss (mL)
a
5.92 (0.63) [4.91 to 6.92]
C0 (µM)
b
42.6 (26.3) [0.8 to 84.5]
A (µM)
b
32.5 (25.2) [0 to 72.5]
B (µM)
b
10.2 (1.2) [8.3 to 12.0]
α (hr
-1
)
b
6.13 (5.88) [0 to 15.5]
β (hr
-1
)
b
0.044 (0.004) [0.037 to 0.051]
t1/2α (hr)
b
0.24 (0.20) [0 to 0.56]
t1/2β (hr)
b
15.8 (1.4) [13.5 to 18.1]
kelimination (hr
-1
)
b
0.17 (0.08) [0.04 to 0.30]
kplasma tissue (hr
-1
)
b
4.61 (5.09) [0 to 12.7]
ktissue plasma (hr
-1
)
b
1.39 (0.90) [0 to 2.82]
AUC (µmol*h)
b
239 (36) [182 to 296]
CL (mL/h)
b
0.28 (0.04) [0.22 to 0.35]
Vd
a
(mL)
b
1.92 (0.77) [0.69 to 3.15]

60

2.4.6 α-CD99-A192 reduces leukemia burden and extends survival of mice in AML
xenograft model
To observe anti-leukemic effects of α-CD99-A192 nanoparticles in an AML xenograft mouse
model, MOLM-13 xenograft NSG mice were used. The mice were treated with α-CD99-A192 or
A192 for 4 doses on day 7, 10, 13 and 16 post MOLM-13 cell engraftment (n=4). Mice treated
with α-CD99-A192 nanoparticles had smaller spleens that weighed significantly less compared to
mice treated with A192 (113 vs. 180 mg, p<0.001) (Fig. 14A). huCD45 analysis for MOLM-13
engraftment showed that mice treated with α-CD99-A192 had significantly less MOLM-13
engraftment compared with mice treated with α-CD99-A192 in the bone marrow (%huCD45: 29
vs. 53%, p<0.0001; Fig. 14C-D), peripheral blood (%huCD45: 6 vs. 22%, p<0.001; Fig.14 E-F)
and the spleen (%huCD45: 5 vs. 13%, p<0.0001; Fig. 14G-H). The anti-leukemic effects of α-
CD99-A192 were further verified in the MOLM-13 xenograft model through survival analysis. Mice
treated with α-CD99-A192 survived significantly longer than mice treated with A192 (median
survival: 37 vs. 28 Days, p<0.0001; Fig 14I).

61

2.5 Discussion
CD99 is overexpressed in various diseases, such as Ewing sarcoma, ependymomas, T-lineage
acute lymphoblastic leukemia, gliomas, and AML (Ambros et al., 1991; Buxton et al., 2009; Choi
et al., 2001; Chung et al., 2017; Kavalar et al., 2009; Vaikari et al., 2020; Zhang et al., 2000).
CD99 is a surface protein, which makes it a potential target of antibody-based therapies for AML
treatment. In fact, anti-CD99 monoclonal antibodies have shown anti-tumor activities in several
preclinical cancer models, such as inhibition of cell migration and activation of cell apoptosis

Figure 15: Anti-leukemia activity of α-CD99-A192 in MOLM-13 murine model. Fig A-H: 2.5 × 10
6

cells MOLM-13 cells/mouse were engrafted in NSG mice. Mice were treated with 200 µL of 220 µM of
A192 (n=4) or α-CD99-A192 (n=4) on day 7, 10, 13 and 16 post engraftment and euthanized on day
17. A) Spleen images and B) Spleens weights were measured upon euthanization. Spleens of mice
treated with α-CD99-A192 weighed significantly less compared with the A192 treated mice (113 vs. 180
mg, p<0.001). Leukemia engraftment was measured using human CD45 (huCD45) antibody by flow
cytometry in the C-D) peripheral blood (%huCD45: 6 vs. 22, p<0.001), E-F) bone marrow (29 vs. 53,
p<0.0001) and G-H) spleen (%huCD45: 5 vs. 13, p<0.0001). I) 2.5 × 10
6
MOLM-13 cells/mouse were
engrafted in NSG mice and treated with 200 µL of 220 µM A192 (n=7) or α-CD99-A192 on Day 7, 10,
13 and 16 post engraftment and survival was recorded. Kaplan Meier survival analysis showed that
mice in the α-CD99-A192 treatment group survived significantly longer than mice in the A192 group
(median survival: 37vs. 28 Days, p<0.0001).

62

(Cerisano et al., 2004; Seol et al., 2012; Sohn et al., 1998). Although CD99 served as an
oncogene in these studies, it was also found that CD99 behaved as an onco-suppressor in
osteosarcoma, gastric adenocarcinoma, gallbladder and pancreatic carcinomas (Choi et al.,
2004; Goto et al., 2004; Manara et al., 2006). Currently, the dual role of CD99 in malignancies is
not understood well, but it is thought that the different isoforms of CD99 attribute to the dual role
(Manara et al., 2018). the CD99 gene encodes two distinct proteins as consequence of alternative
splicing of the cytoplasmic region, resulting in a wild-type full-length CD99 long isoform and a
truncated short isoform. The extracellular and transmembrane domains are similar between both
the isoforms, but the cytoplasmic domain of the long and short isoform can be different
(Goodfellow et al., 1988).
The Alachkar lab has characterized the two isoforms in AML and has revealed that
overexpression of the long isoform promotes cell proliferation and cell aggregation, which results
in increased cell apoptosis and decreased leukemic burden in xenograft murine models. On the
other hand, overexpression of short isoform has shown minimal to no effect. The existence of
isoforms makes characterization of CD99 complicated, but anti-CD99 monoclonal antibodies
have shown anti-leukemic activities in primary blasts and AML cell lines (Chung et al., 2017;
Vaikari et al., 2020). Anti-CD99 monoclonal antibodies only can target the extracellular domain of
CD99, which is similar in the long and short isoform, so it is speculated that the anti-cancer effect
of the CD99 monoclonal antibodies are not isoform specific.
Anti-CD99 monoclonal antibodies have shown potentials for the clinic, but no anti-CD99
antibodies have been approved by the FDA. Limitations related to their ineffective clustering on
target receptors as well as lack of therapeutic concentrations to target cells can lower the efficacy
of the monoclonal antibodies in the clinic. Our study demonstrates the development of a new
modality, elastin-like polypeptide (ELP) nanoparticles targeting CD99 surface proteins. To

63

formulate the nanoparticles, a high molecular weight ELP, A192, was fused to an α-CD99 scFv,
forming α-CD99-A192.
The fusion of A192 to α-CD99 scFv generated the high yield of α-CD99-A192 (34 mg/L)
in the soluble fraction after bacterial lysis. ELPs undergo reversible temperature-dependent phase
separation, and that characteristic allows chromatography-free protein purification with high purity
without any addition of purification tags that can compromise protein activity (Goel et al., 2000;
Sabaty et al., 2013). Using the reversible temperature-dependent phase separation, the fusion
protein was purified with around 77% purity. It has been reported scFvs have the low stability
(Brockmann et al., 2005; Worn and Pluckthun, 1999, 2001), but the fusion of A192 to α-CD99
scFv enhanced the stability of the scFv, resulting in a soluble fraction in PBS. Notably, the fusion
of A192 to α-CD99 scFv formulated stable nanoparticles for at least three days at 37 °C in PBS.
The half-life and mean residence time were also excellent, which are measured as 15.8 hours
and 21.3 hours, respectively. The long half-life of α-CD99-A192 can overcome the challenges of
using α-CD99 scFv in the clinic. Another potential benefit of ELP nanoparticles is that they get
clustered upon binding to target receptors on the cell membrane, resulting in enhanced
internalization of the receptors. The improved receptor internalization made scFv-ELP
nanoparticles outperform a commercial monoclonal antibody (Aluri et al., 2014). It is not known
whether CD99 surface proteins get internalized upon binding, but if CD99 surface proteins involve
receptor internalization for signaling, we might be able to expect an enhanced therapeutic effect
of α-CD99-A192 nanoparticles compared to anti-CD99 monoclonal antibodies.
The nanoparticles exhibited excellent therapeutic effects both in vitro and in vivo. In
various AML cell lines, α-CD99-A192 nanoparticles showed good binding affinity and specificity
to CD99
+
AML cells. Furthermore, the nanoparticles significantly reduced cell survival by
enhancing cell apoptosis in both AML cell lines and primary AML cells. In the MOLM-13 xenograft

64

murine model, α-CD99-A192 nanoparticles exhibited anti-leukemic activities and significantly
extended survival of the mice.
2.6 Conclusion
In conclusion, this chapter reports the formulation and evaluation of ELP nanoparticles targeting
CD99 proteins. The fusion of A192 to α-CD99 scFv solubilizes the scFv, facilitates purification of
the fusion protein, assembles biologically active nanoparticles, and improves PK profiles of α-
CD99 scFv. As CD99 serves as a therapeutic target for various malignancies, this study suggests
α-CD99-A192 nanoparticles as a new therapeutic agent for AML. As previous studies have
reported anti-CD99 monoclonal antibodies sensitize chemo-resistant cells, α-CD99-A192
nanoparticles can also be applied to combination therapies to enhance the efficacy of
chemotherapies.
2.7 Acknowledgements
This work was made possible by University of Southern California (USC), the Gavin S. Herbert
Professorship, the National Institutes of Health R01 GM114839 to JM, the USC Ming Hsieh
Institute, P30 CA014089 to the USC Norris Comprehensive Cancer Center, P30 EY029220 to the
USC Ophthalmology Center Core Grant for Vision Research, Center of Excellence in
Nanobiophysics at USC, and the Translational Research Laboratory at USC School of Pharmacy.

65

Chapter 3: Formulation of ELP nanoparticles stimulating human granulocyte-
macrophage colony-stimulating factor receptors for post leukemia treatment
3

3.1 Abstract
Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is essential in both innate
and acquired immune systems as it stimulates the survival, proliferation, differentiation, and
functional activation of myeloid hematopoietic cells. hGMCSF is integral to leukemia therapies as
it is approved for use in myeloid reconstitution after autologous and allogeneic bone marrow
transplantation, and bone marrow transplantation failure or engraftment delay. Recombinant
hGMCSF can be purified from Escherichia. coli; however, it has been challenging to purify
hGMCSF from E. coli as it forms inclusion bodies that require solubilization and refolding. In this
chapter, we describe the fusion of an elastin-like polypeptide (ELP) to hGMCSF, which previously
reported as purification tags and solubility enhancers. ELPs are recombinant polypeptides that
undergo reversible temperature-dependent phase separation. Here, we show that the fusion of
hGMCSF to an ELP enables direct purification of hGMCSF fusions from the soluble fraction of
bacterial lysate. In addition to that, these ELP-fusions assemble stable and biologically active
nanoparticles that exhibit pro-mitotic activity of hGMCSF. Here, also we report that nanoparticle
assembly significantly increases the entropic cost of phase separation compared to ELP alone.
3.2 Introduction
Granulocyte-macrophage colony-stimulating factor (GMCSF) is a cytokine that stimulates
proliferation and differentiation of hematopoietic cells into neutrophil, eosinophil, and monocyte
colonies (Armitage, 1998; Schwanke et al., 2009). In addition, GMCSF is essential in the innate
immune system by enhancing the production and survival of neutrophils, eosinophils, and
macrophages (Schwanke et al., 2009). GMCSF is secreted in multiple cell types, such as mast

3
This chapter was referenced to a manuscript, titled “Human Granulocyte-Macrophage Colony-Stimulating Factor Fused to
Elastin-Like Polypeptides Assembles Biologically-Active Nanoparticles” by Park et al.

