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Developing recombinant single chain Fc-dimer fusion proteins for improved protein drug delivery
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Developing recombinant single chain Fc-dimer fusion proteins for improved protein drug delivery
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Content
DEVELOPING RECOMBINANT SINGLE CHAIN Fc-DIMER FUSION
PROTEINS FOR IMPROVED PROTEIN DRUG DELIVERY
By
Li Zhou
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
May 2018
Copyright 2018 Li Zhou
ii
DEDICATION
This dissertation is dedicated to my parents, Weiren Zhou and Youqin Li, who helped me
in all things great and small.
iii
ACKNOWLEDGEMENTS
First of all, I would like to convey my sincere gratefulness to my advisor Dr. Wei-
Chiang Shen. His devotion to science, wisdom and persistence in research and kindness to
people have constantly inspired and motivated me. I really appreciate the freedom and
respect that I experienced during my time in the lab, and the strong support in my career
development.
I would specifically like to thank Dr. Jennica Zaro for the guidance and support in
my research, as well as career development. I appreciate the tremendous effort that she has
spent to promote the development of this project and her help on my manuscript. I can
always recall her trademark smile.
I also want to acknowledge my co-advisor Dr. Curtis Okamoto and my other
committee members, Dr. Yong Zhang, Dr. Kwang Jin Kim and Dr. Kathleen Rogers, who
have generously provided their insights and suggestions to me.
Moreover, I really appreciate the friendship and experimental assistance from my
labmates, Hsuan-Yao (Sean) Wang, Yuqian Liu, Dr. Chunmeng Sun, Dr. Zoe Wagner,
Hsin-Fang Lee, Dr. Shanshan Tong and Yang (Lisa) Su. I also want to thank Dr. Shuxing
Li and Dr. Liang Rong for the help on SPR and SEC, respectively. I am grateful to all of
those whom I have had the pleasure to work with during the past five years.
Last but not the least, I would like to thank my parents for supporting me spiritually
throughout writing this dissertation and my life in general.
iv
TABLE OF CONTENTS
DEDICATION ................................................................................................................. II
ACKNOWLEDGEMENTS ........................................................................................... III
LIST OF TABLES ....................................................................................................... VIII
LIST OF FIGURES ........................................................................................................ IX
ABBREVIATIONS ......................................................................................................... XI
ABSTRACT ................................................................................................................... XII
CHAPTER 1 INTRODUCTION ......................................................................................1
1.1. PROTEIN THERAPEUTICS ....................................................................................................... 1
1.2. NEONATAL FC RECEPTOR (FCRN) AND IMMUNOGLOBULIN G (IGG) .................................. 2
1.2.1. Physiological Properties and Functions ......................................................................... 2
1.2.2. Receptor-mediated Recycling and Transcytosis Pathway ............................................. 4
1.3. FC FUSION PROTEINS ............................................................................................................ 5
CHAPTER 2: DESIGN AND PRODUCTION OF SINGLE CHAIN FC-DIMER
RECOMBINANT FUSION PROTEINS .........................................................................8
2.1. BACKGROUND ....................................................................................................................... 8
2.2. MATERIALS AND METHODS ................................................................................................ 12
2.2.1. Cell Cultures ................................................................................................................. 12
2.2.2. Plasmid Construction ................................................................................................... 12
2.2.2.1. pcDNA3.1+_sc(Fc)
2
............................................................................................................................ 12
2.2.2.2. pcDNA3.1+_hGH-sc(Fc)
2
................................................................................................................... 18
2.2.2.3 pcDNA3.1+_sc(Fc)
2
-hGH .................................................................................................................... 18
2.2.2.4. pcDNA3.1+_hGH-Fc ........................................................................................................................... 19
2.2.2.5. pcDNA3.1+_IHH ................................................................................................................................. 19
2.2.3. Fusion Protein Production ............................................................................................ 19
2.2.4. Fusion Protein Purification .......................................................................................... 20
v
2.2.5. Fusion Protein Analysis ............................................................................................... 22
2.2.5.1. SDS-PAGE .......................................................................................................................................... 22
2.2.5.2. Native Gel ............................................................................................................................................ 22
2.2.5.3. Western Blot ........................................................................................................................................ 22
2.2.5.4. Coomassie Blue Staining ..................................................................................................................... 23
2.2.5.5. BCA Assay .......................................................................................................................................... 23
2.2.5.6. Size Exclusive Column ........................................................................................................................ 23
2.2.6. Formulation Development ........................................................................................... 24
2.3. RESULTS .............................................................................................................................. 25
2.3.1. Design of Fusion Protein .............................................................................................. 25
2.3.2. Small-scale and Large-scale Production of Fusion Proteins ........................................ 27
2.3.3. Strategies to Increase Protein Productivity .................................................................. 36
2.3.4. Formulation Development of Fusion Protein for Long-term Storage .......................... 38
2.4. DISCUSSION ......................................................................................................................... 41
2.5. SUMMARY ........................................................................................................................... 42
CHAPTER 3: CHARACTERIZATION OF IN VITRO BIOLOGICAL
ACTIVITIES OF SINGLE CHAIN FC-DIMER RECOMBINANT FUSION
PROTEINS .......................................................................................................................43
3.1. BACKGROUND ..................................................................................................................... 43
3.2. MATERIALS AND METHODS ................................................................................................ 45
3.2.1. Cell Cultures ................................................................................................................. 45
3.2.2. Proteins ......................................................................................................................... 45
3.2.3. Neonatal Fc Receptor (FcRn) Binding Assay .............................................................. 46
3.2.3.1. Cell-based Binding Assay .................................................................................................................... 46
3.2.3.2. Surface Plasmon Resonance ................................................................................................................ 47
3.2.4. Nb2 Cell Proliferation Assay ....................................................................................... 49
3.2.5. Trancytosis Assay ........................................................................................................ 49
3.2.5.1. T84 Cell Differentiation ....................................................................................................................... 49
vi
3.2.5.2. Transcytosis of hGH-sc(Fc)
2
vs hGH-Fc ............................................................................................. 50
3.3. RESULTS .............................................................................................................................. 51
3.3.1. Receptor Binding Affinity in T84 Cells ....................................................................... 51
3.3.2. Surface Plasmon Resonance ........................................................................................ 54
3.3.3. Nb2 Cell Proliferation Assay ....................................................................................... 57
3.3.4. Transcytosis of Fusion Proteins ................................................................................... 59
3.4. DISCUSSION ......................................................................................................................... 61
3.5. SUMMARY ........................................................................................................................... 63
CHAPTER 4 EVALUATION OF IN VIVO PHARMACOKINETICS AND
PHARMACODYNAMICS OF SINGLE CHAIN FC-DIMER RECOMBINANT
FUSION PROTEINS .......................................................................................................64
4.1. BACKGROUND ..................................................................................................................... 64
4.2. MATERIALS AND METHODS ................................................................................................ 66
4.2.1. Animals ........................................................................................................................ 66
4.2.2. Proteins ......................................................................................................................... 66
4.2.3. Measurement of In Vivo Pharmacokinetics .................................................................. 66
4.2.3.1. hGH-sc(Fc)
2
vs sc(Fc)
2
(i.v.) ................................................................................................................ 66
4.2.3.2. hGH-sc(Fc)
2
vs hGH-Fc (i.v.) .............................................................................................................. 67
4.2.3.3. hGH-sc(Fc)
2
vs hGH-Fc (s.c.) ............................................................................................................. 67
4.2.4. Measurement of In Vivo insulin-like growth factor 1 (IGF-1) Level ........................... 68
4.2.5. Statistical Analysis ....................................................................................................... 68
4.3. RESULTS .............................................................................................................................. 69
4.3.1. Pharmacokinetics ......................................................................................................... 69
4.3.2. Pharmacodynamics ...................................................................................................... 73
4.4. DISCUSSION ......................................................................................................................... 75
4.5. SUMMARY ........................................................................................................................... 76
vii
CHAPTER 5 SUMMARY, CHALLENGES AND FUTURE PERSPECTIVES ......77
5.1. SUMMARY ........................................................................................................................... 77
5.2. CHALLENGES ...................................................................................................................... 78
5.3. FUTURE PERSPECTIVES ....................................................................................................... 80
BIBLIOGRAPHY ............................................................................................................81
viii
LIST OF TABLES
Table 1. hGH-sc(Fc)
2
in Superdex
TM
200 Increase 10/300 GL column by ÄKTA Pure . 34
Table 2. Stability of hGH-sc(Fc)
2
and hGH-Fc in PBS at 4˚C ........................................ 39
Table 3. Stability of hGH-sc(Fc)
2
.................................................................................... 40
Table 4. Apparent K
d
from Surface Plasmon Resonance at pH 6.0 ................................. 56
Table 5. Non-compartmental PK analysis of hGH-sc(Fc)
2
and sc(Fc)
2
after i.v. injection
.................................................................................................................................. 70
Table 6. Non-compartmental PK analysis of hGH-sc(Fc)
2
and hGH-Fc after i.v. injection
.................................................................................................................................. 71
Table 7. Pharmacokinetic profiles of hGH-sc(Fc)
2
and sc(Fc)
2
after s.c. injection ......... 72
ix
LIST OF FIGURES
Figure 1. Crystal structure of human IgG1-Fc. .................................................................. 3
Figure 2. Bifunctional fusion protein strategy to improve pharmacokinestics or enable
oral delivery ................................................................................................................ 7
Figure 3. Schematic structures of current Fc-fusion protein technology and their
disadvantages ............................................................................................................ 10
Figure 4. Illustration of the cloning strategy for sc(Fc)
2
with both hinges. ..................... 14
Figure 5. Illustration of the cloning strategy for sc(Fc)
2
. ................................................. 16
Figure 6. Design of sc(Fc)
2
recombinant fusion proteins ................................................ 26
Figure 7. sc(Fc)
2
production ............................................................................................. 28
Figure 8. Small-scale sc(Fc)
2
expression ......................................................................... 28
Figure 9. Expression test of hGH fusion proteins ............................................................ 29
Figure 10. hGH-sc(Fc)
2
production ................................................................................. 30
Figure 11. Identification of hGH fusion proteins ............................................................. 31
Figure 12. (A) Elusion Profile of Fc, (B) sc(Fc)
2
and (C) hGH-Fc in Superdex
TM
200
Increase 10/300 GL column by ÄKTA Pure ............................................................ 33
Figure 13. Elusion Profile of hGH-sc(Fc)
2
in Superdex
TM
200 Increase 10/300 GL
column by ÄKTA Pure ............................................................................................. 34
Figure 14. Characterization of recombinant human IgG1 Fc fragment. .......................... 35
Figure 17. A hypothetical model for utilizing FcRn-mediated recycling and transcytosis
pathways for improved protein drug delivery with sc(Fc)
2
carrier protein. ............. 43
Figure 18. Short incubation uptake assay of hIgG1-Fc and sc(Fc)
2
to determine FcRn
binding ...................................................................................................................... 53
x
Figure 19. SPR Sensorgams for binding of serial twofold dilutions of Fc, sc(Fc)
2
, hGH-
sc(Fc)
2
, and hGH-Fc to immobilized shFcRn at pH 6.0 ........................................... 55
Figure 20. SPR Sensorgams for binding of different concentrations of Fc to immobilized
shFcRn at pH 7.4. ..................................................................................................... 55
Figure 21. Nb2 cell proliferation stimulated by hGH fusion proteins ............................. 58
Figure 22. Transcytosis assay of hGH-sc(Fc)
2
and hGH-Fc through T84 cells formed
epithelial barrier ........................................................................................................ 60
Figure 23. Pharmacokinetic profiles of hGH-sc(Fc)
2
and sc(Fc)
2
after i.v. injection ...... 70
Figure 24. Pharmacokinetic profiles of hGH-sc(Fc)
2
and hGH-Fc after i.v. injection. ... 71
Figure 25. Pharmacokinetic profiles of hGH-sc(Fc)
2
and hGH-Fc after s.c. injection. ... 72
Figure 26. IGF-1 plasma levels after subcutaneous injection of hGH-fusion proteins in
male CF1 mice. ......................................................................................................... 74
xi
ABBREVIATIONS
CHO Chinese hamster ovary cells
CV column volume
FcRn neonatal Fc receptor
HEK human embryonic kidney cells
hGH human growth hormone
IGF-1 insulin-like growth factor 1
IgG immunoglobulin G
i.v. intravenous administration
Nb2 Rat T-lymphoma cells
NCA non-compartmental analysis
PTM post-translational modifications
RU response unit
s.c. subcutaneous administration
sc(Fc)
2
CH2-CH3-(G
4
S)
13
-CH2-CH3 fusion protein
SEC size exclusion chromatography
SPR surface plasmon resonance
TEER transepithelial electrical resistance
TFF tangential flow filtration
TMDD target-mediated drug disposition
PD pharmacodynamics
PK pharmacokinetics
xii
ABSTRACT
Fc fusion protein technology has been successfully used to generate long-acting
forms of several protein therapeutics. In this dissertation, we describe the design of a novel
single chain Fc-based drug carrier, sc(Fc)
2
, and evaluated sc(Fc)
2
-mediated delivery using
a therapeutic protein with a short plasma half-life as the drug cargo. This novel Fc-based
drug carrier was designed to contain two Fc domains recombinantly linked via a flexible
linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge
region was omitted to further stabilize it against proteolysis and reduce FcγR-related
effector functions. The resultant sc(Fc)
2
candidate preserved the FcRn binding. sc(Fc)
2
-
mediated delivery was then evaluated using hGH as the drug cargo. This novel carrier
protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity
compared to the traditional Fc-based drug carrier. Research focusing on the non-invasive
delivery of therapeutic proteins using the transcytosis pathway mediated by FcRn to
facilitate uptake showed the potential value of Fc in developing novel oral therapies. Our
novel sc(Fc)
2
fusion protein, showed better transcytosis efficiency than traditional Fc
fusion protein in cell-based model system. However, due to the complexity of the transport
mechanism and the obstacles during oral administration, more efforts are needed to
optimize this delivery system. Taken together, sc(Fc)
2
technology has the potential to
greatly advance and expand the use of Fc-technology for improving the pharmacokinetics
of therapeutic proteins and bioavailability in protein oral delivery.