66

cells, macrophages, fibroblast, T cells, and endothelial cells, in response to immune and
inflammatory stimuli (Das et al., 2011), and it regulates the immune responses by surveilling and
activating immune cells (Vanitha et al., 2017). GMCSF is essential in peripheral blood, serum,
and bone marrow. GMCSF increases circulating neutrophil half-life, the number of eosinophils
and monocytes, and decreases platelet concentration in peripheral blood. In the bone marrow,
GMCSF increases the number of eosinophil precursors and cycling progenitor cells, stimulates
neutrophil production rate, and decreases progenitor-cell cycle time. In serum, it increases
aminotransferase and cobalamin and decreases albumin (Lieschke and Burgess, 1992). GMCSF
has been employed to approved therapies, such as monoclonal antibodies against checkpoint
inhibitors, prevention of chemotherapy-induced neutropenia, and chimeric antigen receptors
(Chen et al., 2009; Le et al., 2013; Mayordomo et al., 1995; Pilones et al., 2015; Spear et al.,
2012). In addition, GMCSF has been essential for leukemia treatment as it is approved by the
USFDA for use in rapid myeloid reconstitution after autologous and allogeneic bone marrow
transplantation, bone marrow transplantation failure, or engraftment delay.
Human GMCSF (hGMCSF) is a glycosylated protein that consists of 127 amino acids
including two disulfide bonds formed by four cysteines with a two-stranded antiparallel β-sheet
and a four α-helix bundle (Das et al., 2011; Thomson et al., 2012; Walter et al., 1992). As
endogenous hGMCSF undergoes glycosylation, the USFDA-approved hGMCSF, leukine, has
been produced from Saccharomyces cerevisiae for the glycosylation. Proteins expressed and
purified from Escherichia. coli do not undergo post-translational modification, but many
biologically active proteins purified from bacteria have shown biological activities. Bacterial
fermentation is often faster and more economical and shows less batch-to-batch variation than
eukaryotic expression. It has been reported that hGMCSF purified from E. coli has high biological
activity in vitro despite of the absence of glycosylation (Chaubey and Ghosh, 2013). However,
purification of hGMCSF from E. coli is challenging as hGMCSF forms inclusion bodies, which are

67

protein aggregates of misfolded proteins (Belew et al., 1994). Inclusion bodies can be solubilized
using concentrated urea or guanidinium, and then the resolubilized protein can be refolded with
consecutive dialysis steps to remove denaturants (Arakawa et al., 2007). However, the proteins
tend to form aggregates during refolding, resulting in low yield (Tischer et al., 2010). To enhance
the protein refolding, reducing agents, such as β-mercaptoethanol and dithiothreitol, are added to
dialysis buffer (Vallejo and Rinas, 2004). As described, purification of inclusion bodies involves
multiple steps, and the whole process can be more expensive and difficult to scale. Therefore,
new strategies to generate functional hGMCSF in the soluble fraction of bacterial lysate could be
more economical and easier to scale hGMCSF and other difficult-to-express proteins.
Elastin-like polypeptides (ELPs) are genetically engineered protein-polymers that consist
of a hydrophobic pentameric amino acid repeats, (VPGXG) n, where X is a guest residue that can
be any amino acid (Pastuszka et al., 2012). ELPs undergo reversible temperature-dependent
phase separation, similar to a lower critical solution temperature polymer. ELPs remain soluble in
water below the transition temperature, but above the transition temperature, ELPs form a
secondary aqueous phase (Despanie et al., 2016). In other groups and our own, it has been
shown that fusion of ELPs to insoluble proteins enhances the solubility of the proteins (Aluri et al.,
2014; Meyer and Chilkoti, 1999; Trabbic-Carlson et al., 2004). In addition, ELPs can facilitate
purification by serving as tags using cycles of hot and cold centrifugation, which utilizes
temperature-dependent phase separation to concentrate and purify ELP fusion proteins
(Hassouneh et al., 2010; Hassouneh et al., 2012; Lim et al., 2007). In this Chapter, we show that
a fusion of a high molecular weight ELP, A192, to hGMCSF solubilizes hGMCSF, facilitates its
purification from E. coli, and generates a high yield of hGMCSF. In addition, the fusion protein
assembles into stable nanoparticles that maintain significant biological activity towards a human
cell line, TF-1, responsive to hGMCSF in vitro and in vivo.

68

3.3 Materials and methods
3.3.1 hGMCSF-A192 cloning & purification
The hGMCSF gene was fused to the amino terminus of a high molecular weight ELP, A192, in
the pET-25b(+) vector, encoding hGMCSF-A192. The amino acid sequence of hGMCSF fused to
A192 is:
APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYK
QGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE.
The DNA sequence of the hGMCSF gene is: 5’-
GCTCCGGCTCGTTCTCCGTCTCCGTCTACCCAGCCGTGGGAACACGTTAACGCTATCCAG
GAAGCTCGTCGTCTGCTGAACCTGTCTCGTGACACCGCTGCTGAAATGAACGAAACCGTT
GAAGTTATCTCTGAAATGTTCGACCTGCAGGAACCGACCTGCCTGCAGACCCGTCTGGAA
CTGTACAAACAGGGTCTGCGTGGTTCTCTGACCAAACTGAAAGGTCCGCTGACCATGATG
GCTTCTCACTACAAACAGCACTGCCCGCCGACCCCGGAAACCTCTTGCGCTACCCAGATC
ATCACCTTCGAATCTTTCAAAGAAAACCTGAAAGACTTCCTGCTGGTTATCCCGTTCGACTG
CTGGGAACCGGTTCAGGAA-3’. A diagnostic digest was run with NdeI and BamHI restriction
enzymes (New England Biolabs, MA). After the sequence was confirmed, shuffle T7 competent
E. coli (C3026J, New England Biolabs, MA) was transformed with the hGMCSF-A192 plasmid
using heat shock at 42 °C for 45 seconds. The transformed bacteria were spread on carbenicillin
agar plate (100 µg/mL) and incubated for 16-18 hours at 30 °C. After incubation, a colony was
picked and cultured in 50 mL of Terrific Broth with Glycerol (TB) (C8153, CulGeneX, CA) with 100
µg/mL of carbenicillin at 30 °C for 16-18 hours. The 50 mL of the cultured bacteria were pelleted
at 4,000 rpm for 15 minutes at 30 °C, and the supernatant was discarded. The pellet was
resuspended with fresh 5 mL of TB with 100 µg/mL of carbenicillin, and 1 mL of this was added
to each autoclaved 1 L volume of TB containing 100 µg/mL carbenicillin. 4 L of bacteria were
cultured at 30 °C until the optical density at 600 nm (OD 600) reached between 0.6 and 0.8. After

69

the OD 600 reached the desired number, 1 mL of 400 mM Isopropyl β-D-1-thiogalactopyranoside
(IPTG) was added to each flask to produce a final concentration of 0.4 mM. IPTG induction was
allowed to proceed overnight at room temperature. The next day, bacteria were pelleted at 4,000
rpm for 10 minutes, and the supernatant was discarded. Each pellet was resuspended in 30 mL
of fresh cold PBS, vortexed, and disrupted using a probe-tip Misonix sonicator S-4000 (Misonix,
NY). After disruption, 0.5% of Polyethyleneimine (PEI) was added to each cell lysate, and the cell
lysate was incubated on ice for 5 minutes. The cell lysate was centrifuged at 13,000 rpm for 15
minutes at 4 °C. The clarified supernatant was collected, heated to 37 °C with solid NaCl salt of
the 2M final concentration. This is sufficient to promote rapid and visible phase separation of
hGMCSF-A192. After coacervation, the heated supernatant was pelleted at 4,000 rpm for 15
minutes at 37 °C. The supernatant was discarded, and the pellet was resolubilized with cold PBS
on ice. Re-solubilized hGMCSF-A192 was centrifuged at 13,000 rpm for 15 minutes at 4 °C, and
the supernatant was collected. This cycle of hot and cold centrifugation was repeated three times
as required to obtain pure hGMCSF-A192.
3.3.2 hGMCSF-A192 protein concentration
To measure hGMCSF-A192 protein concentration, the protein was denatured with 6 M guanidine
hydrochloride to disrupt the assembly of nanoparticles, and the concentration was measured
using the Eq. 1. The molar extinction coefficient, ε, was obtained using the Eq. 2. The ε of
hGMCSF-A192 was estimated to be 15,720 M
-1
cm
-1
as ε of hGMCSF and A192 were estimated
to be 14,230 M
-1
cm
-1
assuming both pairs of cysteine residues form cystine disulfide bonds and
1,490 M
-1
cm
-1
, respectively. The A 280 and A 350 for the samples were measured with a NanoDrop
2000 (Thermo Fisher, MA, USA), which has a path length of 0.1 cm.
3.3.3 hGMCSF-A192 purity and transition temperature analysis
The purity of hGMCSF-A192 was determined by SDS-PAGE image analysis using ImageJ (NIH,
MD). To run SDS-PAGE, 10 µg of hGMCSF-A192 was mixed with loading dye, and the sample

70

was boiled at 95 °C for 5 minutes. After heating, the sample was loaded to a 4-20% precast SDS-
PAGE gel (58505, Lonza, Switzerland). The gel was stained with 0.1 g/mL of CuCl2, and the gel
image was captured with a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, CA). To
estimate the purity of the fusion protein, an area of interest was selected for a plot profile and the
area of under the peak of each band on the gel was obtained. The purity was calculated using
the Eq. 3. The transition temperature (Tt) of hGMCSF-A192 was measured using a Beckman
Coulter DU 800 UV/Vis spectrometer (Beckman Coulter, CA). To measure ELP phase behavior,
five concentrations of hGMCSF-A192, 50, 25, 12.5, 6.25, and 3.125 µM, were prepared, filtered
with a 0,22 µm filter, and equilibrated on ice in microcuvettes. Next, the microcuvettes were
heated at a rate of 1 °C/min up to 85 °C. The optical density at 350 nm (OD 350) was measured
every 18 seconds. In units of ºC, Tt was defined where the maximum first derivative of the OD 350
with respect to temperature occurred. This data was fit to the Eq. 4.
3.3.4 Thermodynamics of hGMCSF-A192 phase separation
To estimate ΔHcoacervation and ΔScoacervation of hGMCSF-A192 phase separation, van’t Hoff equation
was employed using the following equation:
𝑙𝑛 [𝐶𝐴𝐶 𝐸𝐿𝑃 ] =
∆𝐻 𝑐𝑜𝑎𝑐𝑒𝑟𝑣𝑎𝑡𝑖𝑜𝑛 𝑅 1
𝑇 𝑡 −
∆𝑆 𝑐𝑜𝑎𝑐𝑒𝑟𝑣𝑎𝑡𝑖𝑜𝑛 𝑅 Eq. 8
Where CACELP is the critical aggregation concentration (μM) at each observed transition
temperature, Tt in units of K. R is the universal gas constant, 0.008314 kjouls/mol· K. By plotting
the natural logarithm of the CACELP vs. the inverse of the transition temperature, a slope and y-
intercept of the graph were calculated and then converted to the enthalpy and entropy of phase
separation, respectively. ELP phase separation from solution into coacervate is an endothermic
process as it requires heat;(Amruthwar et al., 2013; Reguera et al., 2007) therefore, the
ΔHcoacervation is defined as positive and assumed constant over this narrow temperature range. For
spontaneous phase separation into the coacervate phase above Tt, the Gibbs free energy of

71

phase separation should become negative; therefore, the TΔS coacervation must become larger than
ΔHcoacervation at the temperature where phase separation occurs.
3.3.5 Characterization Studies of hGMCSF-A192
To quantify the number of free sulfhydryl groups in purified hGMCSF-A192, Ellman’s assay
(Thermo Fisher, MA, USA) was employed. To plot a standard curve, 1.5, 1.25, 1.0, 0.75, 0.5, 0.25,
0 mM of N-acetyl L-cysteine samples were prepared. After 15 minutes of reaction with Ellman’s
reagent at room temperature, absorbance at 412 nm was measured. With the standard curve, the
number of free sulfhydryl groups in hGMCSF-A192 was quantified. To visualize the shape of
hGMCSF-A192 nanoparticles, negative-staining transmission electron microscopy (TEM) was
employed. To image the nanoparticles, 10 µL of 1 µM hGMCSF-A192 was pipetted onto a Ted
Pella carbon/formvar grid (Ted Pella Inc., CA), and the grid was placed in an incubator for 10
minutes 37 °C. Afterwards, the protein sample was dried with a filter paper, and 10 µL of 2%
uranyl acetate was pipetted to stain the protein sample for 10 minutes in an incubator 37 °C. The
uranyl acetate was removed with a filter paper after incubation and was dried before imaging with
a JEOL JEM-2100 LaB6 microscope (JEOL Ltd., Japan). All images were captured under 200 kV
accelerating voltage, and the images were analyzed with ImageJ (NIH, MD) to measure the radius
of hGMCSF-A192 nanoparticles. To measure the hydrodynamic radius and stability of hGMCSF-
A192 nanoparticles, dynamic light scattering (DLS) was employed. To run the DLS, 25 µM of
hGMCSF-A192 samples were prepared and filtered with a 0.22 µm filter. After the filtration, 60 µL
of hGMCSF-A192 was pipetted to 6 wells in a 384-well plate. Then, 15 µL of mineral oil was added
to prevent evaporation of the solvent. The plate was placed into a DynaPro Plate Reader II (Wyatt
Technology, CA), and the hydrodynamic radius was measured at 37 °C. As a control, the
hydrodynamic radius of A192 was also measured. To measure the stability of hGMCSF-A192,
the hydrodynamic radius of hGMCSF-A192 nanoparticles was measured every 24 hours at 37 °C
for 72 hours. To measure the zeta potential of the nanoparticles, nanoparticle tracking analysis

72

was employed with NTA ZetaView® (Particle Metrix GmbH, Meerbusch, Germany), and the
hydrodynamic radius was also measured with it at 4 °C and 37 °C. To study the secondary
structure of hGMCSF-A192, circular dichroism was run with 6.25 µM of the fusion protein was
prepared and then run with Jasco J-815 spectrometer (Easton, MD, USA). The ellipticity was
monitored from 185 to 260 nm, and the CD data was analyzed with BeStSel, a web server for
accurate protein secondary structure prediction. For the comparison, the secondary structure of
hGMCSF was referenced from BeStSel database. To estimate the average molecular weight of
hGMSF-A192 nanoparticles, size exclusion chromatography-multiangle light scattering (SEC-
MALS) was employed. First, 10 µM of hGMCSF-A192 was prepared in PBS and filtered through
the 0.22 µm pore membrane to remove artifacts. A syringe for loading the sample was washed
10 times with water and 10 times with PBS. A Shodex protein KW-804 (8.0mmI.D. x 300mm)
(Showa Denko America, NY) was equilibrated with PBS for more than 30 minutes before running,
and once the column was equilibrated, a loop for loading samples was washed with water and
PBS 10 times each. The prepared hGMCSF-A192 sample was loaded to the SEC column through
the washed loop, and the elution was observed with three detectors, UV 210 nm detector (SYS-
LC-1200) (Agilent Technologies, CA), multi-angle static light scattering detector (DAWN
HELEOS) (Wyatt Technology, CA), and differential refractometer (Optilab rEX) (Wyatt
Technology, CA). The Data were analyzed by ASTRA 6 software.
3.3.6 WST-1 cell proliferation assay
To evaluate the biological activity of hGMCSF-A192, TF-1 cells (American Type Culture Collection,
VA) were purchased and cultured with RPMI 1640 (10-104-CV, Corning, NY) with 10% FBS (25-
550, Corning, NY), 1X Penicillin-Streptomycin (30-002-CI, Corning, NY), and the final
concentration of 5 ng/mL recombinant hGMCSF (G5035-5UG, Sigma-Aldrich, MO). The cultured
cells were washed twice with RPMI 1640 without 10% FBS to remove residual recombinant
hGMCSF. After the wash, cells were pelleted and then resuspended with fresh RPMI 1640.