CHAPTER 1 INTRODUCTION
1.1. Protein Therapeutics
Therapeutic proteins have claimed their importance during the last three decades.
Compared with small molecule drugs, protein therapeutics are usually exhibiting complex
set of functions that cannot be mimicked by simple chemical compounds, and have a high
specificity that is often less potential to interfere with normal biological processes and
cause adverse effects (Leader, Baca, and Golan 2008). The structure complexity of proteins
not only brings those advantages, but also leads to challenges in formulation and delivery.
The high molecular mass of therapeutic proteins substantially reduces their permeability
across biological barriers, such as mucosal membranes, and their protein nature makes
them vulnerable to enzymatic degradation, which limits the primary mode of
administration to injection (Mitragotri, Burke, and Langer 2014).
The development of recombinant technology provides the possibility to combine
the functions of different protein domains to generate novel protein therapeutics. By fusing
the genes of two proteins together, bifunctional fusion proteins are developed to achieve
improved pharmacokinetic and pharmacodynamic properties, delivery or targeting
purposes (Chen, Zaro, and Shen 2012).
2
1.2. Neonatal Fc Receptor (FcRn) and Immunoglobulin G (IgG)
1.2.1. Physiological Properties and Functions
FcRn was first described for its role in passively transporting IgG from the mother’s
milk to the fetus across the proximal small intestine in rodents (Roopenian and Akilesh
2007). Although FcRn is called a ‘neonatal’ receptor, its expression is also found to be
significant in human adult tissues, such as vascular endothelium, endothelial cells of the
central nervous system and the choroid plexus, eyes, intestines, kidneys and lungs (Xu et
al. 2013; Dickinson et al. 1999; Kuo et al. 2010; Roopenian and Akilesh 2007). These
discoveries make it promising to use FcRn in human therapeutic development.
FcRn, a MHC class I-like transmembrane protein, is a heterodimeric molecule
consisting of a heavy chain containing three extracellular domains (α1, α2 and α3) and a
non-covalently associated common β2-microglobulin subunit (Rath et al. 2015).
The IgG Fc fragment is a homodimer with two C-terminal domains (CH2 and CH3) of the
heavy chains and a flexible hinge. The two chains form a homodimer via two disulfide
bonds in the hinge region and non-covalent interactions between the CH3 domains (Liu et
al. 2008). Crystal structures of Fc homodimer show that CH3 dimer aggregates and CH2
domains are widely separate from each other (Figure 1) (Huber et al. 1976). There is one
glycosylation site at each of the CH2 domains. The crystal structure of an FcRn/Fc complex
revealed FcRn dimers are bridged by homodimeric Fc molecules to create an oligomeric
array with two receptors per Fc (Huber et al. 1993; Burmeister, Huber, and Bjorkman
1994), consistent with the 2:1 FcRn:Fc stoichiometry observed in solution (Martin and
Bjorkman 1999; Sánchez, Penny, and Bjorkman 1999). It indicates a natural preference to
3
the dimeric structure of Fc. However, 1:1 FcRn-Fc complex was also observed in some
studies (Popov et al. 1996; Sánchez, Penny, and Bjorkman 1999). FcRn binds to the CH2–
CH3 hinge region in Fc that is distinct from the binding sites of FcγRs or the C1q
component of complement (Roopenian and Akilesh 2007). The most critical binding sites
proved by site-directed mutagenesis are His 310, His 435 and Ile 253, and His 433. Tyr
436 also play a role in the pH dependent binding to FcRn (Raghavan et al. 1995; Medesan
et al. 1997). The protonation of several histidine residues allows the formation of salt
bridges with acidic residues on FcRn, which explains the strict pH dependency of FcRn-
Fc binding (Rath et al. 2015). Mutations of His 310, Arg 311, His 435, and His 436 on Fc
and Glu 117, Glu 132, and Asp 137 on FcRn, substantially lower the binding affinity either
individually or in conjunction with other residues (Martin et al. 2001).
Figure 1. Crystal structure of human IgG1-Fc.
Yellow and pink ribbon and white tube show the
protein structure. Ball and sticks show the
glycosylation at CH2 domain (Krapp et al. 2003).
4
The Fc part of IgG binds FcRn with high affinity at an acidic pH (<6.5), which is
similar to the normal luminal pH of the duodenum and jejunum of neonatal mice, but the
binding affinity is negligible at physiological pH (7.4) (Raghavan et al. 1995; Rodewald
1976). FcRn plays an important role in IgG homeostasis throughout life by prolonging the
half-life of IgG antibodies in the circulation. This protection model was supported by the
studies showing that the half-life of IgG was dramatically decreased in FcRn-knockout
mice (Israel et al. 1996), and that FcRn affinity related mutations on IgG affected its serum
persistence (Ghetie et al. 1997).
In addition, FcRn also contributes to the serum half-life of albumin as it does for
IgG (Chaudhury et al. 2003). Both albumin and IgG bind to FcRn following a similar pH
dependent manner, but the binding sites are different and the binding of albumin and IgG
can be superimposed due to distinctive binding mechanisms (Anderson et al.).
While sequence homology of rat and mouse FcRn is very high, about 98%, rat and
human FcRn sequences are only 65% identical, which is consistent with the different
binding pattern of rodent and human FcRn (Kuo et al. 2010). Human FcRn only bind to
human, rabbit and bovine IgG, but rodent FcRn are able to bind IgG from various species
(Ober et al. 2001).
1.2.2. Receptor-mediated Recycling and Transcytosis Pathway
The major function of FcRn involves recycling and transcytosis of IgG. When there
is a pH difference, like in small intestines, FcRn favors the binding to IgG due to the low
luminal pH, and then transport from the apical side to the basolateral surface. The pH of
blood, which is approximately 7.4, allows the dissociation of IgG.
5
When there is no pH difference, like in vascular endothelial cells, IgG can be
internalized by fluid-phase processes such as pinocytosis. After internalization, they can
bind to FcRn that presents in the early endosomes. The acidic pH in the endosomes allows
interaction between FcRn and the Fc region of those IgG molecules. Upon sorting, some
FcRn-Fc complexes will go to the recycling endosome and finally get released back to the
blood. The physiologic pH of the blood facilitate the dissociation. However, it is still under
investigation that whether the subsequent event after binding to FcRn is recycling or
transcytosis (Kuo et al. 2010). This bidirectional transcytosis mediated by FcRn across the
human intestinal epithelium has been shown both in vivo and in vitro (Dickinson et al. 1999;
Claypool et al. 2004; Tzaban et al. 2009).
1.3. Fc Fusion Proteins
The strategy of engineering Fc conjugates to use the recycling pathway of FcRn to
prolong plasma half-life has been applied in many therapeutic designs. After being
engineered to include an Fc-domain, those therapeutic agents gain increased affinity
toward FcRn so that their pharmacokinetics are improved without compromising the
specificity of the therapeutic moiety. This allows the drugs to be given less frequently and
provides a better safety profile. Enbrel
®
, an Fc fusion to the extracellular domain of tumor
necrosis factor (TNF) α receptor, is the first recombinant Fc fusion protein for clinical
translation treating rheumatoid arthritis with a mean half-life of 4 days depending on
patient population and dosing regimen. The achieved scientific and economic success of
Enbrel
®
leads to more research efforts focusing on Fc fusions, the most widely used
technique to extend half-lives of small therapeutically promising proteins and peptides.
6
Furthermore, the fact that FcRn can transcytose across mucosal surfaces also allows
for a novel non-invasive delivery of protein therapeutics to the circulation. Attempts have
been made to develop inhaled protein therapeutics, such as hIgG1-Fc conjugated
erythropoietin (Epo), follicle-stimulating hormone, and interferon-α and -β (Bitonti et al.
2006). Compared with Fc fusion dimers, Fc fusion “monomers”, consisting of a dimer of
an Fc with a monomeric therapeutic protein, were found to be superior in transport
efficiency and pharmacokinetics than dimers, in which each chain was fused with one
therapeutic protein (Bitonti et al. 2006; Bitonti et al. 2004). The inhaled Epo-Fc monomer
showed similar bioavailability as subcutaneously injected Epo in non-human primates
(Bitonti et al. 2006). In addition to delivery through the lung, transport of Fc fusion proteins
across intestinal mucosa has also been an interesting field to investigate. Oral protein drugs
are convenient and patient friendly, but there are many significant challenges in the
development of oral dosage forms, including poor lipid solubility and reduced stability,
leading to poor uptake across epithelial barrier of gastrointestinal tract (Amet, Wang, and
Shen 2010). Follicle stimulating hormone-Fc fusion protein gave a half-life of 60 h in orally
dosed newborn rats (Low et al. 2005). The pharmacokinetics of Factor IX-Fc was also
examined in 10-day-old mice after a single oral dose (Bitonti et al. 2006). However, due to
the complexity and limited knowledge of the transport mechanism, more efforts are needed
to optimize this delivery system.
7
Figure 2. Bifunctional fusion protein strategy to
improve pharmacokinestics or enable oral delivery
8
CHAPTER 2: DESIGN AND PRODUCTION OF SINGLE CHAIN Fc-
DIMER RECOMBINANT FUSION PROTEINS
2.1. Background
To date, there are nine FDA-approved Fc-fusion proteins, and many others are at
different stages of clinical and preclinical development. A majority of Fc-fusion protein
drugs consist of a protein drug linked to the N-terminal of an Fc domain that forms a drug-
Fc homodimer (“(drug-Fc)
2
”) along with drug-Fc monomer impurities (Figure 3). In the
(drug-Fc)
2
homodimer configuration, the protein drug domains are adjacent to each other,
often leading to their physical instability and/or decreased bioactivity (Dumont et al. 2006;
Fast et al. 2009). Further, many large protein drugs are not suitable candidates as they
cannot be stably expressed (Peters et al. 2013).
In order to overcome these disadvantages, “Monomeric” Fc-fusion proteins
(Figure 3), containing a protein drug linked to only one of the two Fc domains (“drug-
(Fc)
2
”) have recently been tested and clinically approved (eg. Alprolix
®
and Eloctate
®
).
Studies have shown that these Monomeric Fc-fusion proteins have improved half-lives
and/or bioactivity compared to their homodimeric counterparts (Dumont et al. 2006).
However, their main limitation is production, which requires dual expression plasmids
containing the drug-Fc and the Fc sequences. This production protocol generates a mixture
of multiple fusion products including (drug- Fc)
2
, drug-(Fc)
2
and (Fc)
2
, creating issues of
impurity. Further, formation of homodimers (i.e. (drug-Fc)
2
and (Fc)
2
) are favored over the
Monomeric drug-(Fc)
2
products, resulting in low production yields and instability (Carter
2001). Due to these limitations, this promising technology cannot be applied to all protein
drugs.
9
Current Fc-fusion proteins maintain the hinge region sequence of IgG to link the
two Fc domains via disulfide bonds (Figure 3). Other than linking two Fc domains, this
region is not important for Fc function but introduces potential instability due to disulfide
reduction and enzymatic degradation at several protease cleavage sites present in the hinge
region (Ryan et al. 2008). It may also contribute to protein aggregation due to disulfide
bond scrambling (Wang 1999). Additionally, the lower hinge of IgG Fc plays a crucial role
in its binding to Fcγ receptors (FcγR), initiating effector functions that are out of the
designed mechanism of action (Wines et al. 2000; Shields et al. 2001).
10
Figure 3. Schematic structures of current Fc-fusion protein technology and
their disadvantages. A majority of FDA-approved Fc-fusion proteins exist
as a Fc-homodimer. (A) In this configuration, steric hindrance between the
protein domains leads to physical instability, decreased bioactivity and/or
limitations in the size of protein that can be accommodated. Monomeric Fc
fusion proteins have been recently developed to overcome these
limitations. Both Fc-homodimers and Monomeric Fc fusion proteins are
(B) susceptible to instability in the hinge region via protease digestion and
disulfide reduction, and (C) generate several impurities during recombinant
production.
11
Although most therapeutic proteins on the market are produced by mammalian
Chinese hamster ovary (CHO) and murine myeloma (NSO, Sp2/0) cell lines, there has been
a recent shift towards the use of human cell lines (Dumont et al. 2016). One of the most
important advantages of using human cell lines lies on the full human post-translational
modifications (PTMs). CHO and other murine cell lines are able to add potentially
immunogenic non-human PTMs to therapeutic proteins, such as galactose-α1,3-galactose
and N-glycolylneuraminic acid. Besides, human cell lines are competitively easy to use in
production compared with CHO cells. The only possible disadvantage of using human cell
lines is the potential for human-specific viral contamination, but this risk can be mitigated
by multiple viral inactivation or clearance steps (Dumont et al. 2016).
HEK293 cells are easily transfected and are highly efficient at protein production.
HEK293T cell line, a modified version of HEK293, expresses the simian virus 40 large T
antigen that binds to SV40 enhancers of expression vectors resulting in increased protein
expression level. HEK293 cells have been widely used to produce proteins for research use
for many years, and several therapeutic proteins produced in HEK293 cells, including
drotrecogin α (XIGRIS®; Eli Lilly), recombinant factor IX Fc fusion protein (rFIXFc;
Biogen), recombinant factor VIII Fc fusion protein (rFVIIIFc; Biogen), and dulaglutide
(TRULICITY®; Eli Lily), have been approved by the FDA or the EMA (Dumont et al.
2016). Additionally, glycosylated Fc conjugates produced by mammalian systems showed
better thermal stability and can endure exposure to low-pH better than aglycosylated Fc
conjugates expressed in E. coli (Cao et al. 2014).
12
2.2. Materials and Methods
2.2.1. Cell Cultures
The human embryonic kidney cell line HEK293 and HEK293T were purchased
from ATCC (Manassas, VA). Cell culture media were all from Mediatech (Manassas, VA).
HEK293 and HEK293T cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) with 2.5 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) and
50 units/mL of penicillin/50 µg/mL streptomycin (Invitrogen). HEK 293 and HEK 293T
cells were trypsinized with 0.05% trypsin-EDTA for regular passage. The doubling time
of both cell lines was about 24 h. All mammalian cell lines were maintained in a humidified
incubator at 37°C with 5% CO
2
.
2.2.2. Plasmid Construction
2.2.2.1. pcDNA3.1+_sc(Fc)
2
The commercial plasmid pFUSE-hIgG1-Fc2 (Invivogen, San Diego, CA) was used
as the PCR template of native hIgG1-Fc sequence, and pcDNA3.1+ (Invitrogen, Carlsbad,
CA) was used as the expression vector. Two sets of primers were designed to engineer
expression plasmid of the first sc(Fc)
2
with hinges. The underline highlights the restriction
enzyme recognition sites designed for sub-cloning purpose.
Primers for PCR of IL2-MCS-hIgG1-Fc:
Forward (HindIII): 5’-TTTAAGCTTGCCACCATGTACAGGATGCAA-3’
Reverse (XhoI): 5’-ATTTCTCGAGTTTACCCGGAGACAGGGAGA-3’
Primers for PCR of Fc:
13
Forward (XhoI-BamHI):
5’-TAAACTCGAGGGATCCGACAAAACTCACACATGCCCA-3’
Reverse (XbaI): 5’-ATTTCTAGATCATTTACCCGGAGACAGGGA-3’
IL2-MCS-hIgG1-Fc sequence (MCS: multiple cloning site) in pFUSE-hIgG1-Fc2
was amplified by PCR using Q5 High High-Fidelity DNA Polymerase (New England
Biolabs, Ipswich, MA), digested by restriction enzymes and ligated at HindIII and XhoI
sites of pcDNA3.1+ plasmid. Similarly, Fc sequence with stop codon was inserted between
XhoI and XbaI sites, and a BamHI site after the XhoI site was introduced for linker
sequence insertion by this cloning step. The customized plasmid containing linker
sequence coding (G
4
S)
8
with codon optimization for human cell production and restriction
enzyme cutting sites for sub-cloning was purchased from Genscript. (G
4
S)
8
sequence was
inserted between XhoI and BamHI sites of the engineered pcDNA3.1+ plasmid to yield the
expression plasmid coding IL2-Fc-(G
4
S)
8
-Fc. The cloning strategy is illustrated in Figure
4.
14
Figure 4. Illustration of the cloning strategy for sc(Fc)
2
with both hinges.
15
The stop codon TGA and XbaI site at the end of the second Fc sequence
(TGATCTAGA) created a substrate sequence for dam methylase. The E.coli strain used in
cloning was DH5α, a dam+ strain, so the cleavage with XbaI was blocked because of
methylation at the cutting site. The primers for 2
nd
Fc (no hinge) was used to remove the
hinge sequence in the second Fc and modify the restriction enzyme sites at the end of the
fusion protein sequence to allow future insertions at the C-terminal of this carrier protein.
The primers for 1
st
Fc (no hinge) was used to remove the hinge sequence and the unused
MCS in the first Fc. The cloning strategy is summarized in Figure 5.
Primers for 2
nd
Fc (no hinge):
Forward (BamHI): 5’-TAAAGGATCCGCACCTGAACTCCTGGGG-3’
Reverse (XbaI-TGA-ApaI):
5’-TTGGGCCCTCATCTAGATTTACCCGGAGACAGGGAGA-3’
Primers for 1
st
Fc(no hinge):
Forward (EcoRI): 5’-AAAAGAATTCGGCACCTGAACTCCTGGGG-3’
Reverse (XhoI): 5’-ATTTCTCGAGTTTACCCGGAGACAGGGAGA-3’
16
Figure 5. Illustration of the cloning strategy for sc(Fc)
2
.
17
To adding more (G
4
S) repeats after the initially inserted (G
4
S)
8
linkers, two
different strategies were used. By applying long forward linkers, 5’-
AAAGGATCCGGCGGCGGCGGCAGCGCACCTGAACTCCTGGGG-3’ and 5’-
AAAGGATCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGCACCTGAAC
TCCTGGGG-3’, sc(Fc)
2
with (G
4
S)
9
or (G
4
S)
10
linkers were engineered. To generate
longer linker sequences, equal molar commercially synthesized sense and antisense DNA
sequence of (G
4
S)
3
, 5’-
AAAGGATCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGC
GGATCCAAA-3’ and 5’-
TTTGGATCCGCCGCCGCCGCTGCCGCCGCCGCCGCTGCCGCCGCCGCCG
GATCCTTT-3’, was annealed in annealing buffer (New England Biolabs, Ipswich, MA)
according to the following protocol:
95 °C 5 min
93-84 °C 3 min/°C
83 °C 10 min
82-70 °C 3 min/°C
4 °C ∞
The resulting double strand DNA was inserted into the BamHI site with different
copy numbers. sc(Fc)
2
with (G
4
S)n (n=11-14) linkers were obtained by this cloning process.
General cloning protocol:
1. The plasmids and inserts obtained from PCR or annealing were digested by
the corresponding restriction enzymes.
18
2. The linearized plasmids were separated by 1-1.5% agarose gel
electrophoresis, and then extracted from the gel by using the Qiagen Gel
Extraction Kit.
3. The DNA concentrations were measured by NanoDrop
TM
One Microvolume
UV-Vis Spectrophotometers (Thermo Scientific).
4. Linearized vector and insert were ligated by T4 DNA ligase (New England
Biolabs, Ipswich, MA) for 1-2h at room temperature or 16°C overnight.
5. Transformation was performed with Z-Competent
TM
(DH5α) cells (Zymo
Research, Irvine, CA).
6. Plasmid sequences were all confirmed by sequencing using T7 and BGHR
primers.
2.2.2.2. pcDNA3.1+_hGH-sc(Fc)
2
The pcDNA3.1+_sc(Fc)
2
was double digested with HindIII and EcoRI restriction
enzymes (New England Biolabs, Ipswich, MA) to replace the IL2 secretion signal sequence
with the hGH sequence, which has its own secretion signal, allowing for the expressed
fusion protein to be collected from the culture medium.
2.2.2.3 pcDNA3.1+_sc(Fc)
2
-hGH
The pcDNA3.1+_sc(Fc)
2
was digested with XbaI restriction enzymes (New
England Biolabs, Ipswich, MA) to insert the hGH sequence at the end of sc(Fc)
2
sequence.
19
2.2.2.4. pcDNA3.1+_hGH-Fc
The pcDNA3.1+ expression vector harboring hGH-transferrin was double digested
with XhoI and XbaI restriction enzymes (New England Biolabs, Ipswich, MA) to replace
the transferrin fragment with the Fc fragment.
2.2.2.5. pcDNA3.1+_IHH
Fc mutant (I253A/H310A/H435A, IHH) showed no binding to FcRn at both
physiology and acidic pH (Qiao et al. 2008). Three targeted amino acid substitutions on
the Fc fragment was introduced one by one using Q5 Site-Directed Mutagenesis Kit (New
England Biolabs, Ipswich, MA) with the following primer pairs:
I253A_F: 5’-CACCCTCATGgccTCCCGGACCC-3’
I253A_R: 5’-TCCTTGGGTTTTGGGGGG-3’
H310A_F: 5’-CACCGTCCTGgccCAGGACTGGC-3’
H310A_R: 5’-AGGACGCTGACCACACGG-3’
H435A_F: 5’- TCTGCACAACgccTACACGCAGAAGAGC-3’
H435A_R: 5’- GCCTCGTGCATCACGGAG-3’
2.2.3. Fusion Protein Production
Small-scale production was carried out in 6-well plates, while large-scale
production used T-175 flasks. HEK293 cells were seeded two days before transfection and
reached ~80% confluence at the time of transfection. Fusion proteins were produced in
serum-free CD293 medium (Life Technologies, Grand Island, NY) with 4 mM L-
glutamine by transient transfection of HEK293 cells using polyethylenimine (PEI)
20
(Polysciences, Warrington, PA). For one T-175 flask, 66 µg DNA in 1.5 mL 150 mM NaCl
was vigorously mixed with 260 µl PEI (0.646 g/L) in 1.5 mL 150 mM NaCl. The mixture
was left on the bench for 15 min before being diluted in DMEM. After 6 h transfection,
medium was replaced by serum-free CD 293 medium supplemented with 4 mM L-
glutamine. The medium was harvested at post-transfection day 4 and day 7. The harvested
medium containing the desired protein was centrifuged at 3000 g for 20 min at 4°C
immediately after collection to remove cell debris.
2.2.4. Fusion Protein Purification
The combined media containing target fusion proteins were concentrated and
buffer-exchanged to Buffer A (0.02 M NaH
2
PO
4
⋅H
2
O, 0.15 M NaCl, pH 7.0) using a
tangential flow filtration system (TFF) (Millipore, Billerica, MA). Protein A-
Sepharose
®
4B (Sigma, St. Louis, MO) was used to purify the expressed Fc proteins from
the TFF concentrated media. The target Fc fusion proteins were eluted from Protein A
column by Buffer B (0.2 M Na
2
HPO
4
, 0.1 M citric acid, pH 3.2-3.5).
Standard protocol for Protein A column purification:
1. Pour suspension of resin into column. Allow column to flow as it is settling.
After it has settled, wash with 20 column volumes (CV) of Buffer A.
2. Apply sample after centrifugation. Collect the flow-through in a tube, and
reapply the flow-through once to maximize binding.
3. Wash with 10 CV of buffer A.
4. Elute with 10 CV of Buffer B. Collect fractions. Neutralize the eluate with
21
150 uL 1 M Tris-HCl, pH 9.0 per 250 ul elute.
5. Assay the eluates.
6. Re-equilibrate the column with 20-30 CV of Buffer A.
7. Remove precipitated or denatured substances by washing the column with 2
CV of 6 M guanidine hydrochloride. Immediately re-equilibrate with at least 5
CV of Buffer A. Remove strongly bound hydrophobic proteins, lipoproteins
and lipids by washing the column with 70% ethanol and let it stand for 12
hours. Re-equilibrate with at least 5 CV of Buffer A.
8. Store resin in Buffer A with a preservative (20% ethanol) at 4°C.
The combined elutions after column purification were concentrated by
ultrafiltration using Microsep
TM
advanced centrifugal devices with molecular weight cutoff
(MWCO) of 10kD (Pall Corportation, Ann Arbor, MI). The concentrated protein solutions
were dialyzed (MWCO: 12-14 kD, Spectrum Laboratories, Rancho Dominguez, CA) in
freshly prepared PBS, pH 7.4 at 4°C. The dialyzed protein solutions were filtered through
a 0.22 µm membrane, and stored at 4°C for future analysis.
22
2.2.5. Fusion Protein Analysis
2.2.5.1. SDS-PAGE
Purified protein samples were mixed with reducing or non-reducing protein loading
dye, boiled for 5-10 min, and then loaded onto standard polyacrylamide gels (10-13%).
2.2.5.2. Native Gel
Polyacrylamide gels without SDS were used to examine native protein samples
mixed with bromophenol blue dye. Electrophoresis was carried out in native
electrophoresis buffer (3.0g Tris base, 14.4g glycine, adjust volume to 1L with ddH
2
O,
final pH = 8.3).
2.2.5.3. Western Blot
The protein samples were resolved by PAGE, and transferred to 0.2 μm
polyvinylidene fluoride membranes. The membrane was blocked with 5% non-fat milk
before primary antibody incubation. For Fc fragment detection, the membranes were
incubated with goat anti-hIgG (Fc specific) antibody (1:3000 dilution) (Sigma, St. Louis,
MO) at 4°C overnight. Then, the membranes were washed with Tris-buffered saline
containing 0.05% Tween-20 (TBS-T) for 3 times, and incubated with horseradish
peroxidase (HRP)-conjugated anti-goat secondary antibody (1: 10,000 dilution) (Bio-Rad,
Hercules, CA) at room temperature for 1 h. After washing with 0.05% TBS-T buffer for 3
times, the blots were incubated with ECL western blotting detection reagents (GE
Healthcare, UK) and imaged by ChemiDoc
TM
Touch. The band intensities were quantified
and analyzed by ImageLab
TM
software (Bio-Rad, Hercules, CA).