73

hGMCSF-A192, A192, and recombinant hGMCSF were prepared in 1.7 mL microcentrifuge tubes
using 1 to 5 serial dilutions. The prepared concentrations of hGMCSF-A192 were: 47, 9.3, 1.9,
0.37, 0.075, 0.015, 0.0030, 0.00060, 0.00012, and 2.4×10
-5
nM. The prepared concentrations of
rhGMCSF were: 34, 6.8, 1.4, 0.27, 0.054, 0.011, 0.0022, 0.00044, 8.7×10
-5
, and 1.7×10
-5
nM. The
prepared concentrations of A192 were: 41, 8.3, 1.7, 0.33, 0.066, 0.013, 0.0027, 0.00053, 0.00011,
2.1×10
-5
nM. After the sample preparation, 90 µL of the resuspended TF-1 cells (<10,000 cells)
were pipetted to wells of a 96-well plate. Then, 10 µL of prepared protein samples were pipetted
to the 90 µL of cells. The 96-well plate was incubated at 37 °C with 5% CO 2 for 72 hours, and 10
µL of Cell Proliferation Reagent WST-1 (5015944001, Sigma-Aldrich, MO) was pipetted to each
well. The plate was incubated for another 2 hours, and the plate was placed into Biotek Synergy
H1 Hybrid Multi-Mode Reader (Biotek, VT). Absorbance at 450 nm and 690 nm was measured
with the plate reader, and the absorbance at 690 nm was subtracted from the absorbance at 450
nm to analyze the biological activity of proteins at each concentration. To fit the data with a curve,
the following equation was employed:
OD
450
− OD
690
= Baseline + C
𝑝𝑟𝑜𝑡𝑒𝑖𝑛 ×
Plateau−Baseline
𝐸𝐶
50
+C
𝑝𝑟𝑜𝑡𝑒𝑖𝑛 Eq. 9
3.3.7 TF-1 mouse engraftment study In vivo
Animal protocols were approved by the Institution for Animal Care and Use Committee of the
University of Southern California. For mouse xenograft experiment, 4- to 6-week-old male NOD-
scid /Il2rg
-/-
(NSG) mice were purchased from Jackson Laboratory (Bar Harbor, ME). 10 million

TF-1 cells were administered via tail vein injection into mice for the engraftment. Engrafted mice
were treated with 200 µL of 100 nM A192 (n=3) or hGMCSF-A192 (n=3) via tail vein on Day 1, 3,
and 5 post engraftments. Mice were euthanized on Day 16 and the blood and bone marrow were
collected and stained for huCD45. The staining was analyzed using flow cytometry.

74

3.3.8 Statistical Analysis
Student’s t-test was used to verify whether the difference between hGMCSF EC 50 and hGMCSF-
A192 EC 50 is statistically significant. Also, to compare huCD45 engraftment in mice treated with
A192 and hGMCSF-A192, Student’s t-test was used to determine whether the difference is
statistically significant.
3.4 Results
3.4.1 hGMCSF-A192 is highly expressed in E. coli, and the fusion of A192 to hGMCSF
serves as a solubilizing enhancer and a purification tag
To construct a hGMCSF-A192 fusion protein, a gene encoding hGMCSF was synthesized and
purchased, and the gene was fused with a gene encoding A192 into a pET-25b(+) vector to
express hGMCSF-A192 in E .coli (Fig. 15A). To confirm the ligation, a diagnostic digestion was
(Fig. 15B) run, and the result showed that the cloning of hGMCSF-A192 was consistent with the
results of DNA sequencing. To block the formation of inclusion bodies, A192 was fused to provide
steric stabilization of the fusion protein, resulting in the formation of nanoparticles (Fig. 15E). As
intended, hGMCSF-A192 was successfully purified with high purity using temperature-dependent
phase separation (Fig. 15C). The purity of hGMCSF-A192 was estimated by two SDS-PAGE gel
images (Fig. 15D). The estimated purity was 94.6 ± 7.6% (mean ± SD, n=2).

75

Figure 16. ELP fusion enables temperature-dependent purification of hGMCSF fusions from the
soluble fraction of bacterial lysate. A) A pET25b+ vector was modified to express a seamLess fusion
of hGMCSF and the ELP known as A192. A high molecular weight polymer, A192 has an amino acid
sequence of (VPGAG)192 and a phase transition temperature above physiological temperature (Table
6). B) A double restriction digestion (NdeI/BamHI) was used to characterize plasmids encoding
hGMCSF-A192 and A192 alone. C) hGMCSF-A192 was purified by the technique described above: (1)
ELP-mediated phase separation is promoted by adding solid NaCl. (2) The mixture is centrifugated at
37 °C, and the supernatant is discarded. (3) The ELP coacervate is resuspended in cold PBS. (4) The
resuspended ELP coacervate is centrifugated at 4 °C to remove any impurity. (5) The supernatant is
collected. D) After 3 cycles of ELP-mediated purification, SDS-PAGE was performed and developed
with copper chloride. A192 molecular weight is expected to be 73.7 kDa, and hGMCSF-A192 molecular
weight is expected to be 88.1 kDa, which appears as a major band between 75 and 100 kD markers.
The protein purity of hGMCSF-A192 was estimated using image analysis to be ~100%. E) A schematic
of the hGMCSF-A192 where hGMCSF is fused directly to the N-terminus of A192. The high molecular
weight A192 sterically stabilizes nanoparticles with a core of hGMCSF, which remain stable colloids at
physiological conditions.

76

The yield of the fusion protein purified from the soluble fraction was 109 ± 13 mg/L (n=3).
hGMCSF is known to form inclusion bodies when produced from E. coli (Belew et al., 1994;
Chaubey and Ghosh, 2013; Thomson et al., 2012), however, when tagged with A192, hGMCSF-
A192 was expressible in the soluble fraction post cell lysis, resulting in the high yield. To analyze
the transition temperature of hGMCSF-A192, the phase diagram was further evaluated using
optical density (OD) measurements as a function of ELP concentration and temperature. Cloud
point measurements (OD at 350 nm) showed that the fusion of hGMCSF to A192 shifted the
transition temperature of A192 downward (Fig. 16A and 16B). At 25 µM in PBS, the transition
temperature of hGMCSF-A192 was measured to be 44.5 °C while A192 undergoes the phase
separation at 59.9 °C. The ELP concentration dependence of phase diagram was quantified from
the slope of Eq. 4 (Table 6). The quantified slope indicated that the concentration dependence of
the phase diagram for hGMCSF-A192 became lower compared to A192. This significant alteration
in the phase behavior for A192 in the fusion protein suggested that the hGMCSF domain affects
the peptide conformation, resulting in the change of phase behavior for the ELP.

77

Figure 17. Fusion to hGMCSF shifts the phase diagram and thermodynamics of A192 phase
separation. The phase behavior of A192 and hGMCSF-A192 was characterized by monitoring UV/VIS
spectrometry as a function of temperature and concentration. A) Compared to A192, hGMCSF-A192
started at a higher baseline optical density, which may be consistent with scattering of light from
nanoparticles. Both constructs show an abrupt onset in O.D.350 consistent with ELP-mediated phase
separation; however, hGMCSF-A192 shifted this phase separation to a lower temperature. B) Most
often, ELP-phase diagrams have been reported as a log-linear relationship between the transition
temperature and concentration (Eq. 4), whereby an increase in concentration decreases the transition
temperature. This analysis confirms that over a range of concentrations, fusion to hGMCSF reduces
the apparent magnitude and slope of the transition temperature (Table 6). C) To characterize their
thermodynamic relationship, both phase diagrams were replotted for van’t Hoff analysis, which predicts
a linear relationship between the critical aggregation concentration (CAC, µM) and temperature (K)
according to Eq. 8. The slope of a linear fit is ΔHcoacervation/R, and the intercept of a linear fit is -
ΔScoacervation/R. The positive slopes of each curve are consistent with an endothermic phase separation
defined by moving ELP from solution into the coacervate phase. Relative to A192, the steeper slope of
hGMCSF-A192 suggests an increase in the enthalpy of coacervation induced by fusion to hGMCSF.
The best-fit line is indicated (solid) with a 95% confidence interval (dashed lines).

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3.4.2 Enthalpy and entropy of hGMCSF-A192 coacervation are higher for unmodified
A192
As the fusion of hGMCSF to A192 substantially changes the phase diagram relative to free A192
(Fig. 16A and B, Table 6), their phase diagrams were reanalyzed using a modification of the
van’t Hoff equation (Eq. 8) to estimate the enthalpy and entropy of the phase separation. The
natural log of concentration was plotted vs. the inverse temperature (Fig. 16C), and the phase
diagrams for each peptide appear well-fit by a straight line. The slope of the van’t Hoff line
provides an estimate of the enthalpy of phase separation ΔH coacervation/R. Based on its steeper
slope, the ΔHcoacervation for hGMCSF-A192 remains endothermic, requiring 947 kJ/mol [95% CI:
589 to 1,305 kJ/mol], which is 3.6 fold greater than for A192, requiring only 265 kJ/mol [95% CI:
225 to 306 kJ/mol] to coacervate. Thus, the attachment of hGMCSF increases the enthalpy of
coacervation substantially compared to unmodified A192. Since coacervation of both peptides
remains endothermic, to yield a negative Gibbs free energy ΔGcoacervation upon heating requires an
increase in the entropic component of TΔScoacervation. The y-intercept for Eq. 8 provides an estimate
of the entropy for -ΔScoacervation /R. Based on its more negative intercept, the ΔS coacervation for
hGMCSF-A192 is estimated at 2.96 kJ/mol K [95% CI: 1.83 to 4.09 kJ/mol K], which is 3.8 fold
greater than for A192 that 0.77 kJ/mol K [95% CI: 0.65 to 0.89 kJ/mol K] (Table 6). In total, the
attachment of hGMCSF to A192 favors both higher enthalpic and entropic costs for phase
separation compared to A192.

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Table 6. Sequence and biophysical characterization of A192 and hGMCSF-A192.
Label
Amino acid
a

sequence
MW
(kDa)
b

Rh
(nm)
c

Phase diagram
d
Thermodynamics
e

m
(°C/log10[µM])
b
(°C)
ΔHcoacervation
(kJ/mol)
ΔScoacervation
(kJ/mol K )
A192 G(VPGAG)192Y

73.6 7.5 ±
0.3
-8.04
[-9.20 to -6.88]
70.8
[69.5 to 72.2]
265
[225 to 306]
0.77
[0.65 to 0.89]
hGMCSF-
A192
hGMCSF-
G(VPGAG)192Y
88.1 48.5
± 0.7
-1.96
[-2.70 to -1.22]
47.0
[46.1 to 47.8]
947
[589 to
1,305]
2.96
[1.83 to 4.09]
a
hGMCSF:MAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGS
LTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE
b
MW based on open reading frame, as confirmed by SDS-PAGE.
c
Rh was measured using dynamic light scattering at 25 µM in PBS. Mean ± SD (n=3).
d
Slope and intercept fit to Eq. 4. mean [95% confidence interval].
e
Slope and intercept fit to Eq. 8. mean [95% confidence interval].

3.4.3 hGMCSF-A192 forms disulfide bonds and stable nanoparticles
Disulfide bonds in hGMCSF play crucial roles for biological activity, so free sulfhydryl groups in
hGMCSF-A192 were quantified using Ellman’s reagent. Based on the assay, the percent of free
sulfhydryl groups was estimated to be 8.6 ± 1.2% (mean ± SD, n=3). As it is observed that
hGMCSF substantially changed the phase behavior of A192 coacervation, we hypothesized that
hGMCSF may facilitate the assembly of nanoparticles similar to those observed previously for
other ELP fusions.(Aluri et al., 2014; Pastuszka et al., 2014b; Wang et al., 2015; Wang et al.,
2014). To image the structure of hGMCSF-A192, negative-staining transmission electron
microscope (TEM) was employed. TEM images revealed that the fusion protein formed spherical
nanoparticles in various sizes (Fig. 17A). Image analysis of the TEM image by ImageJ indicated
that the average radius of the dried nanoparticles was measured to be 13.4 ± 3.01 nm (mean ±
SD, n=70) (Fig. 17B). DLS data also suggested that the hGMCSF-A192 formed nanoparticles
whose average hydrodynamic radius was measured to be 48.5 ± 0.7 nm (mean ± SD, n=3) (Fig.
17C). The polydispersity of this population was measured to be 11.4 ± 0.2 nm (mean ± SD, n=3)
by the DLS. To analyze the secondary structure of A192 and hGMCSF-A192, circular dichroism
(CD) and BeStSel were employed (Micsonai et al., 2018; Micsonai et al., 2015). The CD analysis
indicated that the CD of hGMCSF-A192 was dominated by A192, so net CD ellipticity of hGMCSF-
A192 was calculated and analyzed assuming that A192 on the fusion protein has the same

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structure as plain A192 (Fig. 17D). The CD of hGMCSF-A192 analyzed with BeStSel indicated
only 0.5% of α-helix structure was observed. On the other hand, when the net difference of CD
between hGMCSF-A192 and A192 was analyzed with BeStSel, 22.7% of α-helix was observed,
which is closer to the secondary structure of hGMCSF (Fig. 17E).

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Figure 18. Fusion of A192 to hGMCSF results in nanoparticles. A) To determine if nanoparticles
were formed by hGMCSF-A192, negative-stain TEM imaging was performed. This showed a clear
distribution of spherical hGMCSF-A192 particles of various sizes. B) The distribution of radii of
hGMCSF-A192 nanoparticles identified by TEM imaging was measured using image analysis. The
average radius of dried hGMCSF-A192 was 13.4 ± 3.0 nm. C) To measure the hydrodynamic radius of
solvated hGMCSF-A192, dynamic light scattering (DLS) was employed. The fusion of hGMCSF to A192
substantially increased the hydrodynamic radius and polydispersity relative to A192. hGMCSF-A192
has an average hydrodynamic radius of 48.5 ± 0.7 nm, while the hydrodynamic radius of free A192 was
7.5 ± 0.3 nm. D) Circular Dichroism was run with 6.25 µM of A192 and hGMCSF-A192, and the
experimental data were fitted using BeStSel, a web server for accurate protein secondary structure
prediction. Blue, red, and purple solid lines represent fitted curves for A192, hGMCSF-A192, net
hGMCSF-A192 CD molar ellipticity, respectively. E) The CD data were analyzed with BeStSel to predict
the secondary structure of A192 and hGMCSF-A192. The secondary structure information of hGMCSF
was obtained from the BeStSel database. Based on the analysis, hGMCSF-A192 significantly lacks α-
helix conformation compared to hGMCSF. On the other hand, it exhibited a much higher proportion of
β-sheet conformation compared to hGMCSF. However, when the net hGMCSF-A192 molar ellipticity
was analyzed a much higher similarity to hGMCSF was observed.

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DLS was employed to measure the stability of nanoparticles for three days at 37 °C (Fig.
18A). The data revealed that the hydrodynamic radius of hGMCSF-A192 was constant for three
days, suggesting that hGMCSF-A192 forms stable nanoparticles at the physiological temperature.
In addition, the distribution of the hydrodynamic radius of hGMCSF-A192 did not change much
over three days (Fig. 18B). Nanoparticles Tracking Analysis (NTA) was also employed to
measure the hydrodynamic radius and zeta potential. After 4 days of incubation in PBS at 4 °C
and 37 °C, the hydrodynamic radius was measured to be 84.1 ± 1.7 nm (mean ± SD, n=3) and
78.1 ± 0.3 nm (mean ± SD, n=3), respectively. The zeta potential of the nanoparticles in 1/100
dilution of PBS at 4 °C and 37 °C was measured to be -28.5 ± 0.6 mV (mean ± SD, n=3) and -
28.0 ± 4.2 mV (mean ± SD, n=3), respectively. Overall, NTA analysis suggests that the
nanoparticles are stable colloids and they have negative surface charges.

To measure the average molecular weight of hGMCSF-A192 nanoparticles, size exclusion
chromatography with multi-angle light scattering (SEC-MALS) was used. As a control, A192 was
run through the SEC column, and it eluted at 8.5 to 9.0 min. A192 was only observed at the

Figure 19. hGMCSF-A192 nanoparticles are stable colloids at physiological temperature. To
measure the stability of hGMCSF-A192, 25 µM of hGMCSF-A192 was incubated at 37°C for three days
in PBS. Every 24 hours, the hydrodynamic radius of hGMCSF-A192 was measured with DLS. A) The
average size of hGMCSF-A192 did not change substantially over 72 hours. B) Similarly, the size
distribution of hGMCSF-A192 did not change much over 72 hours.

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molecular weight of a monomer in PBS (Fig. 19A). In contrast, hGMCSF-A192 eluted more rapidly
in the void volume between 5.5 to 6.0 min, and its average molecular weight was estimated to be
2.72 ± 0.06 ×10
7
Da (Fig. 19B). Based on this number, the average number of hGMCSF-A192
fusions forming a nanoparticle in solution was calculated to be 306 molecules. The radius of
gyration (Rg) of hGMCSF-A192 was measured to be 55.9 ± 0.03 nm from SEC-MALS.

3.4.4 hGMCSF-A192 nanoparticles are biologically active in vitro.
To verify whether the purified hGMCSF-A192 is biologically active, TF-1 cells were treated with
either recombinant hGMCSF, A192, or hGMCSF-A192. As shown in Fig. 20A, hGMCSF-A192
stimulated cell proliferation as recombinant hGMCSF did. On the other hand, A192 was not able
to stimulate cell proliferation. The result suggests that hGMCSF-A192 nanoparticles are
biologically active. EC 50 of hGMCSF-A192 and recombinant hGMCSF were measured to be 0.29
± 0.07 nM and 0.015 ± 0.008 nM, respectively (Fig. 20B).

Figure 20. hGMCSF-A192 nanoparticles consist of several hundred fusion proteins. To measure
the absolute molecular weight of hGMCSF-A192 nanoparticles, size exclusion chromatography-
multilight angle scattering (SEC-MALS) was employed. A) The absolute molar mass of A192 was
calculated to be 8.07×10
4
Da, which agrees closely with the expected molecular weight of monomeric
A192. B) In contrast, the absolute molar mass of hGMCSF-A192 nanoparticles was 2.72×10
7
Da, which
was much higher and eluted much earlier by SEC compared to A192. This mass is equivalent to about
306 hGMCSF-A192 molecules per nanoparticle.

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3.4.5 hGMCSF-A192 enhances engraftment of TF-1 cells in xenograft murine model
To verify whether hGMCSF-A192 has similar effects on TF-1 cells as seen in vitro, NSG mice
were injected with 10×10
6
TF-1 cells. Mice were then randomized into two treatment groups. Mice
were treated with 71.1 µg/kg/mouse with either A192 or hGMCSF-A192 on Day 1, 3, and 5 post
leukemia engraftments. Mice were euthanized on day 16 and organs were evaluated for TF-1
engraftment. Notably, flow cytometry analysis of hCD45 for TF-1 engraftment indicated that mice
treated with hGMCSF-A192 had significantly higher engraftment compared with the A192 mice in
the peripheral blood (%huCD45: 27.4 vs. 0.26, p=0.0042; Fig. 21A, 21B) and the bone marrow
(%huCD45: 5.09 vs. 2.55, p=0.001; Fig. 21C, 21D).

Figure 21. hGMCSF-A192 nanoparticles are biologically active. It was unknown if hGMCSF-A192
nanoparticles retained the ability to stimulate human GMCSF receptors; therefore, a human TF-1 cell
line was used to evaluate GMCSF-induction of proliferation. A) The WST-1 cell proliferation assay
was conducted with hGMCSF-A192 nanoparticles, recombinant hGMCSF, and free A192. As a
negative control, A192 proved unable to stimulate TF-1 cell proliferation at any concentration. In
contrast, both hGMCSF-A192 nanoparticles and recombinant hGMCSF stimulated TF-1 cell
proliferation to a similar magnitude. B) By fitting to Eq. 7 The EC50 of hGMCSF-A192 and hGMCSF
were measured to be 0.29 ± 0.07 nM and 0.015 ± 0.008 nM respectively (n=3, p=0.007), indicating that
the potency of hGMCSF-A192 is about 20 times less than for recombinant hGMCSF. While consistent
with steric hindrance of A192 coated hGMCSF-A192, these nanoparticles still retain sub nanomolar
activity.

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3.5 Discussion
hGMCSF is an important therapeutic cytokine to treat neutropenia and aplastic anemia after
chemotherapy or bone marrow transplantation (Das et al., 2011). Although mammalian cells are
ideal to produce the biologically active hGMCSF with post-translational modifications, they
frequently show the low level of protein expression (DeLamarter et al., 1985). hGMCSF purified
from E. coli has shown the high yield and the biological activity despite the absence of post-
translational glycosylation, but it has been reported that hGMCSF produced from E. coli formed
protein aggregates, known as inclusion bodies (Belew et al., 1994; Chaubey and Ghosh, 2013).

Figure 22. An in vivo experiment verifies activity of hGMCSF-A192 in a mouse model. To
demonstrate proof-of-principle bioactivity, this nanoformulation was assessed in promoting the
engraftment of human TF-1 cells into NSG mice. 10×10
6
cells TF-1 cells were engrafted in each NSG
mice on day 1. Mice were injected with 200 µL of 100 nM of A192 (n=3) or hGMCSF-A192 (n=3) on
days 1, 3, 5 and euthanized on Day 16. TF-1 cell engraftment was measured specifically using anti-
human CD45 (huCD45) antibody by flow cytometry. A, B) hGMCSF-A192 dramatically enhanced
engraftment of human cells in the peripheral blood compared to A192 alone (%huCD45: 27.4 vs. 0.26,
p=0.0042). C, D) Similarly, hGMDSF-A192 significantly enhanced engraftment of TF-1 cells in the bone
marrow (%huCD45: 5.09 vs. 2.55, p=0.001).

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To solubilize the inclusion bodies, advanced purification methods, such as denaturing and
refolding proteins, are required, and the entire process can be expensive and laborious. Also, as
it involves multiple additional steps, the protein yield can decrease (Thomson et al., 2012).
To resolve these problems, our strategy was to fuse A192 to hGMCSF, and the fusion of
A192 to hGMCSF increased the solubility of the protein. After the purification of hGMCSF-A192,
the concentration of the purified protein was measured to be 469 µM, but the protein solution
remained soluble, indicating that A192 can serve as a solubilizing enhancer. The protein yield
was calculated to be 109 ± 13 mg/L, resolving the low expression level observed in mammalian
cells. Another benefit of fusion of A192 is that the fusion protein can be purified using reversible
temperature-dependent phase separation without the need for any additional affinity tag, such as
poly-histidine. To purify the ELP fusion protein, our lab employed multiple cycles of hot and cold
centrifugation, a purification method of ELPs introduced by Dr. Chilkoti and coworkers (Meyer and
Chilkoti, 1999). With the method, hGMCSF-A192 was selectively isolated (Fig. 15D). When two
SDS-PAGE gel images were assessed with ImageJ, the purity of hGMCSF-A192 measured to be
89.2% and 100%. This demonstrates how ELP’s temperature-dependent phase separation can
be used to eliminate the necessity of purification tags or affinity chromatography while recovering
a high yield of protein. Based on the Tt of hGMCSF-A192 and A192, phase separation of both
proteins is a non-spontaneous at the physiological temperature and endothermic reaction as the
ELPs require the heat to undergo phase separation (Fig. 16A and B).
To quantify the thermodynamics of ELP’s phase separation, we employed the van’t Hoff
equation to reanalyze the temperature-concentration phase diagram. Based on the van’t Hoff
plotting, it was discovered both ΔHcoacervation and ΔScoacervation of hGMCSF-A192 phase separation
were greater than for A192 alone (Table 6), indicating that nanoparticle formation increases both
enthalpic and entropic cost of ELP’s phase separation. The entropic cost of coacervation is the
sum of the entire system of water and ELP, which includes both a decrease in entropy due to
establishment of a boundary between the ELP coacervate and bulk water as well as an increase