23
For hGH fusion proteins, the membranes were incubated with goat anti-hGH
specific antibody (1:1000 dilution) (R&D Systems, Minneapolis, MN) at 4°C overnight
followed by incubation with HRP-conjugated anti-goat secondary antibody (1: 10,000
dilution) at room temperature for 1 h. The immunoreactive bands were detected by ECL
or
ECL Prime western blotting detection reagents.
2.2.5.4. Coomassie Blue Staining
The PAGE gels were stained with Coomassie blue staining buffer (0.1% G-250 or
R-250, 10% acetic acid, 40% methanol) for 0.5-1 h, and de-stained with primer destain
buffer (10% acetic acid, 40% methanol) for 1 h followed by secondary de-stain buffer (10%
acetic acid, 10% methanol) overnight at room temperature. The gels were imaged by
ChemiDoc
TM
Touch, and the band intensities were quantified and analyzed by ImageLab
TM
software.
2.2.5.5. BCA Assay
The concentrations of purified protein samples were determined by Pierce
TM
BCA
or microBCA protein assay kits (Thermo Scientific, Rockford, IL) according to
manufacturer’s instructions.
2.2.5.6. Size Exclusive Column
Protein A purified protein samples were analyzed or further purified by ÄKTA pure
(GE Healthcare) and Superdex
TM
200 Increase 10/300 GL column using PBS as the fluid
phase. OD
280
was measured for each fraction to obtain the elusion profile.
24
2.2.6. Formulation Development
The fusion proteins were analyzed for degradation following storage in two
different buffer systems, PBS (140 mM NaCl, 10 mM Na
2
HPO
4
) and mannitol phosphate
buffer (4 mM K
2
HPO
4
, 1 mM KH
2
PO
4
, 88 mM mannitol), at different temperatures.
Preparation of 1X PBS (2L):
16.24 g NaCl
2.84 g Na
2
HPO
4
QS adjust volume to 1 L with ddH
2
O
Adjust pH to 7.4 at room temperature
Preparation of 10X mannitol phosphate buffer:
Prepare 50 mM K
2
HPO
4
in 200 ml ddH
2
O by dissolving 1.8 g K
2
HPO
4
in 200 ml
ddH
2
O (0.05 M x 174.2 g/mol x 0.2 L = 1.8 g). Prepare 50 mM KH
2
PO
4
in 200 ml
ddH
2
O by dissolving 1.36 g KH
2
PO
4
in 200 ml ddH
2
O (0.05 M x 136 g/mol x 0.2 L =
1.36 g). Mix 50 mL of KH
2
PO
4
with 200 mL K
2
HPO
4
. Prepare 880 mM mannitol (10x)
in 10x phosphate buffer by dissolving 40.1 g of mannitol in 250 mL 10x phosphate buffer
(0.88 M x 0.25L x 182.17 g/mol = 40.1 g).
10X buffer mannitol phosphate buffer was diluted at 1:10 ratio with ddH
2
O, and
the pH of the 1X buffer was adjusted to 7.32 at room temperature. Purified fusion protein
solutions were buffer exchanged into different storage buffer systems by dialysis.
25
2.3. Results
2.3.1. Design of Fusion Protein
In this study, we have designed a novel type of Fc fusion protein by using a long,
flexible glycine-serine (GS) linker (Chen, Zaro, and Shen 2013) to link two Fc chains, with
the hinge sequence removed, to create a single chain Fc-dimer, sc(Fc)
2
. A flexible, rather
than a rigid, linker was used to allow the two Fc domains to interact properly with each
other. Linkers can be classified into three different categories: flexible, rigid, and cleavable
(Chen, Zaro, and Shen 2013). Only a flexible linker with enough length can allow the two
Fc moieties to form proper interaction with each other. Flexible linkers are generally
composed of small, non-polar or polar residues such as Gly, Ser and Thr, among which the
(G
4
S)n linker is the most commonly used. The addition of a polar residue serine reduces
linker-protein interactions and preserves protein functions compared with polyglycine
linkers. This type of linker is utilized in a variety of fusion proteins, including the well-
established single chain antigen binding proteins (i.e. scFv) (Chen, Zaro, and Shen 2013;
Shen et al. 2008). Different lengths (n = 8-14) of (G
4
S)n were used to link two Fc chains
to generate a single chain Fc-dimer proteins (Figure 6).The use of this novel design allows
for the advantages of a monomeric Fc-fusion protein, without the issues of production
impurities and requirement of the hinge region.
26
Figure 6. Design of sc(Fc)
2
recombinant fusion proteins. (A) Plasmids coding sc(Fc)
2
.
were constructed in pcDNA3.1+ vector. (B) Schematic structure of sc(Fc)
2
.
A
B
CH2 –
linker –
CH3 –
27
2.3.2. Small-scale and Large-scale Production of Fusion Proteins
HEK293 cell line was used for fusion protein production. In a small-scale
expression test of sc(Fc)
2
with various linkers, the expression levels increased with the
increasing linker length, where the highest expression level was achieved when n = 12 or
13 (Figure 7A). The small molecular weight impurity, which was positive in anti Fc-
Western blot, did not increase after 7-day incubation with the conditioned medium of
HEK293 cell culture, suggesting it was a product generated during the intracellular process
of the fusion protein rather than the degradation in the medium. Based on band intensity,
the calculated percentage of monomer impurity showed that the (G
4
S)
13
linker provided
the highest expression purity as well as highest expression level. The phenomenon was
confirmed in a separate expression test (Figure 8). Therefore, the (G
4
S)
13
linker was
selected for further use in all of the subsequent studies for the sc(Fc)
2
fusion proteins,
including both sc(Fc)
2
and hGH-sc(Fc)
2
.
sc(Fc)
2
, single chain-Fc dimer with a (G
4
S)
13
linker, was expressed in large-scale
and purified by Protein A affinity chromatography (Figure 7B). The dominant band
showed > 80% abundance. Due to the glycosylation on the CH2 domains, the apparent
molecular weight of sc(Fc)
2
under reducing condition shown on the SDS-PAGE gel was
higher than its calculated molecular weight (54 kDa).
28
Figure 7. sc(Fc)
2
production. Conditioned media from transiently transfected HEK293
cells was collected and (A) analyzed by non-reducing SDS-PAGE and Western blot
probed with goat anti-hIgG Fc specific antibody (1:3000 dilution) (B) sc(Fc)
2
(n=13)
purified by Protein A-Sepharose® 4B followed by reducing SDS-PAGE with
Coomassie blue staining.
(G
4
S)
n
linker, n = 8 9 10 11 12 13 14
55 kDa –
36 kDa –
A
55 kDa –
36 kDa –
B
0
200000
400000
600000
800000
1000000
1200000
11 12 13 14
Adj. Vol./INT*mm2
number of (G
4
S)repeat
Intact Protein Yield
0%
2%
4%
6%
8%
10%
12%
14%
11 12 13 14
number of (G
4
S)repeat
Impurity%
Figure 8. Small-scale sc(Fc)
2
expression. Conditioned media from
transiently transfected HEK293 cells was collected and analyzed by non-
reducing SDS-PAGE and Western blot probed with goat anti-hIgG Fc
specific antibody (1:3000 dilution).
29
Human growth hormone (hGH) was fused to sc(Fc)
2
to evaluate the protein drug
delivery properties of this novel carrier protein. The same expression system using vector
pcDNA3.1+ and the HEK293 cell line was adapted to express hGH fusion proteins. hGH-
sc(Fc)
2
and sc(Fc)
2
-hGH were expressed in small-scale, and the collected media were
examined by reducing SDS-PAGE followed by Western Blotting. The expression level of
hGH-sc(Fc)
2
was about 3-fold higher than that of sc(Fc)
2
-hGH based on band intensity
measurement (Figure 9), so fusion at N-terminal of sc(Fc)
2
was chosen for further
functional studies.
Figure 9. Expression test of hGH
fusion proteins. Anti-hGH antibody
recognized the target bands on the
Western blot membrane of the
expression samples of hGH-sc(Fc)
2
and sc(Fc)
2
-hGH. The expression
level of hGH- sc(Fc)
2
was about 3-
fold higher than that of sc(Fc)
2
-
hGH.
1: hGH-sc(Fc)
2
2: sc(Fc)
2
-hGH
1 2
95 kDa-
72 kDa-
55 kDa-
43 kDa-
34 kDa-
26 kDa-
30
Both of hGH-sc(Fc)
2
and hGH-Fc fusion proteins were expressed in large-scale and
purified with Protein A affinity chromatography. The purity of hGH-sc(Fc)
2
, calculated
based on Coomassie blue stained gels under reducing condition, was over 80% (Figure
10A).
Figure 10. hGH-sc(Fc)
2
production. (A) Conditioned media from transiently
transfected HEK293 cells was collected and purified by Protein A-Sepharose
®
4B
followed by reducing or non-reducing SDS-PAGE with Coomassie blue staining.
(B) Schematic structure of hGH-sc(Fc)
2
.
Reducing Non-reducing
55 kDa-
72 kDa-
43 kDa-
95 kDa-
hGH-sc(Fc)
2
hGH-Fc
A B
Fc –
linker –
hGH –
N-terminal
C-terminal
31
The identity of both fusion proteins was determined by the recognition of anti-hGH
and anti-hIgG (Fc specific) antibodies in Western blot (Figure 11).
Figure 11. Identification of hGH fusion proteins. Both anti-hIgG
(Fc specific) antibody and anti-hGH antibody recognized the
target bands on the Western blot membrane of purified samples
of hGH-sc(Fc)
2
and hGH-Fc.
1&3: hGH-sc(Fc)
2
2&4: hGH-Fc
anti-Fc anti-hGH
1 2 3 4
32
Size exclusion chromatography (SEC) was able to remove the small amount of
impurities in the Protein A purified stock of each recombinant proteins. As expected, in the
SEC column, recombinant Fc fragment of IgG1 existed mainly in the form of dimers with
a small portion of monomers (Figure 12A), which is consistent with native gel data
(Figure 14C). However, the ratio of the dimers and monomers obtained from the two
analytical methods were different, indicating that the dimer formation is at equilibrium that
could be influenced by concentration and other factors. The small molecular weight
impurity in Protein A purified sc(Fc)
2
sample was well separated from the major peak
representing the intact protein and can be easily removed by SEC to further increase the
purity (Figure 12B). When fused with hGH at the N-terminal of Fc, hGH-Fc still persist
dimeric structure in the storage solution (Figure 12C). The SEC elution profile of hGH-
sc(Fc)
2
showed several peaks, representing hGH-sc(Fc)
2
monomer, dimer and higher
orders of aggregation (Figure 13 and Table 1). The hGH-sc(Fc)
2
monomer was well
separated from aggregated forms.
33
Figure 12. (A) Elusion Profile of Fc, (B) sc(Fc)
2
and (C) hGH-Fc in Superdex
TM
200
Increase 10/300 GL column by ÄKTA Pure.
A
B
C
34
Table 1. hGH-sc(Fc)
2
in Superdex
TM
200 Increase 10/300 GL column by ÄKTA Pure
Peak Retention (mL) Area (ml*mAU) Area% Fraction(s) Volumn (mL)
A 9.831 5.360 12.15 17-22 3.0
B 10.844 3.930 8.91 23-24 1.0
C 11.990 7.009 15.89 25-27 1.5
D 13.993 27.82 63.06 29-35 3.5
Figure 13. Elusion Profile of hGH-sc(Fc)
2
in Superdex
TM
200
Increase 10/300 GL column by ÄKTA Pure.
35
Figure 14. Characterization of recombinant human IgG1 Fc fragment.
Conditioned media containing Fc from transiently transfected HEK293
cells was collected and (A) analyzed by non-reducing SDS-PAGE and (B)
reducing SDS-PAGE followed by Western blot probed with goat anti-hIgG
Fc specific antibody (1:3000 dilution), or (C) purified by Protein A-
Sepharose® 4B followed by native gel with Coomassie blue staining.
55-
70-
25-
35-
55-
70-
25-
35-
A B C
36
2.3.3. Strategies to Increase Protein Productivity
HEK293 and HEK293T cell lines were compared for protein production. Although
the expression level of target protein was much higher in HEK293T cells, other protein
expression levels were also elevated. Moreover, HEK293T showed stronger tendency to
detach from the flask bottom during transfection.
pFUSE and pcDNA3.1+ plasmids were also compared for protein production.
hIgG1-Fc with signal peptide IL2 was inserted into pcDNA3.1+ between HindIII and XhoI
sites. Primers used in this cloning step are listed below:
Forward (HindIII): 5’-TTTAAGCTTGCCACCATGTACAGGATGCAA-3’
Reverse (XhoI): 5'-ATTTCTCGAGTCATTTACCCGGAGACAGGGA-3'
A small-scale expression test in a 6-well plate was carried out. The Fc concentration
in the well transfected with pFUSE-hIgG1-Fc was significantly higher than the well
transfected with pcDNA3.1+_Fc (Figure 15C).