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in entropy due to the release of structured water surrounding the hydrophobic moieties on A192.
Since the length of A192 is the same with and without hGMCSF, one might expect that the free
energy of coacervation remains similar between A192 and hGMCSF-A192. However, the van’t
Hoff analysis showed that below about 38.4 °C the ΔG coacervation for A192 phase separation is lower
than that of hGMCSF-A192 (Eq. 8). Between 38.4 and 46.9 °C, ΔG coacervation of hGMCSF-A192
remains positive, albeit lower than for A192 alone. During this temperature interval, both ELP and
the fusion remain in solution. Between 46.9 °C and 71.1°C, only the hGMCSF-A192 nanoparticles
have negative ΔGcoacervation, thus the ELP fusion protein undergoes phase separation. Above
71.1 °C, the ΔGcoacervation for A192 also becomes negative, and both ELPs undergo phase
separation. The fact that fusion of A192 to hGMCSF increases both the enthalpic and entropic
costs of phase separation compared to A192 alone, we attribute to the effect of nanoparticle
assembly. The van’t Hoff equation is a useful method to predict thermodynamic properties of any
phase change of molecules without employing differential scanning calorimetry (DSC). In a
research paper published in 2018, Zai-Rose et al. showed the van’t Hoff equation could predict
ΔH of an ELP phase change accurately with DSC analysis of the ELP’s phase separation using
the transition temperature of the ELP at various concentrations between 1 and 200 µM. In the
paper, ΔH of the ELP phase separation from the van’t Hoff equation and DSC was measured to
be 333 kJ/mol and 318 ± 8 kJ/mol (mean ± SD, n =2), respectively (Zai-Rose et al., 2018). Their
study suggested that the van’t Hoff equation is a valid way to predict thermodynamic properties
of phase change of proteins in different concentrations.
Multiple studies by our group have revealed that ELP fusion proteins form nanoparticles
(Aluri et al., 2014; Janib et al., 2014). This is conjectured that because the high molecular weight
of A192 sterically stabilize nanoparticles assembled around a core of oligomerized protein (Fig.
15E). This assumption is supported by SDS-PAGE run with non-reduced and hGMCSF-A192.
Non-reduced hGMCSF-A192 was unable to run through SDS-PAGE gel, suggesting some
disulfide bonds are formed intermolecularly and contribute nanoparticles formation. At

88

physiological temperature, A192 does not undergo phase separation and lack cysteine thiols
capable of covalent crosslinking. Since only hGMCSF domains contain cysteine thiols that form
intermolecular disulfide bonds, it seems the hGMCSF domains form the core of the nanoparticles.
Nanoparticle assembly was verified using a transmission electron microscope (TEM), where TEM
images showed diverse sizes of round-shape hGMCSF-A192 nanoparticles (Fig. 17A). This
observation was supported by DLS, which also revealed that hGMCSF-A192 formed stable
nanoparticles at 37 °C in PBS (Fig. 17C, 18A, and 18B). SEC-MALS was employed to estimate
the molecular weight of the nanoparticles, and the data showed that the molecular weight of a
nanoparticle is equivalent to be 306 hGMCSF-A192 molecules (Fig. 19A) while A192 remained
a monomer at 37 °C (Fig. 19B). The Rg/Rh ratio was employed to predict the shape of the
nanoparticles, and the ratio was determined to be 1.15, whereby a ratio of 0.775 is expected for
a compact sphere and ratios above 1 are associated with coil or rod-like structure. However,
based on the NTA analysis, the Rh of hGMCSF-A192 nanoparticles were measured differently,
and the Rg/Rh ratio was measured to be 0.72, which is consistent with spherical nanoparticles.
Although TEM imaging showed spherical shape nanoparticles, but it is possible that the
nanoparticle shapes were affected by the drying process of TEM imaging. To verify the shape of
nanoparticles, cryogenic electron microscopy should be employed.
The biological activity of nanoparticles was verified with a cell proliferation with TF-1 cells
that require hGMCSF to proliferate. The results showed that the hGMCSF-A192 nanoparticles
retained the biological activity of hGMCSF (Fig. 20A). The EC 50 of hGMCSF-A192 nanoparticles
and a recombinant hGMCSF was measured to be 0.29 nM and 0.015 nM, respectively (Fig. 20B).
While the potency of recombinant hGMCSF was 20 times greater than that of hGMCSF-A192,
the EC 50 of hGMCSF-A192 remains in the sub-nanomolar range. It was suspected that the
reduced potency might be relevant to the lack of disulfide bonds or the secondary structure of
hGMCSF-A192 as observed in the CD analysis and Ellman’s assay. Ellman’s assay results
indicated that 8.6 ± 1.2% (mean ± SD, n=3) of cysteine was in reduced form. The CD of hGMCSF-

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A192 showed significant differences of the secondary structure compared to hGMCSF (Fig. 17E).
However, it was observed that the CD of hGMCSF-A192 was dominated by A192. To remove the
effect, the net CD ellipticity was obtained by subtracting the CD ellipticity of A192 from that of
hGMCSF-A192 in order to predict the secondary structure of hGMCSF fused to A192. The net
CD ellipticity was analyzed, and the predicted secondary structure was similar to the hGMCSF.
Still, it significantly lacked the α-helix structure and contained a high proportion of the β-sheet
structure. It seems the difference in the secondary structure resulted in decreased potency
compared to a recombinant hGMCSF.
In vivo activity was investigated using TF-1 cell engraftment in a murine model. As
expected, injection of TF-1 cells with A192 resulted in poor TF-1 cell engraftment in both
peripheral blood and bone marrow. On the other hand, injection of TF-1 cells with hGMCSF-A192
resulted in significantly enhanced TF-1 engraftment in both peripheral blood and bone barrow
(Fig. 21A, B, C, and D). These studies confirmed that hGMCSF-A192 nanoparticles are
biologically active both in vitro and in vivo.
3.6 Conclusion
In this chapter, we report that the fusion of A192 to hGMCSF generates soluble ELP fusion protein,
and the fusion protein assembles biologically active nanoparticles in vitro and in vivo. Also, the
fusion of A192 to hGMCSF results in a change in entropy and enthalpy of A192 phase separation
and the formation of stable nanoparticles.
3.7 Acknowledgements
This work was supported by R01 GM114839 and R01 EY026635 to J.A.M., P30 EY029220 to the
USC Ophthalmology Core Grant in Vision Research, P30 CA014089 to the USC Norris
Comprehensive Cancer Center, P30 DK048522 to the Liver Histology Core of the USC Research
Center for Liver Diseases, the L.K. Whittier foundation, the Gavin S. Herbert Endowed Chair of
Pharmaceutical Sciences, the USC Nano Biophysics Core Facility, the Translational Research

90

Laboratory at USC School of Pharmacy, and the USC Cell and Tissue Imaging Core. Thank you
to Y. Kolodji for performing NTA analysis.

91

Chapter 4: Design of an activatable anti-CD19 chimeric antigen receptor using elastin-like
polypeptides for B-cell acute lymphoblastic leukemia treatment
4.1 Abstract
Chimeric antigen receptor (CAR) T-cell therapy has been successfully translated from the
laboratory to the clinic and has shown great potentials in cancer therapy. CAR-T cells specific to
the CD19 antigen have yielded excellent outcomes in B-cell acute lymphoblastic leukemia,
boasting a complete remission rate close to 90%. However, CAR-T cell therapies accompany
severe side effects, such as cytokine release syndrome (CRS), which requires therapeutic
interventions, such as corticosteroids or interleukine-6 receptor blockade to suppress the CRS.
Abortion of CAR-T cell therapy due to irreversible inhibition of CAR T-cell activation can be costly
and can hamper the therapeutic progress of patients. In addition, reliable indicators for the CRS
have not well established, and the therapeutic interventions are not readily available as the
biomarkers require lab study. Therefore, it is essential to introduce a tool that can modulate the
activation of CAR T-cells easily and reversibly and promptly respond to the CRS. Elastin-like
polypeptides (ELPs) are artificial protein polymers that consist of aliphatic pentameric amino acid
repeats, (VPGXG)n, where “X” is a guest amino acid, and they undergo reversible temperature-
dependent phase separation. In this chapter, we show a design of an activatable anti-CD19 CAR
T-cells using the ELP’s reversible temperature-dependent phase separation and observe the
effects of ELP-driven phase separation of CAR-ELP on T cell signaling transduction. If successful,
the CAR-ELP will promptly activate or inactivate in response to ambient temperature in a
reversible way by ELP’s phase separation. If the ELP phase separation inhibits activation of CAR,
The CAR-ELP may be able to minimize adverse effects from the CRS by quickly responding to a
fever induced by the CRS.

92

4.2 Introduction
Chimeric antigen receptor (CAR) is a fusion transmembrane receptor protein that consists of
extracellular target binding single-chain variable fragment (scFv) of antibody, spacer domain,
transmembrane domain, and intracellular signaling domain of CD3ζ linked with a costimulatory
domain, such as CD28. In the past several years, CAR T-cell therapy targeting the CD19 antigen
for B-cell acute lymphoblastic leukemia (B-ALL) treatment has yielded outstanding outcomes,
boasting high complete remission (CR) rates of 70~94% (Wang et al., 2017). Despite the excellent
therapeutic outcomes, CAR T-cell therapies have been challenging as they accompany severe
side effects, such as cytokine release syndrome (CRS) (Davila et al., 2014). To mitigate the CRS,
therapeutic interventions, such as corticosteroids or interleukin-6 receptor (IL-6R) blockade. High
dose of steroids rapidly reverses CRS toxicities, but at the same time, the medical intervention
incapacitates 19-28z CAR T-cells. The IL-6R monoclonal antibody (mAb) tocilizumab ameliorates
CRS toxicities and less compromises 19-28z CAR T-cells (Davila et al., 2014), but the additional
mAb treatment along with CAR T-cell therapies can be an economic burden for patients. In
addition, reliable indicators for the CRS have not been established well (Davila et al., 2014), and
the biomarkers require a lab study, which can hinder a prompt medical intervention when needed.
Therefore, to manage the CRS more effectively, it is important to modulate the activation of CAR
T-cells easily and reversible and respond to the CRS promptly.
To devise a new generation of CAR T-cells that can switch on and off the activation upon
a fever induced by the CRS, elastin-like polypeptides (ELPs) are employed to a CAR. ELPs are
artificial protein polymers that consist of aliphatic amino acid repeats, (VPGXG) n, where “X” is a
guest amino acid that can be any amino acid, and they undergo reversible temperature-
dependent phase separation (Despanie et al., 2016). Below the transition temperature, ELPs
remain soluble, but above the transition temperature, they coacervate and form puncta in human
cells (Pastuszka et al., 2014a). By using this characteristic, previously our group generated a

93

molecular switch of epidermal growth factor receptor (EGFR) using ELPs. The ELP fused to
EGFR switched on and off EGFR activation upon temperature change without EGF ligands (Li et
al., 2018). In this chapter, we show our design of a new generation of anti-CD19 CAR that can
turn on and off the activation by responding to temperature change by fusing ELPs and investigate
the effect of ELP coacervation on the CAR activity. Our preliminary data show that this new CAR
is successfully expressed in human cell lines. If successful, this project will present a new
generation of CAR that can reversibly modulate the activity of CAR T-cells in response to
temperature change. If the ELP coacervation inhibits the CAR activation, the ELP-CAR will serve
as a new generation of CAR that can reversibly modulate the activation in response to a fever
triggered by the CRS, which can be more efficient and economical compared to current medical
interventions, such as steroids and IL-6R monoclonal antibodies.
4.3 Materials and methods
4.3.1 α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP cloning
The second generation of the α-CD19 CAR gene in the pLVX vector was generously given by Dr.
Epstein’s lab. GFP gene was fused to pLVX-α-CD19 CAR by using XbaI and NotI restriction
enzymes, to construct α-CD19 CAR-GFP. The amino acid sequence of α-CD19 CAR-GFP is:
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT
VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITG
GGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEW
LGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWG
QGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT
CGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA
DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE
AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLTRSRVATMVSKGEELFTG
VVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRY

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PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG
HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS
TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSGLTGAIS.
Similarly, to construct α-CD19 CAR-GFP-V72, GFP-V72 gene was inserted into pLVX-α-CD19
CAR by using XbaI and NotI restriction enzymes. The amino acid sequence of α-CD19 CAR-
GFP-V72 is:
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT
VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITG
GGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEW
LGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWG
QGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT
CGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA
DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE
AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLTRSRVATMVSKGEELFTG
VVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRY
PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILG
HKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS
TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSGLTGAMGVPGVGVPGVGVPGVGVPG
VGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVP
GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGV
PGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGV
GVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG
VGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGY.

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The cloning was confirmed with restriction digest by using XbaI and NotI. After the insertion of
GFP and GFP-V72 was confirmed, the plasmids were sent for sequencing. After the sequence
was confirmed, the plasmids were purified with a Qiagen Maxi kit (Qiagen, Hilden, Germany) for
transient transfection and lentivirus production to make stable cell lines.
4.3.2 Transient expression with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP
To verify whether α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP can express in human cells,
293T cells (ATCC, VA, USA) were transfected with the plasmids. To transfect the cells, 1 million
of 293T cells were seeded on MatTek 35 mm glass bottom dishes (MatTek Corp., MA, USA).
Next day, the media of the cells were changed to Opti-MEM (Gibco, MA, USA), and 2 µg of α-
CD19 CAR-GFP-V72 or α-CD19 CAR-GFP were added to the cells with Lipofectamine 3000
Transfection Reagent (Thermo Fisher, MA, USA). The cells were incubated for 3 days at 30 °C
with 5% CO2.
4.3.3 Live cell imaging with a temperature ramp
After the transfection, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS)
(Thermo Fisher, MA, USA) three times, and 1 mL of live cell imaging solution (Thermo Fisher,
MA, USA) was added to the cells. To observe the expression and temperature-dependent phase
separation of α-CD19 CAR-GFP and α-CD19 CAR-GFP-V72, a DIAPHOT epifluorescence
microscope equipped with a DS digital camera (Nikon Instruments, NY, USA) was used with
increasing temperature from 4 °C to 70 °C at a rate of 4 °C/min on a temperature-controlled
microscope stage (Linkam Scientific, Tadworth, UK). The medium temperature was monitored by
using a thermometer probe (Sper Scientific, AZ, USA). For every 1 °C increment of the stage
temperature, the medium temperature was recorded, and cell images were captured.
4.3.4 Production of lentivirus with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP gene
To generate stable cell lines expressing α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP, lentiviral
transduction was used. To make lentivirus with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP

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gene, HEK293 cells were grown in complete Dulbecco’s Modified Eagle’s Medium (DMEM)
(DMEM, 10% fetal bovine serum, and 1% of 10,000 U/mL penicillin-streptomycin) and maintained
in T-150 flasks. A day prior to transfection, 17 to 18 million HEK293 cells were transferred to new
T-150 flasks, and 30 mL of complete DMEM medium was added to the flasks. On the day of
transfection, the medium was changed with fresh and warm complete DMEM medium supplement
with 25 µmol/mL chloroquine. Three plasmids, α-CD19 CAR-GFP-V72 or α-CD19 CAR-GFP,
psPAX2, and pMD2.G, were mixed in 2:1:1 ratio of mass, and the DNA mixture was mixed with
Xfect Transfection Reagent (Takara Bio, Kusatsu, Japan). The transfection reagent mixture was
added to HEK293 cells, and the cells were incubated at 37 °C for two days. Each day, the medium
was collected, and the T-150 flasks were filled with fresh complete DMEM medium. After two
days, the collected media were spun down at 3,000 rpm for 10 minutes, and the supernatants
were filtered with 0.45 µm polyethersulfone (PES) membrane filters. The filtered supernatants
were centrifuged at 20,000 rpm for 2 hours to concentrate the virus. Supernatants were discarded,
and the virus was resuspended with 100 µL of phosphate-buffered saline (PBS) containing 1%
bovine serum albumin (BSA) and 7% trehalose.
4.3.5 Lentiviral transduction with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP gene to
make stable cell lines
To make stable cells lines expressing α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP, a non-
tissue treated 24-well plate was coated with 15 µg of retronectin in 300 µL PBS at 4 °C overnight.
The plate was washed with PBS, and then 1 mL of sterile filtered PBS with 2% BSA to block the
plate. The plate was incubated at room temperature for 30 minutes. Lentivirus with α-CD 19 CAR-
GFP-V72 and α-CD19 CAR-GFP was mixed with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) buffer and LentiBlast Kit (Oz Biosciences, CA, USA). The lentivirus mixture was
added to the coated 24-well plate, and the plate was centrifuged at 3,000 rpm for 1 hour at room
temperature. 293T or Jurkat cells were added to the plate, and the plate was spun down at 1,200

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g for 45 minutes. After the spinning, the plate was incubated at 37 °C with 5% CO 2 overnight.
Next day, the cells were washed, and fresh complete medium was added to the plate.
4.3.6 Confocal microscope imaging
To image stable cells expressing α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP with high
resolution, LSM880 laser scanning confocal microscope with Airyscan (Carl Zeiss, Oberkochen,
Germany) was used. For images, a 63× magnification lens was used, the stage temperature was
set to room temperature.
4.4 Results
4.4.1 α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP express in 293T and Jurkat cells
To confirm whether α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP (Fig. 23A) can express in
human cell lines, 293T and Jurkat cells were transiently transfected, and the expression was
confirmed with an epifluorescence microscope imaging. Both α-CD19 CAR-GFP-V72 and α-CD19
CAR GFP expressed in both 293T and Jurkat cells. However, imaging with temperature ramp did
not clearly show the difference between α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP in
response to temperature increase.
4.4.2 Lentivirus with α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP is producible, and 293T
and Jurkat cells are transducible with the lentivirus
To produce stable cell lines expressing α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP, lentiviral
transduction was employed. Lentivirus was producible by transfecting HEK293 cells with α-CD19
CAR-GFP-V72 and α-CD19 CAR-GFP along with the second-generation packaging vector and
VSV-G envelop vector. 293T cells and Jurkat cells were transduced with the lentivirus, and both
cells stably expressed α-CD19 CAR-GFP-V72 and α-CD19 CAR (Fig. 22).

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Figure 23. α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP are expressible in 293T and Jurkat cells
and transducible with lentivirus. A) The gene of α-CD19 CAR-GFP (left) and α-CD19 CAR-GFP-V72
(right) were inserted into pLVX vector to express the proteins in human cells. B) To generate stable cell
lines expressing α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP, lentivirus was transfected with α-CD19
CAR-GFP-V72 and α-CD19 CAR-GFP, and the transfected lentivirus was used to transduce 293T and
Jurkat cells. Both α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP were expressible and transducible in
293T and Jurkat cells. To image the transduced cells, an epifluorescence microscope was used.

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4.5 Discussion
In the past several years, CAR T-cell therapy targeting the CD19 antigen for B-cell acute
lymphoblastic leukemia (B-ALL) treatment has been promising as the therapy shows high
complete remission (CR) rates of 70~94% (Wang et al., 2017). Nevertheless, CAR T-cell
therapies have faced several challenges as they develop severe adverse effects, such as cytokine
release syndrome (CRS) (Davila et al., 2014). There are several medical interventions to mitigate
the CRS, such as corticosteroids and interleukin-6 receptor blockade, but these interventions can
incapacitate the function of CAR T-cells, resulting in early termination of CAR-T cell therapy
(Davila et al., 2014). In addition, biomarkers of the CRS are not certain (Davila et al., 2014), and
detecting the biomarkers requires a lab study, which can delay a therapeutic intervention to
mitigate the CRS. In the light of these challenges in current CAR T-cell therapies, we have
designed activatable CAR receptors using ELPs in response to temperature change, which is
frequently observed symptom of the CRS. Temperature-sensitive CAR-ELP fusion proteins are
expected to change the biological activity of CAR upon ELP’s phase separation in response to
temperature increase. In this chapter, we tried to investigate whether ELP’s phase separation can
either switch on or off the CAR-T signaling pathway.
First, α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP were cloned to visualize the behavior
of temperature-dependent phase separation of α-CD19 CAR-ELP in comparison with α-CD19
CAR. Expression of α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP was first confirmed in 293T
cells and Jurkat cells by using a transient transfection using Lipofectamine.
To find the transition temperature of α-CD19 CAR-GFP-V72, the expressed proteins were
observed with an epifluorescence microscope along with the temperature ramp at a rate of
4°C/min. Unfortunately, it was difficult to determine the transition temperature of α-CD19 CAR-
GFP-V72 using the microscope because both α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP
formed clusters at low temperature. Thus, it was challenging to differentiate the puncta formed by

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ELP coacervation from the cluster originally formed. In the future, α-CD19 CAR-V72, α-CD19
CAR-V96, and α-CD19 CAR-A96 will be cloned to reduce the clustering that can be enhanced by
the GFP. Then, to estimate the transition temperature of α-CD19 CAR-V72, α-CD19 CAR-V96,
and α-CD19 CAR-A96, transfected cells will be incubated at several temperatures, such as 4, 25,
37 °C, fixed, and probed with an anti-ELP antibody.
After the confirmation of the expression of both α-CD19 CAR-GFP-V72 and α-CD19 CAR-
GFP, stable cell lines expressing α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP were generated
with lentiviral transduction. The lentiviral transduction with 293T and Jurkat cells was successful,
indicating that both α-CD19 CAR-GFP-V72 and α-CD19 CAR-GFP can be transducible with the
lentivirus in human cells (Fig. 22). In the future, after defining the transition temperature of α-
CD19 CAR-V72 and α-CD19 CAR-V96 using fixed-cell imaging, the effects of ELP’s phase
separation on CAR activity will be investigated. To do so, the IL-2 level increase or decrease can
be studied with stable Jurkat cells expressing α-CD19 CAR-V72 and α-CD19 CAR-V96. If the
ELP coacervation enhances the T-cell activity, CAR-ELP construct can be applied to solid tumor
targeting CAR T-cells. Solid tumor-targeting CAR T-cells have not been successful unlike the one
targeting hematologic malignancies (Newick et al., 2017). If we can locally enhance the CAR T-
cell activity by applying the mild heat to the tumor site, the CAR-ELP construct might be able to
overcome the current challenges in CAR T-cells targeting solid tumors. If the ELP coacervation
inhibits the T-cell activity, CAR-ELP construct can be applied to α-CD19 CAR therapy. Although
α-CD19 CAR has been successful to eradicate hematologic malignancies, the safety of the
therapy has been a concern as the CRS can be life-threatening. If the CAR-ELP coacervation
inhibits CAR T-cell activation reversibly in response to temperature change, the new generation
of CAR will be able to modulate the activation upon the fever induced by the CRS without any
medical intervention, resulting in the better safety.

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4.6 Conclusion
In this chapter, we have shown that α-CD19 CAR-GFP-V72 can be expressed in 293T cells and
Jurkat cells by using lentiviral transduction. In the future, the transition temperature of multiple
CAR-ELPs will be determined using fixed-cell imaging at multiple temperatures, and the effects
of ELP coacervation on CAR T-cell activation will be investigated.
4.7 Acknowledgements
This work was made possible by University of Southern California (USC), the MacKay lab, Dr.
Alan Epstein, Dr. Peisheng Hu, and Larry (Long) Zheng.

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Chapter 5: Formulation of elastin-like polypeptide nanoparticles targeting cytotoxic T-
lymphocyte antigen-4 to harness the immune system for acute myeloid leukemia
treatment
5.1 Abstract
Acute myeloid leukemia (AML) is a form of cancer that exhibits uncontrolled proliferation of
abnormal or poorly differentiated hematopoietic cells, and its prognosis has remained poor for
decades. As currently available AML treatment is limited to chemotherapy and bone marrow
transplantation, new modalities of AML therapy are desperately needed. Immunotherapies
targeting either cytotoxic T-lymphocyte antigen-4 (CTLA4) or programmed cell death protein 1
(PD-1) have been successful to treat several types of cancer, especially metastasized tumors,
and they have become promising therapies along with chimeric antigen receptor T-cell therapy.
In Chapter 1 and Chapter 2, we have proposed targeted therapies for AML treatment with elastin-
like polypeptide (ELP) nanoparticles targeting a known target, FMS-like tyrosine kinase 3 (FLT3),
and a novel target, CD99. In this chapter, we discuss the formulation of ELP nanoparticles fused
to anti-CTLA4 scFv. Here, we show the characterization study of the fusion protein, α-CTLA4-
A192, supporting the formation of nanoparticles. The ELP nanoparticles are expected to show a
long half-life, biological activity in vitro and in vivo, and better efficacy through reticuloendothelial
uptake.
5.2 Introduction
Acute myeloid leukemia (AML) is a form of blood cancer that exhibits uncontrolled proliferation of
abnormally or poorly differentiated hematopoietic cells (Döhner et al., 2015; Tallman et al., 2005).
Its prognosis has been poor for several decades, and therapeutic options for AML are mainly
limited to chemotherapy and hematopoietic stem cell transplantation (Lamble and Lind, 2018;
Liao et al., 2019). As a new modality of AML therapy, tyrosine kinase inhibitors (TKIs) have been
studied for AML treatment, and one of the TKIs, midostaurin, was approved by the FDA in 2017

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(Levis, 2017). However, TKIs may not be entirely specific to FLT3 receptor tyrosine kinase, and
the treatment developed resistance mechanisms involving secondary mutations in the kinase
domain and upregulation of FLT3 wild type or the FL-ligand in the bone marrow microenvironment
(Daver et al., 2015; Daver et al., 2019; Williams et al., 2013). Recent development of immune
checkpoint inhibitors (ICIs), such as anti-cytotoxic T-lymphocytic antigen 4 (CTLA4) and anti-
programmed cell death protein 1 (PD-1), has focused on the reinvigoration of a host’s immune
system to induce immune-mediated elimination of tumor cells that frequently evade
immunosurveillance (Darvin et al., 2018). Currently, ipilimumab (anti-CTLA4) is approved by the
USFDA for metastatic melanoma treatment, and nivolumab (anti-PD-1) and pembrolizumab (anti-
PD-1) are approved by the USFDA for the treatment of metastatic melanoma and non-small cell
lung cancer (NSCLC) (Friedman et al., 2016). Mechanisms of anti-CTLA4 and anti-PD-1 are
similar in reinvigorating the immune response. CTLA4 receptors on T-cells inhibit T-cell
maturation and differentiation by competing with the costimulatory receptor CD28 for B7.1 and
B7.2 on antigen presenting cells (Liu et al., 2019). It is expected that anti-CTLA4 binds CTLA4
receptors, promoting CD28 to bind B7.1 and B7.2. Similarly to CTLA4, PD-1/PD-L1 signaling
suppresses T-cell activation after T-cell receptor/MHC engagement and CD28 activation (Liu et
al., 2019). Therefore, it is expected that anti-PD-1 interrupts PD-1/PD-L1 binding, resulting in
enhanced T-cell activation. Although anti-CTLA4 and anti-PD-1 monoclonal antibodies have
shown great clinical results in metastatic melanoma and non-small cell lung cancer, they have
exhibited disappointing results in AML as monotherapies in early-phase clinical trials (Stahl and
Goldberg, 2019). These results from the clinical trials suggest a new approach to using ICIs in
AML is necessary.
To effectively deliver ICIs to immune cells in order to increase the number of activated T-
cells, we have employed elastin-like polypeptides (ELPs) to formulate nanoparticles targeting
CTLA4. ELPs are artificial protein polymers that consist of hydrophobic amino acid repeats,