37
pcDNA3.1+_Fc pFUSE-hIgG1-Fc2
Non-reducing
Reducing
Figure 15. Production of hIgG1 Fc fragment in pcDNA3.1+ and pFUSE expression
vector systems. (A) Map of pcDNA3.1+_Fc (B) Map of pFUSE-hIgG1-Fc2 (Invivogen)
(C) The productivity of the two expression vectors were tested follow small-scale
expression protocol. Equal volume of conditioned media from transiently transfected
HEK293 were collected and analyzed by non-reducing and reducing SDS-PAGE and
Western blot probed with goat anti-hIgG Fc specific antibody.
A B
C
38
2.3.4. Formulation Development of Fusion Protein for Long-term Storage
Proteins are usually stored at low temperature or freeze-dried into powder form for
long term storage due to stability issues. Various factors, such as temperature, formulation
pH, absorption to surfaces and interfaces, salts, metal ions, chelating agents, shaking and
shearing, protein denaturants, non-aqueous solvents, protein concentration, source and
purity of proteins, can affect protein stability (Wang 1999). Special formulation and careful
handling are important for proteins storage to ensure optimal biological activities. Liquid
aqueous formulations are preferred by clinicians because they are ready to use and do not
need rehydration (Bye, Platts, and Falconer 2014). Storing proteins in aqueous solutions is
also the first choice in a research setting due to the convenience in preparation and handling,
if the proteins can maintain acceptable stability for a desirable time length.
Mannitol-phosphate buffer, a liquid formulation for hGH, has been adopted for the
storage of hGH and hGH-Transferrin fusion proteins by our lab. However, fusing with
different carrier proteins leads to different physical and chemical properties. A long-term
storage study was conducted to select the optimal formulation.
The fusion proteins were stable for up to 2 months at 4°C in PBS, as measured by
SDS-PAGE/Coomassie blue staining for protein purity and by Nb2 cell proliferation assay
(experimental details described in CHAPTER 3) for confirming activity (Figure 16 and
Table 2). Storage of hGH-sc(Fc)
2
in mannitol phosphate buffer showed similar stability at
4°C compared with PBS, but provided better protection during freeze-and-thaw cycle
(Table 3). Significant hGH activity lost was observed during storage of hGH-sc(Fc)
2
in
PBS at -80°C.
39
Table 2. Stability of hGH-sc(Fc)
2
and hGH-Fc in PBS at 4˚C
Sample
Lane #
Gel A Gel B Gel C
Sample
Days of
storage
Sample
Days of
storage
Sample
Days of
storage
1
hGH-Fc
7
hGH-
sc(Fc)
2
6
hGH-Fc
7
2 17 16 17
3 28 27 28
4 35 34 35
5
hGH-
sc(Fc)
2
6 47 48
6 27 59 60
7 16 73 74
8 34 88 89
B A C
Figure 16. Degradation analysis of hGH-Fc and hGH-sc(Fc)
2.
The hGH-Fc and hGH-
sc(Fc)
2
fusion proteins were analyzed for degradation following storage in PBS at 4˚C by
SDS-PAGE/Coomassie blue staining. Sample identities are listed in the table blow.
40
Table 3. Stability of hGH-sc(Fc)
2
Storage Conditions
Residual Activity*
Duration Temp. Buffer
0 month -- PBS --
1 month 4˚C PBS 106%
2 months 4˚C PBS 94%
1 month -80˚C PBS 74%
1 month 4˚C Mannitol phosphate buffer 94%
1 month -80˚C Mannitol phosphate buffer 94%
*Activity was determined by the Nb2 cell proliferation assay and the ED
50
values were compared.
41
2.4. Discussion
Using HEK293 cells that have human glycoforms, we have produced, purified, and
analyzed the stability of the sc(Fc)
2
fusion proteins. Although glycosylation is not
necessary for FcRn binding, it has been shown to enhance the stability of Fc (Simmons et
al. 2002). As shown in Figure 7A, sc(Fc)
2
was designed to contain (G
4
S)
n
linkers with
different repeats from n=8 to 14, and it was found that the GS linker length affected the
production yield and formation of stable dimers. Shorter linkers had a lower production
yield and larger percentage of impurities, while the longest repeat tested, n=14, resulted in
a relatively lower production yield. Our preliminary data (Figure 14) showed that the
majority of hIgG1-Fc in the expressing medium existed as dimers. This result is consistent
with the general knowledge of the naturally favored dimeric structure of IgG as well as the
2:1 ratio of FcRn-IgG complexes observed in crystal structures (Krapp et al. 2003;
Oganesyan et al. 2014) and surface plasmon resonance biosensor assays (Abdiche et al.
2015). By adding a long flexible linker, the dimeric structure of Fc is expected to be
protected, especially in in vivo conditions. Different linker lengths allow different levels of
flexibility in protein folding, and influence the production level at the same time. The major
impurity, which can be recognized by anti-hIgG1-Fc antibody and cannot be purified by
Protein A column, may result from improper folding near the linker region. The (G
4
S)
13
linker in sc(Fc)
2
provides the best balance between expression level and purity. The binding
of these sc(Fc)
2
to Protein A indicates their proper folding at the CH2-CH3 interface that
is also the region interacting with FcRn (Rath et al. 2015; Oganesyan et al. 2014; Wines et
al. 2000).
42
In traditional Fc-fusion proteins, therapeutic proteins are fused at the N-terminal of
an intact Fc sequence. We tested both N-terminal and C-terminal fusions, however the
expression level of hGH-sc(Fc)
2
is about 3-fold higher than that of sc(Fc)
2
-hGH. Fusing
hGH at the N-terminal promotes the desired folding of the fusion protein, which is
consistent with the properties revealed from structure of Fc fragment that C-terminal is
buried inside the hydrophobic core of CH3 domain and N-terminal is used to link with the
hinge region relatively exposed to the outside.
Purity and stability of the fusion proteins and productivity of the production system
were assessed before moving forward to functional tests of the fusion proteins. If large
production scale is needed, for example for in vivo oral delivery studies, we can switch
expression vector and production cell line to increase productivity.
2.5. Summary
In this chapter, a single chain Fc dimer recombinant fusion protein has been
designed and produced in HEK 293 mammalian systems. An optimized linker length was
selected to allow proper folding of two linked Fc moieties and good production yield.
Human growth hormone (hGH) was fused with sc(Fc)
2
and Fc, and the resulting fusion
proteins were produced and evaluated. Large scale production process including
expression, purification, and storage was characterized and established.
43
CHAPTER 3: CHARACTERIZATION OF IN VITRO BIOLOGICAL
ACTIVITIES OF SINGLE CHAIN Fc-DIMER RECOMBINANT
FUSION PROTEINS
3.1. Background
Bifunctional fusion proteins with one side binding to the therapeutic target and the
other side aiming at improving pharmacokinetics of the whole protein using innate
pathways like FcRn-mediated recycling and transcytosis pathway is a potentially effective
delivery system to achieve prolonged half-life and non-invasive delivery (Figure 17).
Human Growth hormone (hGH) is a 191 amino acid long, single-chain polypeptide
that is primarily synthesized, stored, and secreted by the somatotrophic cells in anterior
pituitary gland. Cells and tissues other than the pituitary and its somatotrophs, such as
lymphoid tissues, also produce hGH (Clark 1997). hGH deficiency is associated with
Figure 17. A hypothetical model for utilizing FcRn-mediated
recycling and transcytosis pathways for improved protein drug
delivery with sc(Fc)
2
carrier protein.
44
several clinical indications, including short stature, Turner syndrome, chronic kidney
disease, HIV-associated wasting and abnormal metabolism (Amet, Wang, and Shen 2010).
The first recombinant hGH for human therapy was developed by Genentech in 1981. hGH
has a very short plasma half-life of 3.4 h after subcutaneous injection, and 0.36 h after
intravenous injection in human (Webster et al. 2008). Therefore, current treatment of hGH
deficiency is limited to needle injection of hGH several times a week, which is not favored
by patients, especially children and seniors (Amet, Wang, and Shen 2010). The non-
compliance rate of pediatric patients to adhere to a GH treatment regimen could be as high
as 23-50%, especially in long term treatments (Kapoor et al. 2008; Hou et al. 2016).
Additionally, several studies comparing injection and slow infusion of GH or slow release
GH formulations indicated long exposure of GH led to better therapeutic effects (Clark
1997).
Therefore, there is an unmet need in long-acting hGH that requires less frequent
dosing and hGH that can be administered non-invasively through oral or pulmonary routes.
We chose to use hGH as the model drug cargo to evaluate our novel delivery system.
45
3.2. Materials and Methods
3.2.1. Cell Cultures
The human colon carcinoma cell line T84 were purchased from ATCC (Manassas,
VA), and Nb2 cells derived from rat T lymphoma cells were purchased from Sigma (St.
Louis, MO). Cell culture media were all from Mediatech (Manassas, VA). T84 cells were
cultured in 1:1 mixture of Ham's F12 medium and DMEM with 2.5 mM L-glutamine, 10%
FBS, and 50 units/mL of penicillin/50 µg/mL streptomycin. T84 cells were trypsinized
with 0.5% trypsin-EDTA for regular passage. Nb2 cells grow in suspension. They were
cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2
mM L-glutamine, 10% FBS, 10% horse serum, 50 units/mL of penicillin/50 µg/mL
streptomycin, and 50 mM 2-mercaptoethanol. All mammalian cell lines were maintained
in a humidified incubator at 37°C with 5% CO
2
.
3.2.2. Proteins
Recombinant hIgG1-Fc produced from HEK293 (ACROBiosystems, Newark, DE)
and hGH produced from HEK293 (Sigma, St. Luise, MO) were used in the assays. Purified
Human IgG isolated from pooled normal human serum by fractionation and ion-exchange
chromatography was purchased from Sigma (Saint Louis, Missouri). Fc, sc(Fc)
2
, hGH-
sc(Fc)
2
, and hGH-Fc fusion protein was produced in large-scale in HEK 293 cells as
described in CHAPTER 2.
46
3.2.3. Neonatal Fc Receptor (FcRn) Binding Assay
3.2.3.1. Cell-based Binding Assay
3.2.3.1.1. Iodination
PBS (0.2 mL) was added to a new bottom of
125
I-Na. The solution was mixed
thoroughly by pipetting. Lyophilized hIgG1-Fc from ACROBiosystems was reconstituted
according to manufacture’s instruction. Reconstituted hIgG1-Fc (1 mg/mL) was mixed
well with 50 µl of
125
I-Na (0.5 mCi, Perkin Elmer) in a glass tube. Chloramine-T (8 mg/mL),
sodium metabisulfite (4.8 mg/mL), and potassium iodine (10 mg/mL) solutions were
freshly prepared in PBS. The reaction was started by adding 50 µl of chloramine-T solution
to the mixture. The reaction mixture was incubated on ice for 10 min with frequent swirling
during the incubation. The reaction was stopped by adding 50 µl of sodium metabisulfite
solution followed with a 5 min incubation on ice. Then, 100 µl of potassium iodine solution
was added, and the mixture was left on ice for another 2-5 min. After the chemical reactions,
the whole mixture was loaded onto G-50 size exclusion column with 10 mL resin bed pre-
balanced with PBS. The column was washed with PBS, and ten fractions (1.0 mL/tube)
was collected and counted by a γ-counter (Packard, Downers Grove, IL). The concentration
of the fractions containing
125
I-hIgG1-Fc was calculated based on the counts. The fraction
with the highest counts were stored in small aliquots at 4°C for the experiment on the
following day or -20°C for future use.
3.2.3.1.2 Surface Binding Assay (Saturation Dose)
T84 cells were seeded in 6-well plates at a density of 2.5×10
6
/well, and cultured for
1-2 weeks post-confluence to allow for differentiation. The cells were dosed with
47
ascending concentrations of
125
I-hIgG1-Fc and incubated at 4 °C for 1 h in serum-free
media adjusted to pH 6.0. After incubation, the cells were washed 3 times with cold PBS
(pH 6.0) and dissolved in 2.5 ml 1N NaOH/well. The radioactivity of the cell lysates was
measured using a γ-counter. The data were normalized by the total protein concentration
determined by microBCA kit.
3.2.3.1.3 Short Time Course Uptake Assay
T84 cells were seeded in 6-well plates at a density of 2.5×10
6
/well, and cultured for
1-2 weeks post-confluence to allow for differentiation. Serum-free medium was prepared
by adding 10 mM citric acid/salt into NaHCO
3
-free RPMI medium and filtered through a
0.2 µm membrane. The cells were dosed with 120 nM of
125
I-hIgG1-Fc and ascending
concentrations of unlabeled hIgG1-Fc or sc(Fc)
2
, and incubated at 37°C for 15 min in
serum-free media adjusted to pH 7.4 for the control group or pH 6.0 for the remaining
treatment groups. After incubation, the cells were washed 3 times with cold PBS followed
by trypsinization. The cells were collected in PBS and centrifuged (3000 rpm, 3 min, 25°C),
and the radioactivity in the cell pellets was measured using a γ-counter. The data were
analyzed by sigmoidal curve fitting in GraphPad Prism 5.
3.2.3.2. Surface Plasmon Resonance
Surface Plasmon Resonance (SPR) measurements were performed by using a
Biacore T100 instrument. Lyophilized shFcRn (ACROBiosystems, Newark, DE) was
reconstituted with sodium acetate/acetic acid buffer (pH 4.5-5). The immobilization
process involved activation of carboxymethyl groups on the dextran surface of a Series S
48
Sensor Chip CM5 (GE Healthcare, Pittsburgh, PA) at pH 4.5-5 by 1- ethyl-(3-
dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide
(NHS), followed by covalent bonding of shFcRn to the chip surface via amide linkage.
Residual sites on the dextran were blocked with 1 M ethanolamine hydrochloride (pH 8.5).
A control flow cell was blocked with ethanolamine for reference subtraction. Experiments
were performed in running buffer (50 mM phosphate, 100 mM sodium chloride, and 0.01%
vol/vol Tween 20, pH 6.0 or 7.4). FcRn was coated to a final density of ~9 ,000 response
units (RU). Dilutions of recombinant Fc, sc(Fc)
2
, hGH-Fc and hGH-sc(Fc)
2
in running
buffer were injected over the shFcRn-CM5 chip at 20 µL/min for 2 min (except 1 min for
Fc group). The proteins were then dissociated from the chip for 2.5 min with running
buffer. Remaining protein was removed from the chip with regeneration buffer (10 mM
HEPES, 150 mM NaCl, and 0.01% vol/vol Tween 20, pH 7.4) at 30 µL/min for 30 s.
Sensorgrams were generated and analyzed by Biacore T100 Evaluation Software (version
2.0.2). The equilibrium RU observed for each injection was plotted against the
concentration of protein. The equilibrium K
d
values were derived by analysis of the plots
using the steady-state affinity model.
49
3.2.4. Nb2 Cell Proliferation Assay
Nb2 cells growing at log-phase were washed 3 times with serum-free RPMI 1640
medium, re-suspended in serum-free assay medium, and counted using Z1 Coulter particle
counter (Beckman, Fullerton, CA). Nb2 cells were then seeded (15,000/well) in 96-well
plates in 200 µL FBS-free assay medium (RPMI1640 medium supplemented with 2 mM
L-glutamine, 10% horse serum, 50 units/mL of penicillin/50 µg/mL streptomycin, and 50
mM 2-mercaptoethanol) and cultured for 24 h. The fusion protein samples and commercial
recombinant hGH (Somatropin, LGC Standard USP1615708, United States Pharmacopeia
Convention, Rockville, MD) were diluted into 10 mL PBS, and varying doses were added
to the serum-starved Nb2 cells. After 4-day incubation, cells were incubated overnight with
20 µL of resazurin solution (Biotium, Hayward, CA). The absorbance was measured at 560
nm and 595 nm using a Genios microplate reader (Tecan, San Jose, CA) and normalized
against the vehicle control. The data were collected as duplicates and analyzed by
sigmoidal curve fitting in GraphPad Prism 5.
3.2.5. Trancytosis Assay
3.2.5.1. T84 Cell Differentiation
Differentiation of T84 cells was carried out in transwell 6-well plates. Cells were
seeded on 0.4 µm polycarbonate membrane of 24 mm inserts in 6-well transwell plates
(Corning, Kennebunk, ME). The cell growth area of the insert is 4.67 cm². Upon 100%
confluence, cells were further cultured for another 3-4 weeks for differentiation forming
steady tight junctions, during which cells were fed with fresh medium every other day. The
50
transepithelial electrical resistance (TEER) was measured with an epithelial voltage ohm
meter (World Precision Instruments, Sarasota, FL).
3.2.5.2. Transcytosis of hGH-sc(Fc)
2
vs hGH-Fc
Assay medium A (DMEM/F12 50/50 with L-Gln, 50 mM MES, 0.1% ovalbumin
pH 6.0) and assay medium B (DMEM/F12 50/50 with L-Gln, 50 mM HEPES, 0.1%
ovalbumin, pH 7.4) were prepared and filtered through a 0.2 µm membrane. The cells were
dosed with 120 nM of hGH-Fc, hGH-sc(Fc)
2
, or hGH-sc(Fc)
2
with 100X human serum IgG
at the apical chambers in assay medium A. Assay medium B was added to the basolateral
chambers. The cells were incubated at 37 °C for 2 hours. The medium from the basolateral
chambers were collected and analyzed by Human Growth Hormone Quantikine ELISA Kit
(R&D Systems, Minneapolis, MN).
51
3.3. Results
3.3.1. Receptor Binding Affinity in T84 Cells
In adult human intestine, the expression pattern showed an increasing proximal to
distal gradient and the highest FcRn expression was found in the proximal colon (Hornby
et al. 2014). The colon carcinoma cell line T84 expresses FcRn in a similar pattern as what
has been found in crypt enterocytes of normal human small intestine and expresses a higher
level of FcRn than the other commonly used colorectal carcinoma cell line Caco-2
(Dickinson et al. 1999). Also, this cell line can form well-differentiated polarized
monolayers that have high electrical resistances. Therefore, T84 cells can be a good mimic
of the in vivo situation in human intestine and will give higher signal in the binding assay
as compared with Caco-2 cells.
However, FcRn in T84 cells was reported to mainly reside close to the apical
membrane at or just below the level of the tight junctions, which means the amount of
receptors present on the cell surface is limited (Dickinson et al. 1999). The surface binding
showed a linear correlation with dose indicating that the amount of receptors present on
the cell surface was not high enough to perform surface binding assay, which is also
consistent with the literature that the cell surface expression of FcRn in T84 cells at steady
state display strong basolateral polarity (Claypool et al. 2004).
Incubation at 18°C has been demonstrated to block certain membrane trafficking
events and experimental data showed that such incubation could lead to the accumulation
of hFcRn at the apical surface of transfected MDCK cells (Claypool et al. 2004). Thus, we
tried to perform a pre-incubation at 18°C overnight to induce the relocation of FcRn to the
52
apical surface of the T84 cells before the surface binding assay at 4°C, but the data quality
had not been improved by pre-treatment.
Although the amount of FcRn present at the apical surface was undetectable in the
immunoprecipitation assay, FcRn was found to be able to move transiently to the apical
membrane and bind to its ligands in the apical reservoir (Claypool et al. 2004). Therefore,
a short time course uptake assay could be an alternative way to compare the binding
affinities of sc(Fc)
2
and control protein hIgG1-Fc to FcRn. The cells were dosed with 120
nM of
125
I-hIgG1-Fc and ascending concentrations of unlabeled hIgG1-Fc or sc(Fc)
2
to
compete. The data indicated that the internalization was specific, which is consistent with
what had been found in MDCK cells expressing recombinant FcRn (Tesar, Tiangco, and
Bjorkman 2006). The internalization was inhibited in a dose-dependent manner by both
hIgG1-Fc and sc(Fc)
2
(Figure 18). The IC
50
values for hIgG1-Fc and sc(Fc)
2
were 220.7
nM and 344.0 nM, respectively.
53
Figure 18. Short incubation uptake assay of hIgG1-Fc and sc(Fc)
2
to
determine FcRn binding. Confluent T84 cells were incubated with 120
nM
125
I-hIgG1-Fc and the indicated concentrations of unlabeled hIgG1-
Fc or sc(Fc)
2
, and incubated at 37°C for 15 min in serum-free medium
adjusted to pH 6.0. Cells were then washed with PBS, trypsinized, and
the radioactivity in the cell pellets was measured. The data were
analyzed by sigmoidal curve fitting in GraphPad Prism 5, where it was
determined that the IC
50
of hIgG1-Fc was 220.7 nM and of sc(Fc)
2
was
344.0 nM. Each data point represents mean ± SD (n = 3).
54
3.3.2. Surface Plasmon Resonance
SPR is now one of the most popular label-free methods to characterize protein-
protein interaction in both industry and academia (Maynard et al. 2009a; Maynard et al.
2009b). This technology typically works by immobilizing one binding partner onto a thin
gold film coated chip. To measure equilibrium and binding kinetics, a solution containing
ligand flows across the surface. When ligand binds the immobilized binding partner, the
mass of material bound to the surface increases. This change is detected as a change in the
angle of polarized light reflected from the bottom surface of the chip.
FcRn was immobilized to the sensor surface by amine coupling. The binding of Fc,
sc(Fc)
2
, hGH-Fc and hGH-sc(Fc)
2
to shFcRn was measured based on the experimental
conditions developed by Mezo et al. (Mezo et al. 2008). At pH 6.0, Fc showed a K
d
of 44.9
nM, and sc(Fc)
2
showed a weaker K
d
of 285 nM. After fusing hGH to the N-terminal of
the carrier proteins Fc or sc(Fc)
2
, the K
d
dropped to 1.43 µM (hGH-Fc) or 400 nM (hGH-
sc(Fc)
2
) (Figure 19 and Table 4). No binding was observed at pH 7.4 (example data shown
in Figure 20).
55
A
C
B
D
Figure 19. SPR Sensorgams for binding of serial twofold dilutions of Fc (A, 6.25 nM
to 400 nM), sc(Fc)
2
(B, 3,200 nM to 6.25 nM), hGH-sc(Fc)
2
(C, 6,400 nM to 50 nM),
and hGH-Fc (D, 6,400 nM to 6.25 nM) to immobilized shFcRn at pH 6.0.
Figure 20. SPR Sensorgams for binding of
different concentrations of Fc to immobilized
shFcRn at pH 7.4.
56
Table 4. Apparent K
d
from Surface Plasmon Resonance at pH 6.0
Sample KD (M) SE (KD)
Rmax
(RU)
SE
(Rmax)
offset
(RU)
SE
(offset)
Chi
2
(RU
2
)
Fc 4.493E-08 4.9E-09 61.76 1.4 9.650 1.6 1.17
sc(Fc)
2
2.85E-07 4.0E-08 49.17 1.6 3.675 1.1 2.85
hGH-Fc 1.429E-06 2.0E-07 62.64 3.0 0.5985 0.6 3.04
hGH-
sc(Fc)
2
4.007E-07 5.8E-08 50.95 1.6 1.122 1.7 1.73
57
3.3.3. Nb2 Cell Proliferation Assay
hGH bioactivity was measured using a well-established in vitro assay, the Nb2 cell
proliferation assay, where suspension culture shows a dose-dependent proliferation when
exposed to exogenous hGH (Tanaka et al. 1980). This bioassay is listed in the USP
Somatropin monograph as a standard assay to determine hGH potency. Following
treatment of Nb2 cells with various concentrations of the fusion proteins or hGH control,
cell growth was stimulated in a dose-dependent manner (Figure 21). The bioactivity of
free hGH and hGH-sc(Fc)
2
were similar, with EC
50
values of 166 pM and 284 pM,
respectively. However, the activity of the hGH-Fc fusion protein was approximately 3-fold
lower (EC
50
= 1064 pM). This assay was repeated several times to confirm the relative
difference in EC
50
among hGH, hGH-sc(Fc)
2
and hGH-Fc.
58
Figure 21. Nb2 cell proliferation stimulated by hGH fusion proteins.
Nb2 cells were serum-starved for 24 h, and then treated with the
indicated concentrations of fusion proteins. Cell proliferation was
determined by the resazurin assay after a 4-day incubation. The data
were collected as duplicates and analyzed by sigmoidal curve fitting in
GraphPad Prism 5, where it was determined that the EC
50
of hGH, hGH-
sc(Fc)
2
and hGH-Fc were 166 pM, 284 pM and 1064 pM, respectively.
59
3.3.4. Transcytosis of Fusion Proteins
Culture of T84 cells on porous filters fosters in vivo-like morphology and
functionality. Because of the optical properties of the filter materials and the columnar
shape of the cells, cell quality is hard to assess via light microscopy, so transepithelial
electrical resistance (TEER) measurement becomes a widely accepted quantitative
technique to monitor the integrity of tight junction dynamics in cell culture models of
endothelial and epithelial monolayers (Chen, Einspanier, and Schoen 2015; Marvin-Guy
et al. 2008; Claypool et al. 2004; Srinivasan et al. 2015). The TEER value of T84
monolayer reached a plateau after 3-4 weeks of culture. The average values of TEER*cell
growth area showed low variability within experiments, but relatively high variability
between experiments, ranging from 3,000 to 4,500 Ω*cm
2
.
The normalized amount of hGH-sc(Fc)
2
transcytosed through T84 monolayer was
significantly higher than hGH-Fc group based on two-tail student t-test (p < 0.05). When
competed with 100X human serum IgG, the normalized amount of hGH-sc(Fc)
2
transcytosed decreased, indicating the involvement of FcRn-mediated transcytosis
pathway.
60
Figure 22. Transcytosis assay of hGH-sc(Fc)
2
and hGH-Fc through T84 cells formed
epithelial barrier. Each bar represents mean ± SEM (n = 3). The amounts of
transcytosed fusion proteins determined by hGH ELISA kit were normalized by the
molecular weight of the respective fusion proteins.
Transcytosis Assay
pg/kDa
hGH
hGH-Fc
hGH-sc(Fc)
2
hGH-sc(Fc)
2
+ 100X IgG
0
5
10
15
20
hGH
hGH-Fc
hGH-sc(Fc)
2
hGH-sc(Fc)
2
+ 100X IgG
61
3.4. Discussion
The binding of sc(Fc)
2
and Fc to FcRn was evaluated in both cell-based assay and
SPR. Due to the nature of receptor localization pattern, the cell-based assay is an indirect
assay to compare binding affinity to the receptor, but it provides a better mimic of the in
vivo situation. The absolute value obtained from the two assays are not comparable, but
they showed a consistent trend. Amine coupling that works through lysine resides and the
N-terminal amino group was used in receptor immobilization onto the chip surface in SPR
assay. The site of immobilization and the orientation of receptors on the chip is not
controllable by this type of immobilization process, but minimal influence on binding is
assumed because the key binding residues on both FcRn and Fc region do not have lysine.