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(VPGXG)n, where X is a guest amino acid that can be any amino acid. ELPs undergo reversible
temperature-dependent phase separation. Above the transition temperature, ELPs coacervate
and form a secondary aqueous phase. Below the transition temperature, ELPs become soluble
(Despanie et al., 2016). In previous chapters, it has been shown that a high molecular weight,
A192, fused to scFvs form soluble and stable nanoparticles. To formulate ELP nanoparticles
targeting CTLA4, A192 was fused to anti-CTLA4 single-chain variable fragment (scFv).
Formulation of ELP nanoparticles targeting CTLA4 is expected to improve T-cell activation by the
enhanced cellular uptake of the nanoparticles and increased localization of the fusion protein to
the lymphatic system by reticuloendothelial transport, where immune cells reside (Cai et al., 2011),
resulting in enhanced efficacy of the anti-CTLA4. In this chapter, we show the fusion of A192 to
anti-CTLA4 assembles stable nanoparticles. Then, we will discuss future studies to verify whether
the ELP nanoparticles targeting CTLA4 have the enhanced efficacy of anti-CTLA4 compared to
ipilimumab.
5.3 Methods
5.3.1 Cloning and purification of α-CTLA4-A192
To construct an immune check point inhibitor fused to an elastin-like polypeptide (ELP), a α-
CLTA4 scFv gene was fused to the amino terminus of an ELP called A192, in the pET-25b(+)
vector, encoding α-CTLA4-A192. The α-CTLA4 scFv DNA sequence was generously given by Dr.
Spangler’s lab at Johns Hopkins University, and the sequence is:
ATGGGTGGTGGGGGCTCCGGTGGcGGAGGTAGCGGcGGcGGAGGTTCTGCTAGTCAAGTT
CAGCTTCAACAATCTGGTCCAGGGCTGGTAAAGCCCTCCCATACTTTATCGCTTACATGTG
TTGTTAGCGGGGACTCTGTGTCCTCCAGCTCAGCCGCGTGGCATTGGATTCGTCAGTCTC
CTAGTCGTGGATTGGAATGGTTGGGCAAGACTTACTACCGTAGCAAGTGGTACAATGACTA
TGCAGTAAGCGTTAAAAGTCGCATCACTATTAACACAGATACGTCTAAGAACCAGTTCTCGT
TGCAACTTAATTCAGTAACTCCTGAGGATACAGCGGTGTACTACTGTGCCCGCGAGTCATA

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TTCCTATGACAGCAGCGGTTTTTATGAGTATTACGGGTTGGACGTCTGGGGACAGGGCAC
CACCGTAACTGTTTCGAGTGGAATCCTTGGTTCGGGTGGTGGCGGATCAGGGGGTGGTG
GTAGTGGCGGCGGGGGTTCGCAACCGGTGCTTACACAGAGTCATAGCGTGTCTGAGTCC
CCAGGGAAAACGGTTACAATTAGTTGTACACGCTCCAGCGGGTCTATTGCTAGTAATTACG
TACAGTGGTACCAGCAACGCCCCGGGAGCGCACCAACGACTGTCATCTATGAGGACAATC
AGCGTCCCTCGGGGGTACCCGATCGTTTTAGCGGAAGCATCGACAGTTCGTCTAACTCTG
CATCGCTTACTATTAGTGGATTGAAAACCGAAGATGAGGCTGACTATTATTGCCAAAGCTAC
GACTCCAATAACCACGCAGTCTTCGGCGGTGGTACGCAGCTGACAGTATTATCCGGCATTT
TA.
The α-CTLA4 scFv sequence was purchased from Integrated DNA Technologies, IA, USA,
and was cloned into an empty pET-25b(+) vector cut using NdeI and BamHI restriction enzymes
(New England Biolabs, MA, USA). After the sequence of α-CTLA4 scFv in pET-25b(+) vector was
confirmed, BseRI and BssHII restriction enzymes were used to digest pET-25b(+)-α-CTLA4 scFv
and pET-25b(+)-A192 to construct pET-25b(+)-α-CTLA4-A192. After the ligation, a diagnostic
DNA digestion was performed using NdeI and BamHI restriction enzymes followed by
electrophoresis on a 1% agarose gel in order to confirm whether the ligation was successful. After
the diagnostic DNA digestion, the plasmid was sent for DNA sequencing with the T7 promoter
that is specific for TAATACGACTCACTATAGGG and T7 terminator that is specific for
GCTAGTTATTGCTCAGCGG, which confirmed in-frame insertion into the pET-25b(+). To
produce the fusion protein, Shuffle® T7 competent cells (New England Biolabs, MA, USA) were
transformed with the α-CTLA4-A192 plasmid. After transformation, colonies were picked and
cultured in 60 mL of Terrific Broth with Glycerol (TB) (C8153, CulGeneX, CA, USA) with 100
µg/mL of carbenicillin at 30 °C for 16-18 hours. For each autoclaved 1 L of TB with 100 µg/mL
carbenicillin, 10 mL of the bacteria culture was added. A total of 6 L of bacteria was cultured at
30 °C until the optical density (OD) at 600 nm reached between 0.6 and 0.8. Once the measured

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OD 600 reached above 0.6, 400 µL of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) was added
to each 1 L flask to bring the final concentration to 400 µM IPTG. IPTG induction was done for
overnight at room temperature. The next day, bacteria were recovered by centrifugation at 4,000
rpm for 15 minutes, and the supernatant was discarded. Each pellet was resuspended with 30
mL of cold PBS, vortexed, and disrupted using a probe-tip Misonix sonicator S-4000 (Misonix,
NY, USA). After sonication, 0.5 % of polyethyleneimine (PEI) was added to each cell lysate, and
the cell lysate was incubated on ice for 5 minutes. Then, the cell lysates were pelleted at 13,000
rpm for 15 minutes at 4 °C. The supernatant was recovered, and solid NaCl was added to the
supernatant to reach a final concentration of 2 M. Each supernatant with 2 M of NaCl was placed
in the water bath at 37 °C until phase separation was observed. After the phase separation, the
supernatant was centrifuged at 4,000 rpm for 15 minutes at 37 °C, and the supernatant was
discarded. The pellet was resolubilized with cold PBS on ice, and the solubilized pellet was
centrifuged at 13,000 rpm for 15 minutes at 4 °C. The supernatant was collected, and this process,
hot and cold centrifugation, was repeated three times with a decreasing volume of PBS and NaCl
concentration to remove impurity and increase the purity of α-CTLA4-A192.
5.3.2 α-CTLA4-A192 protein concentration measurements
As forming nanoparticles, the fusion protein was denatured with 6 M guanidine hydrochloride to
disrupt the assembly of nanoparticles. Then, the protein concentration was measured using the
Eq. 1. Using the Eq.2, ε of α-CTLA4 scFv was estimated to be 58,580 L mol
-1
cm
-1
assuming all
pairs of cysteine residues are oxidized to form cystine. ε of A192 was estimated to be 1,490 L
mol
-1
cm
-1
as it has only one tyrosine at the end of A192 sequence. Therefore, ε of α-CTLA4-A192
was estimated to be 60,070 L mol
-1
cm
-1
. The optical absorbance at 280 and 350 nm was
measured with a NanoDrop 2000 (Thermo Fisher, MA, USA), which has a path length of 0.1 cm.

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5.3.3 α-CTLA4-A192 purity and transition temperature analysis
The purity of α-CTLA4-A192 was determined by an SDS-PAGE gel image quantified using
ImageJ. To run SDS-PAGE, purified α-CTLA4-A192 was loaded to a 4-20% precast SDS-PAGE
gel (4561095, Bio-Rad Laboratories, CA, USA). The gel was stained with Gelcode
TM
Blue Safe
Protein Stain (24596, Thermo Fisher, MA, USA). After the staining, the gel was imaged with a
ChemiDoc Touch Image System (Bio-Rad Laboratories, CA, USA), and the image was analyzed
with ImageJ (NIH, MD, USA) to measure the purity of the purified fusion protein. A whole lane
was plotted to obtain the area under each peak, and the Eq. 3 was used to calculate the purity of
the protein. The transition temperature (Tt) of the fusion protein was measured using Beckman
Coulter DU 800 UV/VIS spectrometer (Beckman Coulter, CA, USA). Four different concentrations
of α-CTLA4-A192, 3.125, 6.25, 12.5, and 25 µM were prepared, and the samples were heated at
a rate of 1 °C/min starting from 20 °C to 85 °C. OD 350 was measured every 18 seconds, and the
Tt was determined where the maximum first derivative of the OD 350 with respect to temperature
occurred. The data was fit to the Eq.4 to plot the Tt change with respect to a concentration of the
ELP fusion protein.
5.3.4 α-CTLA4-A192 protein refolding
As α-CTLA4-A192 has four cysteines, the fusion protein was refolded to maximize the biological
activity. α-CTLA4-A192 in PBS was mixed with the same volume of 8 M urea buffer with 10 mM
β-mercaptoethanol (BME). After mixing with the 8 M urea buffer, the mixture was dialyzed to 3 M
urea buffer with 2 mM glutathione (GSH) and 0.4 mM oxidized glutathione (GSSH) for 24 hours
at 4 °C. Then, the protein was dialyzed to 1 M urea buffer with 2 mM GSH and 0.4 mM GSSH,
0.5 M urea buffer, 0 M urea buffer, and then 2 times of PBS for 24 hours for each step at 4 °C.
5.3.5 Measurements of the hydrodynamic radius of α-CTLA4-A192 and colloidal stability
The hydrodynamic radius and stability of α-CTLA4-A192 was measured using dynamic light
scattering (DLS). To measure the hydrodynamic radius, 25 µM of α-CTLA4-A192 was prepared,

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filtered with a 0.22 µm filter, and 60 µL of the protein was added to each well of a 384-well plate.
To prevent evaporation of the protein, 15 µL of mineral oil was added to each sample. The
hydrodynamic radius was measured with DynaPro Plate reader II (Wyatt Technology, CA, USA)
at 37 °C. The hydrodynamic radius of A192 was also measured to observe the difference between
the fusion protein and A192 in the hydrodynamic radius. The stability of α-CTLA4-A192
nanoparticles was measured with the same sample using DLS. The DLS plate was incubated at
37 °C, and every 24 hours, the hydrodynamic radius of α-CD99-A192 was measured until 120
hours.
5.3.6 Measurement of the molecular weight of α-CTLA4-A192 nanoparticles
The molecular weight of α-CTLA4-A192 nanoparticles was measured with size exclusion
chromatography-multi-angle light scattering (SEC-MALS). To run SEC-MALS, 10 µM of α-
CTLA4-A192 was prepared, and the sample was filtered with 0.22 µm filter. A Shodex protein
KW-804 (8.0mmI.D.x300mm) (Showa Denko America, NY,USA) was equilibrated with PBS, and
the fusion protein was loaded to the column. The fusion protein elusion was observed with three
detectors, UV 210 mm detector (SYC-LC-1200) (Agilent Technologies, CA, USA), multi-angle
static light scattering detector (DAWN HELEOS) (Wyatt Technology, CA), USA, and differential
refractometer (Optilab rEX) (Wyatt Technology, CA, USA). The data were analyzed by ASTRA 6
software.

109

5.4 Results
5.4.1 α-CTLA4-A192 is highly expressed in E. coli, and the fusion protein purified using
temperature-dependent phase separation is soluble and pure.
To generate α-CTLA4-A192, information of a gene encoding α-CTLA4 scFv was generously given
by the Spangler lab at Johns Hopkins University, and the gene was inserted into a pET25-25b(+)
vector to express α-CTLA4-A192 in Shuffle T7 competent cells (Fig. 24A). After three cycles of
hot and cold centrifugation, the purity of α-CTLA4-A192 was estimated by SDS-PAGE gel analysis
using ImageJ and measured to be 94.7 %.

Figure 24. Cloning and purification of α-CTLA4-A192 A) F8-2 scFv, targeting CTLA4, was fused to
the N-terminus of A192 to construct α-CTLA4-A192. B) α-CTLA4-A192 was purified by using three
cycles of hot and cold temperature with mild salts. Lane 1: protein ladder Lane 2: α-CTLA4-A192 after
one cycle Lane 3: α-CTLA4-A192 after two cycles Lane 4: α-CTLA4-A192 after three cycles. The
expected molecular weight of α-CTLA4-A192 is 103.4 kDa.