Since we always compared the binding within the same assay platform, the trends
obtained were reliable. Based on the results of two binding assays, we can conclude that
sc(Fc)
2
maintained part of the binding affinity of Fc to FcRn. When hGH was fused to the
N-terminal of the carrier proteins, the binding affinities decreased. The binding affinity
drop was more dramatic in hGH-Fc fusion protein, which might be the result of the steric
hindrance of hGH disrupting stable dimeric structure formation that is essential to maintain
FcRn binding.
The activity of the hGH-sc(Fc)
2
fusion protein was determined by the Nb2
proliferation assay (Amet et al. 2010; Amet, Wang, and Shen 2010). In this assay, hGH-
dependent proliferation is induced by hGH binding to prolactin receptor, rather than growth
hormone receptor. Our production system, HEK293 cell line, is free of prolactin, thus the
Nb2 cell proliferation assay can accurately reflects in vitro cell proliferation activity of
hGH, hGH-sc(Fc)
2
and hGH-Fc fusion proteins. As shown in Figure 21, the hGH-sc(Fc)
2
62
fusion protein maintained 58% biological activity compared to free hGH, and was
approximately 3.7-fold higher than the bioactivity of hGH-Fc. An explanation for this
phenomenon could be that when two hGH-Fc molecules form a dimeric structure, the two
hGH domains are sterically hindered, reducing GH receptor binding.
Proteins are not administered orally due to their degradation by digestive enzymes
in the intestinal lumen and low intestinal permeation caused by large molecular size and
high polarity. Formulation, encapsulation, macromolecular conjugation and chemical
modification are the general approaches currently being studied for improving protein
absorption in the GI tract (Shen 2003). In neonatal rodents, high levels of FcRn expression
and IgG transport in intestine were reported during suckling time (Benlounes et al. 1995;
Kliwinski et al. 2013). However, FcRn is expressed throughout adult life in primates,
making it a potential target for oral protein delivery. Transcytosis of mAb was studied in
adult human intestinal segments mounted in Ussing-type chambers, Caco-2 monolayer and
cynomolgus monkeys with regional intestinal delivery (Hornby et al. 2014). They found
that neither apical surface pH nor mAb-FcRn affinity affected internalization or
transcytosis at saturating concentrations, indicating that non-receptor mediated process was
involved. This observation was consistent with our transcytosis assay data that human
serum IgG competition could not completely abolish the transcytosis of tested fusion
proteins (Figure 22). The transcytosis of hGH fusion proteins may be influenced by both
GHR and FcRn bindings. However, because of the detection method used in this assay,
hGH competition was not included as a control. The kit sensitivity was not evaluated
towards fusion proteins, so the transcytosis amounts of fusion proteins could be
underestimated.
63
3.5. Summary
In this chapter, the functions of purified fusion proteins were evaluated by in vitro
binding assays, including radiolabeled cell-based competition assay and real time label-
free SPR, and in vitro potency assay using Nb2 cells. The transcytosis property of our
sc(Fc)
2
carrier protein was also investigated in the in vitro transwell system using
differentiated human carcinoma cell monolayer.
64
CHAPTER 4 EVALUATION OF IN VIVO PHARMACOKINETICS
AND PHARMACODYNAMICS OF SINGLE CHAIN FC-DIMER
RECOMBINANT FUSION PROTEINS
4.1. Background
When two different proteins are genetically fused together, the PK and PD of the
fusion protein usually are not a simple superimposition of the properties of its parent
proteins.
Protein therapeutics are usually administered invasively, e.g. intravenously or
subcutaneously. Due to the large molecular weight, fusion proteins have very limited
permeability to get into blood capillary system and are likely to be absorbed via lymphatic
draining. However, the exact mechanism is still poorly understood. The large size and high
hydrophilicity of fusion proteins also contributes to the limited tissue penetration ability
resulting in small apparent volume of distribution, approximately equal to or slightly higher
than the total plasma volume. If the binding targets of fusion proteins distributed in certain
tissues with high expression level and turn-over rate, active tissue uptake may exist
(Carnemolla et al. 2002).
Pharmacokinetics of fusion proteins usually can be well characterized by one, two
or multiple compartment models comprised of central and peripheral compartments where
central compartment primarily represents the vascular space and the interstitial space of
well-perfused organs, while the peripheral compartment represents the interstitial space of
poorly perfused tissues (Chen, Zaro, and Shen 2012).
In addition to FcRn recycling discussed in CHAPTER 1, routes of Fc fusion protein
clearance could be influenced by target-mediated drug disposition (TMDD), renal filtration,
65
anti-drug antibodies, and nonspecific catabolism. Different from small molecule drugs that
experience extensive elimination in the liver by CYP450 enzymes, most fusion proteins do
not have significant hepatic elimination, and the typical elimination mechanism is the
proteolysis followed by fluid-phase or receptor-mediated endocytosis. This elimination
mechanism can happen in all accessible tissues. TMDD refers to the receptor-mediated
endocytosis and subsequent intracellular metabolism that is unique and critical for the
elimination of many proteins. It is the dominant reason for the often observed non-linear
pharmacokinetics of protein therapeutics, and this fast clearance mechanism tends to
dominate the effect of FcRn recycling or renal filtration (Wu and Sun 2014).
The cut-off size for glomerular filtration are generally considered to be 50-60 kDa.
Apart from size, other factors, such as charge, lipophilicity, functional groups, sugar
recognition, vulnerability to protease degradation, aggregation to particles and formulation
of complexes with spsonization factors, all play a role in renal filtration (Wu and Sun 2014).
The secretion of GH is pulsatile and can be affected by many intrinsic and extrinsic
factors including sleeping, feeding and so on, therefore direct measurement of GH levels
could be misleading. The circulating IGF-1, which is primarily induced by GH stimulation
and mediates the direct and indirect effect of GH on peripheral tissue to promote linear
growth, is often used as an PD and safety indicator of GH (Wu et al. 2014; Wu, Ji, and Hu
2015; Lee et al. 2015; Hou et al. 2016) and is the most important biomarker for diagnosis
and monitoring of GH deficiency (Mukherjee and Shalet 2009).
66
4.2. Materials and Methods
4.2.1. Animals
CF1 mice (male, 6 weeks, ~30 g, Charles River Laboratories, Wilmington, MA)
were utilized for all of the in vivo assays in this study. All animal studies were performed
in accordance with the NIH guidance in “Guide for the Care and Use of Laboratory
Animals” and approved by the University of Southern California Institutional Animal Care
and Use Committee. Animals were housed at 12 h light/12 h dark cycles under standard
conditions (room temperature at 22 ± 3°C and relative humidity at 50 ± 20%) with access
to regular rodent chow (Labdiet, St. Louis, MO) and water.
4.2.2. Proteins
Commercial recombinant hGH (Somatropin USP1615708, LGC) was used in the
in vivo pharmacodynamics assay. Fc, sc(Fc)
2
, hGH-sc(Fc)
2
, and hGH-Fc fusion proteins
were produced in large-scale in HEK 293 cells as described in CHAPTER 2.
4.2.3. Measurement of In Vivo Pharmacokinetics
4.2.3.1. hGH-sc(Fc)
2
vs sc(Fc)
2
(i.v.)
Purified hGH-sc(Fc)
2
and sc(Fc)
2
were injected into the tail vein of mice. At 5 min,
30 min, 1 h, 4 h, and 8 h post-injection time points, blood samples were collected from the
saphenous vein. At 16 h after injection, blood samples were collected from the heart. Blood
samples were immediately mixed with concentrated heparin to avoid coagulation and then
67
centrifuged at 2,000 rpm for 20 min at 4°C to generate plasma samples. The plasma
samples were immunoprecipitated by Protein A-Sepharose
®
4B beads, and then tested by
Western Blot using anti-hIgG (Fc specific) antibody. Fusion proteins with known
concentration are loaded in serial concentrations to serve as the standards. The blots were
imaged by ChemiDoc
TM
Touch and quantitatively analyzed by ImageLab
TM
software.
4.2.3.2. hGH-sc(Fc)
2
vs hGH-Fc (i.v.)
Purified hGH-sc(Fc)
2
and hGH-Fc were injected into the tail vein of mice. At 5
min, 30 min, 1 h, 4 h, and 8 h post-injection time points, blood samples were collected
from the saphenous vein and mixed with concentrated heparin to avoid coagulation. Blood
samples were then centrifuged at 2,000 rpm for 20 min at 4°C to generate plasma samples.
The plasma samples were analyzed by Double Antibody Human Growth Hormone RIA kit
(MP Biomedicals, Costa Mesa, CA).
4.2.3.3. hGH-sc(Fc)
2
vs hGH-Fc (s.c.)
Purified hGH-sc(Fc)
2
and hGH-Fc were injected subcutaneously into male CF1
mice. The doses were normalized to the hGH portion of each fusion protein as 1 mg/kg
hGH. At pre-injection and 1 h, 4 h, 8 h, 16 h, 28 h, 48 h post-injection time points, blood
samples were collected from the saphenous vein. At 72 h after injection, blood samples
were collected from the heart. Blood samples were immediately mixed with concentrated
heparin to avoid coagulation and kept on ice. Next, blood samples were centrifuged at
2,000 rpm for 20 min at 4°C to generate plasma samples. The plasma samples were
analyzed by Double Antibody Human Growth Hormone RIA kit.
68
4.2.4. Measurement of In Vivo insulin-like growth factor 1 (IGF-1) Level
Purified hGH-sc(Fc)
2
, hGH-Fc, commercial recombinant hGH and PBS control
were injected subcutaneously into male CF1 mice. The doses were normalized to the hGH
portion of each fusion protein as 1 mg/kg hGH. At pre-injection and 1 h, 4 h, 8 h, 16 h, 28
h, 48 h post-injection time points, blood samples were collected from the saphenous vein.
At 72 h after injection, blood samples were collected from the heart. Blood samples were
immediately mixed with concentrated heparin to avoid coagulation and kept on ice. Next,
blood samples were centrifuged at 2,000 rpm for 20 min at 4°C to generate plasma samples.
The plasma samples were analyzed by Mouse/Rat IGF-I Quantikine ELISA Kit (R&D
Systems, Minneapolis, MN). The absorbance at 450 nm and 540 nm were measured using
a Synergy H1 Hybrid Plate Reader (BioTek, Winooski, VT). 4-Parameter logistic
regression was used in curve fitting (elisaanalysis.com).
4.2.5. Statistical Analysis
Non-compartmental analysis (NCA) was conducted on PK data in Phoenix 7.0 with
linear trapezoidal linear/log interpolation method and weighting scheme of 1/y^2. The two-
tailed Student's t-test was applied to determine differences between data sets, where P <
0.05 was considered statistically significant.
69
4.3. Results
4.3.1. Pharmacokinetics
In male CF1 mice, hGH-sc(Fc)
2
showed a half-life of 5.62 h, while the half-life of
the carrier protein sc(Fc)
2
was 23.42 h (Figure 23 and Table 5). The systemic error of
quantitative Western Blot was relatively large due to the multiple pretreatment steps, this
method allowed us to quantify the amount of intact fusion proteins. The half-life of hGH-
sc(Fc)
2
(2.79 h) was approximately 2-fold longer than that of hGH-Fc (1.49 h) (Figure 24
and Table 6), and over 12-fold longer than the previously reported half-life of hGH (less
than 15 min) in male CF1 mice (Chen et al. 2011). The plasma concentrations of both
fusion proteins reached their peak concentrations at 8 h time point with almost two-fold
difference in their Cmax, AUC and apparent clearance CL/F (Figure 25 and Table 7). The
apparent half-lives after s.c. injection were much longer than half-lives obtained after i.v.
injection.
70
Table 5. Non-compartmental PK analysis of hGH-sc(Fc)
2
and sc(Fc)
2
after i.v. injection.
Sample T1/2 (h) AUC (h*ng/uL)
V (uL/g) CL (uL/h/g)
hGH-sc(Fc)
2
5.62 202.78 51.94 6.41
sc(Fc)
2
23.42 611.86 49.71 1.47
Figure 23. Pharmacokinetic profiles of hGH-sc(Fc)
2
and sc(Fc)
2
after i.v.
injection. Male CF1 mice were injected via the tail vein with 1.30 mg/kg hGH-
sc(Fc)
2
or 0.90 mg/kg sc(Fc)
2
, and blood samples were collected from the
saphenous vein at the indicated time points. The plasma concentration (Cp) at
different time points was determined by quantitative Western blot.
71
Table 6. Non-compartmental PK analysis of hGH-sc(Fc)
2
and hGH-Fc after i.v. injection.
Sample T1/2 (h) AUC (h*ng/uL)
V (uL/g) CL (uL/h/g)
hGH-sc(Fc)
2
2.79 130.01 40.19 10.00
hGH-Fc 1.49 33.73 50.98 23.72
Figure 24. Pharmacokinetic profiles of hGH-sc(Fc)
2
and hGH-Fc after i.v.
injection. Male CF1 mice were injected via the tail vein with 1.30 mg/kg hGH-
(Fc)
2
or 0.80 mg/kg hGH-Fc, and blood samples were collected from the saphenous
vein at the indicated time points. The Cp was determined by double antibody hGH
RIA kit.