110

5.4.2 The fusion of α-CTLA4 scFv to A192 decreases the transition temperature of A192
by increasing ΔHcoacervation and ΔScoacervation
The phase diagram that shows optical density measurements as a function of concentration and
temperature was analyzed in order to estimate the transition temperature of the fusion protein. A
maximum point of the first derivative was considered as the transition temperature of ELPs, and
the transition temperature of A192, crude α-CTLA4-A192, and refolded α-CLTA4-A192 were
measured to be 59.9, 41.2, and 44.2 °C, respectively at 25 µM of ELP concentration (Fig. 25A).
While α-CTLA4-A192 remained soluble at 37 °C, the fusion of α-CTLA4 scFv to A192 substantially
decreased the transition temperature of A192. Interestingly, protein refolding process slightly
increased the transition temperature of A192 compared to that of crude α-CTLA4-A192. In
addition, the concentration dependence of the phase diagram was quantified from the slope of
Eq. 4 (Table 7). This significant alteration in the phase behavior for A192 suggested that the α-
CTLA4 domain exerts a substantial effect on the peptide conformation and thus phase behavior
for the ELP. By using van’t Hoff equation (Eq. 8), ΔHcoacervation and ΔScoacervation were estimated, it
was discovered that α-CTLA4-A192 transition temperature becomes lower due to substantial
increase in both ΔHcoacervation and ΔScoacervation of A192 phase separation in the fusion protein (Table
8).
Table 7. Biophysical characterization of elastin-like polypeptides evaluated in Chapter 5
ELP Amino acid
sequence
M.W.
(kDa)
b

T t
(°C)
c

R h
(nm)
d

R g
(nm)
d

R g/R h Shape
e

A192

MG(VPGAG)192Y 73.6 59.9 7.5
(0.2)
N/A N/A N/A
α-CTLA4-
A192
α-CTLA4
a
-
G(VPGAG)192Y
103.4 44.2 40.4
(0.7)
62.8 (0.1) 1.6 extended
a
the amino acid sequence is:
MGGGGSGGGGSGGGGSASQVQLQQSGPGLVKPSHTLSLTCVVSGDSVSSSSAAWHWIRQSPSRGLEWLGKTYYR
SKWYNDYAVSVKSRITINTDTSKNQFSLQLNSVTPEDTAVYYCARESYSYDSSGFYEYYGLDVWGQGTTVTVSSGIL
GSGGGGSGGGGSGGGGSQPVLTQSHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSG
VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSNNHAVFGGGTQLTVLSGIL-LVPRGS
b
expected molecular weight based on the amino acid sequence and confirmed by SDS-PAGE
c
transition temperature defined as the maximum first derivative of the optical density at 350 nm for 25 µM ELP in PBS
d
values indicate the mean (standard deviation) of n=6
e
the Rg/Rh ratio of α-CTLA4-A192 is consistent with a ‘rod-like’ nanoparticle of an extended aspect ratio as estimated
using DLS and SEC-MALS

111

5.4.3 α-CTLA4-A192 forms stable nanoparticles
To measure the size of α-CTLA4-A192 nanoparticles, DLS was used measure the hydrodynamic
radius of the fusion protein, and the DLS results indicated that α-CTLA4-A192 formed
nanoparticles. The average hydrodynamic radius of α-CTLA4-A192 was measured to be 40.4 ±
0.7 (mean ± SD, n=6) nm (Fig. 25B). The polydispersity index was measured to be 0.17 ± 0.01
(mean ± SD, n=6). The stability of nanoparticles was measured by running DLS at 37 °C for 120
hours (Fig. 25C). The hydrodynamic radius of α-CTLA4-A192 nanoparticles stayed constant over
5 days at 37 °C, indicating that the fusion protein forms stable nanoparticles in PBS.
To measure the average molecular weight of α-CTLA4-A192 nanoparticles, SEC-MALS
was performed. A192 was run as a control, and it was eluted from the SEC column at 8.5 to 9.2
mL. The absolute molecular weight of A192 was equivalent to that of a monomer. On the other
hand, α-CTLA4-A192 was eluted more rapidly in the void volume between 5.2 and 7.0 mL, and
the absolute molecular weight was measured to be 3.2×10
7
Da (Fig. 26A and B). Based on this
number, the average number of α-CLTA4-A192 forming a nanoparticle in solution was estimated

Figure 25. α-CTLA4-A192 has a lower transition temperature than A192, and it forms stable
colloids at 37 °C. A) The transition temperature of A192, crude α-CTLA4-A192, and refolded α-CTLA4-
A192 were measured by using a UV/VIS spectrometer. The fusion of anti-CTLA4 scFv to A192 lowered
the transition temperature of A192. It was observed that the refolding of the protein slightly increased
the transition temperature of α-CTLA4-A192. B) The hydrodynamic radius of A192 and α-CTLA4-A192
was compared by using dynamic light scattering (DLS). The hydrodynamic radius of A192 and refolded
α-CTLA4-A192 was measured to be 7.5 nm and 40.4 nm, respectively (Table 7). C) Stability of refolded
α-CTLA4-A192 in PBS was analyzed by using DLS at 37°C. for 120 hours, the hydrodynamic radius of
refolded α-CTLA4-A192 was observed every 24 hours. It was observed that refolded α-CTLA4-A192
forms stable colloids in PBS at 37 °C.

112

to be 310 molecules. The radius of gyration (Rg) of α-CLTA4-A192 was measured to be 62.8 nm
from SEC-MALS.
Table 8. Thermodynamics of A192 phase separation in scFv-A192
Label
Phase diagram
a
Thermodynamics
b

m
(°C/log 10[µM])
b
(°C)
ΔH coacervation
(kJ/mol)
ΔS coacervation
(kJ/mol K )
A192 -7.57
[-9.43 to -5.72]
70.5
[68.6 to 72.3]
284
[221 to 347]
0.83
[0.64 to 1.01]
α-FLT3-A192 -1.23
[-2.53 to 0.07]
44.0
[42.7 to 45.4]
1390
[-82.7 to 2860]
4.38
[-0.28 to 9.04]
α-CD99-A192 -1.53
[-3.11 to 0.05]
47.6
[46.0 to 49.1]
1120
[-37.3 to 2280]
3.50
[-0.13 to 7.14]
α-CTLA4-A192 -2.09
[-3.23 to -0.96]
47.3
[46.1 to 48.4]
887
[406 to 1370]
2.77
[1.26 to 4.28]
a
Slope and intercept fit to Eq. 4. mean [95% confidence interval].
b
Slope and intercept fit to Eq. 8. mean [95% confidence interval].

5.5 Discussion
AML is the most common type of acute leukemia in adults (Wouters and Delwel, 2016), and its
prognosis has been poor for several decades. Moreover, therapeutic options for AML has been
limited to chemotherapy and hematopoietic stem cell transplantation (Lamble and Lind, 2018;

Figure 26. Size exclusion chromatography with multi-angle light scattering reveals that 310 α-
CTLA4-A192 molecules form a nanoparticle. To measure the absolute molar mass of nanoparticles
formed by α-CTLA4-A192, size exclusion chromatography with multi-angle light scattering (SEC-MALS)
was employed. A) The graph shows the SEC-MALS analysis of α-CTLA4-A192. B) To measure the
molar mass, a major peak was selected, and the molar mass of the peak was plotted. The average
molecuar weight of the peak was measured to be 3.2×10
7
± 0.0 Da, equivalent to 310 α-CTLA4-A192
molecules.

113

Liao et al., 2019). For that reason, it is urgently needed to develop new modalities of AML therapy.
In 2017, a new modality of AML treatment, midostaurin, was approved by the FDA, but
midostaurin has shown several drawbacks, such as the development of drug resistance as
discussed in Chapter 1. To overcome the drawbacks, we formulated ELP nanoparticles targeting
FLT3 that are stable at 37 °C in PBS and have anti-leukemic activities both in vitro and in vivo. In
Chapter 2, we presented another new modality of AML therapy by forming ELP nanoparticles
targeting a novel target, CD99. The ELP nanoparticles exhibited specificity, anti-leukemic activity,
and long half-life. In this chapter, we suggest another way of AML treatment with ELP
nanoparticles. Recent development of ICIs, such as anti-CTLA4 and anti-PD-1 have been
successful to treat metastatic melanoma and NSCLC by harnessing a host’s immune system
(Darvin et al., 2018; Friedman et al., 2016). Nevertheless, anti-CTLA4 and anti-PD-1 monoclonal
antibodies showed disappointing clinical results in AML treatment as monotherapies (Stahl and
Goldberg, 2019), suggesting a new way of approach is needed for AML therapy using ICIs.
To generate a new form of ICIs, we formulated ELP nanoparticles targeting CTLA4. α-
CTLA4 scFv was fused to a high molecular weight ELP, A192, encoding α-CTLA4-A192 (Fig.
24A). By inserting the α-CTLA4-A192 gene into pET-25b vector, expression of the fusion protein
was enabled in E. coli. Many scFvs have been reported to form inclusion bodies when expressed
in E. coli, but when fused to A192, the fusion protein remained in a soluble fraction of bacteria
lysate. In addition, the fusion of A192 facilitated the purification of α-CTLA4-A192. By using three
cycles of cold and hot centrifugation, a high purity of the fusion protein was obtained (94.7%) (Fig.
24B). The fusion of α-CTLA4 scFv to A192 substantially decreased the transition temperature of
A192, similarly to other ELP nanoparticles shown in Chapters 1, 2, and 3. Interestingly, the
refolding of α-CTLA4-A192 slightly increased the transition temperature of A192 (Fig. 25A). It
seems correctly folded α-CTLA4 scFv has less tendency to oligomerize, resulting in a higher A192
transition temperature compared to crude α-CTLA4-A192. The thermodynamic analysis of A192

114

phase separation in α-CTLA4-A192 using van’t Hoff equation indicated that the transition
temperature of A192 in α-CTLA4-A192 decreased due to a substantial increase in both
ΔHcoacervation and ΔScoacervation in the fusion protein. Based on the DLS data, the average
hydrodynamic radius was measured to be 40.4 ± 0.7 nm (mean ± SD, n=6), and the polydispersity
index of the nanoparticles was estimated to be 0.17 ± 0.01 (mean ± SD, n=6). Based on the
polydispersity index, α-CTLA4-A192 nanoparticles are expected to be monodisperse. The
nanoparticles were stable at 37 °C for 5 days in PBS (Fig. 25C), suggesting that the fusion of
A192 to α-CTLA4 scFv formulates stable nanoparticles at the physiological temperature. SEC-
MALS data revealed that the average molecular weight of the ELP nanoparticles was estimated
to be 3.2×10
7
Da, equivalent to 310 α-CTLA4-A192 molecules (Fig. 26B). Based on the DLS and
SEC-MALS data, Rg/Rh ratio was calculated to be 1.6, suggesting that the ELP nanoparticles are
likely to be extended worm-like nanoparticles.
The formation of nanoparticles is expected to have a therapeutic advantage over currently
available anti-CTLA4 monoclonal antibodies. The size of monoclonal antibodies is about 10 nm
(Reth, 2013), and the size allows them to extravasate to normal tissues (Sarin, 2010;
Stylianopoulos, 2013). Most immune cells are located in the lymphatic system (Cai et al., 2011),
and the lymphatic system is in a leaky environment. The diameter of α-CTLA4-A192 nanoparticles
is around 80 nm, and the size prohibits the extravasation of the nanoparticles to normal tissues,
unlike monoclonal antibodies. Still, the ELP nanoparticles can enter the lymphatic system due to
the leaky environment and reticuloendothelial system, so the nanoparticles can be specifically
accumulated to the lymphatic system. Therefore, T-cell activation by α-CTLA4-A192
nanoparticles could be more enhanced compared to anti-CTLA4 monoclonal antibodies. Also, the
fusion of A192 increases the molecular weight of the fusion protein substantially, and it can allow
the fusion protein to evade the glomerular filtration, resulting in longer circulation time in the body.

115

5.6 Conclusion
In this chapter, we formulated ELP nanoparticles targeting CTLA4 by fusing α-CTLA4 scFv to
A192. The fusion of A192 to α-CTLA4 scFv generated the fusion protein in a soluble fraction and
formulated stable ELP nanoparticles at 37 °C for 5 days. Based on SEC-MALS and DLS data,
the average molecular weight of α-CTLA4-A192 nanoparticles was estimated to be 3.2×10
7
Da
with the extended rod-like shape. In the future, the binding and biological activities of α-CTLA4-
A192 nanoparticles will be investigated. To analyze the specific binding to CTLA4 receptors, laser
scanning confocal microscopy and flow cytometry will be employed with α-CTLA4 monoclonal
antibodies. It will be investigated whether α-CTLA4-A192 nanoparticles can activate T-cells in
vitro and in vivo. Then, its therapeutic effects on mice engrafted with AML cells will be studied
and compared with anti-CTLA4 monoclonal antibodies. Finally, PK parameters of α-CTLA4-A192
nanoparticles will be analyzed by using rhodamine-labeled fusion proteins.
5.7 Acknowledgements
This work was made possible by University of Southern California (USC), the MacKay lab, and
the Spangler lab at Johns Hopkins University, USC Translational Research Lab, and USC Center
of Excellence in Nanobiophysics.

116

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Creator Park, Mincheol (author)

Core Title Application of elastin-like polypeptides to therapeutics in leukemia

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 07/26/2020

Defense Date 06/15/2020

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

Tag CD99,CTLA-4,ELP,FLT3,GM-CSF,leukemia,nanoparticle,OAI-PMH Harvest,therapeutic target

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor MacKay, John Andrew (committee chair), Alachkar, Houda (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email mincheol.park88@gmail.com,mincheop@usc.edu

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