72
Table 7. Pharmacokinetic profiles of hGH-sc(Fc)
2
and sc(Fc)
2
after s.c. injection.
a
a
NCA based on the average of normalized Cp of ‘hGH’.
b
Data are shown as mean ± SEM.
Sample T1/2 (h)
AUC
(h*ng/mL)
Cmax
(ng/mL)
b
Tmax (h)
CL/F
(mL/h/kg)
hGH-
sc(Fc)
2
17.46 13228.78
879.54 ±
84.51
8 75.59
hGH-Fc 16.04 7074.99
428.52 ±
43.73
8 141.34
Figure 25. Pharmacokinetic
profiles of hGH-sc(Fc)
2
and
hGH-Fc after s.c. injection. Mice
were injected with 1 mg/kg hGH-
fusion proteins (normalized to
hGH) and blood samples were
collected from the saphenous vein
at the indicated timepoints. The
plasma concentration (Cp) was
determined by double antibody
hGH RIA kit. Each data point
represents mean ± SD (n = 5).
Subcutaneous PK
0 10 20 30 40 50
10
100
1000
10000
t/h
Cp of 'hGH' (ng/ml)
hGH-Fc
hGH-sc(Fc)
2
73
4.3.2. Pharmacodynamics
To evaluate in vivo bioactivity, purified hGH-sc(Fc)
2
, hGH-Fc, commercial hGH
standard, and PBS control were injected subcutaneously into male CF1 mice. As shown in
Figure 26, IGF-1 levels in the PBS control group were maintained at approximately 500
ng/mL throughout the 3-day experimental period. The IGF-1 levels of the hGH group
showed no significant difference from the baseline at this dose, which is consistent with
the same experiments performed in the studies of different long-acting hGH forms (Wu, Ji,
and Hu 2015; Wu et al. 2014). On the other hand, the IGF-1 levels in both hGH-sc(Fc)
2
and hGH-Fc fusion protein groups reached peak levels between 16 h and 28 h. The IGF-1
levels in the hGH-sc(Fc)
2
group were significantly higher than that of the hGH-Fc group
at the 1 – 28 h post-injection time points.
74
Figure 26. IGF-1 plasma levels after subcutaneous injection of hGH-fusion proteins
in male CF1 mice. Mice were injected (s.c.) with 1 mg/kg hGH-fusion proteins
(normalized to hGH) and blood samples were collected from the saphenous vein at the
indicated timepoints. Plasma samples were then isolated and analyzed by Mouse/Rat
IGF-1 Quantikine ELISA. The data at t = 0 were obtained from the blood samples
collected 1 h before injection. Each data point represents mean±SD (n = 4-5). The two-
tail student t test was used to compare the data from the hGH-sc(Fc)
2
and hGH-Fc
groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001
75
4.4. Discussion
The in vivo biological activity of the hGH-fusion proteins was determined by
measuring the increase in IGF-1 secretion following treatment in CF-1 mice (Donovan et
al. 1991; Zhao and Donovan 1995; Zhao, Unterman, and Donovan 1995). As shown in
Figure 25, the IGF-1 levels were significantly higher in the hGH-sc(Fc)
2
treated group
compared to both hGH-Fc and hGH treatment. The half-life of hGH-sc(Fc)
2
(2.79 h) was
about 2-times longer than hGH-Fc (1.49 h) following intravenous injection in CF1 mice.
The much shorter half-life of hGH-sc(Fc)
2
compared to sc(Fc)
2
could be a result of its
lower binding to FcRn observed by SPR (K
d
= 400 nM vs 285 nM, respectively) and hGH
related TMDD. Similarly, the shorter half-life of hGH-Fc compared to hGH-sc(Fc)
2
is also
likely due to its lower FcRn binding affinity (K
d
= 1.43 µM vs 400 nM, respectively), and
hGH-related TMDD reduced the difference in half-lives. Another possible contributing
factor is that in the in vivo situation the dimeric structure of hGH-Fc could be disrupted
forming a higher amount of lower molecular weight monomers of hGH-Fc. Since the
molecular weight of hGH-Fc monomers (~47 kDa) are slightly below the glomerular
filtration molecular weight cutoff (50-60 kDa), the shorter half-life of hGH-Fc could be a
result of higher kidney elimination of the monomers. Our in vivo data of the two control
groups, hGH and hGH-Fc, are consistent with the previously reported
pharmacokinetic/pharmacodynamic studies of hGH-Fc in male Sprague-Dawley rats from
another group (Kim et al. 2015). In their study, hGH-Fc had a much longer plasma half-
life than hGH. After a 14-day daily injection in hypophysectomized rats, the high-dose
hGH-Fc group showed a statistically significant increase in weight gain compared with
hGH group.
76
Absorption after s.c. injection is slow because of convention of the lymphatic
system, passive paracellular diffusion, and receptor-mediated transport (Datta-Mannan et
al. 2012). The s.c. PK data of hGH-sc(Fc)
2
and hGH-Fc indicates that the absorption of
hGH-sc(Fc)
2
is significantly better than hGH-Fc, which might be due to FcRn-mediated
transport. However, the NCA calculated elimination half-lives are different from the i.v.
PK data. This discrepancy may be explained by flip-flop kinetics (Katakam et al. 1997;
Yáñez et al. 2011). The absorption was slow and served as the rate limiting step after s.c.
injection for large fusion proteins, but the instinct elimination rate is high according to the
i.v. PK data. The difference in elimination rates was masked by the slow absorption process.
4.5. Summary
In this chapter, the PK and PD of hGH-sc(Fc)
2
fusion protein were investigated in
mouse models. Compared with hGH-Fc and hGH, hGH-sc(Fc)
2
has significantly prolonged
elimination half-life and stronger PD response, suggesting that sc(Fc)
2
fusion is a better
design compared with traditional Fc fusion. The potential use of sc(Fc)
2
as an oral delivery
carrier still needs further investigation.
77
CHAPTER 5 SUMMARY, CHALLENGES AND FUTURE
PERSPECTIVES
5.1. Summary
Increasing knowledge about the biology of IgG molecules is promoting
mechanism-based optimization of existing protein therapeutics, most of which are based
on the binding properties of Fc domain of IgG to FcRn. Fc fusion protein technology has
been successfully used to generate long-acting forms of several protein therapeutics.
In this dissertation, we describe the design of a novel single chain Fc-based drug
carrier, sc(Fc)
2
, and evaluated sc(Fc)
2
-mediated delivery using a therapeutic protein with a
short plasma half-life as the protein drug cargo. This novel Fc-based drug carrier was
designed to contain two Fc domains recombinantly linked via a flexible linker. Since the
Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted
to further stabilize it against proteolysis and reduce FcγR-related effector functions. The
resultant sc(Fc)
2
candidate preserved the FcRn binding. sc(Fc)
2
-mediated delivery was then
evaluated using hGH as the drug cargo. This novel carrier protein showed a prolonged in
vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based
drug carrier.
Research focusing on the non-invasive delivery of therapeutic proteins using the
transcytosis pathway mediated by FcRn to facilitate uptake shows the potential value of Fc
in developing novel oral therapies. Our novel sc(Fc)
2
fusion protein, showed better
transcytosis efficiency than traditional Fc fusion protein in in vitro model system. However,
due to the complexity of the transport mechanism and the obstacles during oral
administration, more efforts are needed to optimize this delivery system.
78
5.2. Challenges
Currently, we only have evidence indicating the structure of those fusion proteins
in the storage buffer, but in vivo situations are much more complicated. We haven’t found
good tools to study what really happened in vivo.
In our PK studies, we used two different detection method, quantitative Western
blot and RIA. Quantitative Western blot allows us to quantify the amount of intact protein,
but it has relatively high systemic error because of the protein A pull down procedure to
eliminate the influence of high concentrations of blood proteins. hGH RIA can detect hGH
structure which means it can detect hGH fusion proteins with partially cleaved carrier.
According to our preliminary data, the anti-hGH antibody in this RIA kit recognize hGH
fusion proteins with different affinities. Therefore, the concentration of intact fusion
proteins in the plasma were not accurately determined. The emerging mass spectrum-based
protein analytical method may be able to solve this problem.
One of the goals of developing sc(Fc)
2
fusion proteins is to apply them as an oral
delivery carrier. However, finding a proper animal model that can provide meaningful
translational data is a knotty issue and hopefully could be solved in the future with more
advanced technologies.
Due to the variation in the pattern of FcRn expression, transgenic mice expressing
human FcRn, in which hFcRn expression is controlled by the endogenous human promoter
were used as a ‘translational’ model for the assessment of FcRn-based therapeutics
(Roopenian and Akilesh 2007; Petkova et al. 2006; Zalevsky et al. 2010). Similar
expression levels of hFcRn were found within the intestinal epithelium of
hFcRnTg/hβ2mTg/mFcRn-/- mice and native human intestinal epithelial cells (Yoshida et
79
al. 2004). However, the limited interspecies IgG binding of human FcRn leads to the
argument on the lack of endogenous IgG competition in this animal model.
Neonatal rats have been used to study intestinal uptake of IgG after regional
intestinal delivery (Kliwinski et al. 2013). Because the baseline levels of GH and IGF-1
are expected to be high in neonatal rodents, GH-deficient hypophysectomized neonatal rats
may be used as an animal model to test oral hGH-sc(Fc)
2
efficacy. However, the surgery
success rate in adult rats is only about 50% based on our experience. The procedure could
be more difficult in neonatal rats, leading to an even lower success rate. Moreover, the
experimental time frame is very limited due to the expression pattern of FcRn.
When biologics provide more treatment options with various mechanisms, new
challenges come with these newer drugs. Immunogenicity is one of the issues that needs
attention during drug development. The induction of immune responses to administered
therapeutic protein could lead to complexities in its pharmacokinetic and
pharmacodynamic profiles, toxicity and safety. Anti-drug antibodies (ADA) can cause
allergic or anaphylactic reactions and reduction in efficacy. Both binding and neutralizing
ADAs have the likelihood to impact the clearance of therapeutic proteins. The immune
complexes of ADA-therapeutic protein circulating in the bloodstream trigger regular
endogenous elimination processes, constituting an additional elimination pathway for
therapeutic protein. In the case of some fusion proteins, it may result in severe adverse
effect when neutralizing ADAs cross-react with autologous protein antigens. Our Fc-based
fusion proteins contains endogenous protein domains (e.g. hGH) and foreign linker (e.g.
(G
4
S)n) sequences, which makes them potentially facing immunogenicity problems.
Although we have already incorporate the human cell expression system to minimize the
80
risk, other de-risking strategies could also be considered to further optimize our carrier
protein to fulfill long-term treatment need.
5.3. Future Perspectives
Versatile recombinant technology allows us to fuse different types of therapeutic
proteins to our carrier system to achieve improved delivery properties through different
administration routes.
Oral protein delivery faces a lot of challenges and many approaches have been
studied, including use of absorption enhancers and enzymatic inhibitor, mucoadhesive
polymeric systems, formulation vehicles, derivatization or chemical modification of
proteins and peptides, cell penetrating peptides, endogenous cell carrier system, etc
(Muheem et al. 2016). Our study can be classified into the endogenous cell carrier system
approach. To achieve clinically desired oral bioavailability of protein, further optimization
of our current carrier system and incorporation with other types of approaches are needed.
81
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Abstract (if available)
Abstract
Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this dissertation, we describe the design of a novel single chain Fc-based drug carrier, sc(Fc)₂, and evaluated sc(Fc)₂-mediated delivery using a therapeutic protein with a short plasma half-life as the drug cargo. This novel Fc-based drug carrier was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc)₂ candidate preserved the FcRn binding. sc(Fc)₂-mediated delivery was then evaluated using human growth hormone (hGH) as the drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. Research focusing on the non-invasive delivery of therapeutic proteins using the transcytosis pathway mediated by FcRn to facilitate uptake showed the potential value of Fc in developing novel oral therapies. Our novel sc(Fc)₂ fusion protein, showed better transcytosis efficiency than traditional Fc fusion protein in cell-based model system. However, due to the complexity of the transport mechanism and the obstacles during oral administration, more efforts are needed to optimize this delivery system. Taken together, sc(Fc)₂ technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics of therapeutic proteins and bioavailability in protein oral delivery.
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University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Zhou, Li
(author)
Core Title
Developing recombinant single chain Fc-dimer fusion proteins for improved protein drug delivery
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Molecular Pharmacology and Toxicology
Publication Date
03/04/2019
Defense Date
11/28/2017
Publisher
University of Southern California
(original),
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(digital)
Tag
Fc dimer,Fc fusion,FcRn,flexible linker,fusion protein,Growth Hormone,long acting,OAI-PMH Harvest,oral,protein delivery,single chain,trancytosis
Language
English
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(provenance)
Advisor
Shen, Wei-Chiang (
committee chair
), Okamoto, Curtis (
committee member
), Zhang, Yong (
committee member
)
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zhou372@usc.edu
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https://doi.org/10.25549/usctheses-c40-479806
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Tags
Fc dimer
Fc fusion
FcRn
flexible linker
fusion protein
long acting
oral
protein delivery
single chain
trancytosis