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Site-specific PEGylation of recombinant immunotoxin antiFAP-CD8h-PE38 with an expanded genetic code

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Site-specific PEGylation of recombinant immunotoxin antiFAP-CD8h-PE38 with an expanded genetic code

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Site-specific PEGylation of recombinant immunotoxin antiFAP-CD8h-PE38
with an expanded genetic code

by

Qianqian Zhao

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

August 2016

Copyright 2016 Qianqian Zhao
I

Table of Contents
Acknowledgements ..................................................................................................................... III
List of Figures .............................................................................................................................. IV
List of Tables ............................................................................................................................... VI
Abbreviations ............................................................................................................................. VII
Abstract ........................................................................................................................................ IX
1. Introduction ............................................................................................................................... 1
1.1 Microenvironment of the tumor ............................................................................................ 1
1.2 Tumor-associated fibroblasts (TAFs) are a novel target for tumor therapy .......................... 1
1.3 AntiFAP-CD8h-PE38, a novel therapeutic against TAFs in breast cancer ........................... 3
1.4 PEGylation, an effective way to increase circulation half-life ............................................ 10
1.5 Increase the half-life of antiFAP-CD8h-PE38 by site-specific PEGylation using unnatural
amino acids ................................................................................................................................ 11
1.6 Genetic incorporation of unnatural amino acids ................................................................. 13
1.7 Rationale of this project ...................................................................................................... 15
2. Materials and methods ........................................................................................................... 18
2.1 Equipment and Reagents ..................................................................................................... 18
2.2 TAG mutations on antiFAP-CD8h-PE38 gene by site-directed mutagenesis ..................... 19
2.3 Construction of pEVOL plasmid containing pAcFRS/tRNA pair ...................................... 22
2.4 Expression, purification and refolding of antiFAP-CD8h-PE38 ........................................ 24
II

2.5 Further purification of antiFAP-CD8h-PE38 by size exclusion chromatography (SEC) ... 26
2.6 PEGylation of antiFAP-CD8h-PE38 ................................................................................... 26
2.7 Purification of PEGylated antiFAP-CD8h-PE38 ................................................................ 26
3. Results ...................................................................................................................................... 27
3.1 Four mutation sites on antiFAP-CD8h-PE38 gene and mutation primers .......................... 27
3.2 Expression and purification of wild-type and pAcF incorporated antiFAP-CD8h-PE38 ... 30
3.3 Further purification of antiFAP-CD8h-PE38 by SEC......................................................... 36
3.4 PEGylation of antiFAP-CD8h-PE38 ................................................................................... 46
3.5 Purification of PEGylated antiFAP-CD8h-PE38 ................................................................ 49
4. Discussion................................................................................................................................. 52
References .................................................................................................................................... 54

III

Acknowledgements
I hereby would like to express my heartfelt gratitude to my mentor, Dr. Jianming Xie, for his
guidance and advice during my master’s study in the University of Southern California (USC). It
has been a great experience for me to work in his laboratory, from which I learned not only research
skills but also scientific thinking.
Moreover, I would like to show my appreciation to Dr. Pin Wang for his guidance on the project
and advice on my master thesis.
I also would like to thank one of my committee members, Dr. Curtis T. Okamoto, for his
encouragement and guidance during my graduate studies in USC and for his suggestions on the
thesis.
Finally, I also want to thank Dr. Liang Rong and Rebecca Lim for their help and advice in my
experiments and this thesis. I additionally thank my other lab mates—Ting Fu, Ze Wang and Tao
Ma —for their support.

IV

List of Figures
Figure 1. Diagram of construction of immunotoxin antiFAP-CD8h-PE38.
Figure 2. The crystal structure of PE (PDB:1IKQ).
Figure 3. Antitumor effect of antiFAP-CD8h-PE38 in mice.
Figure 4. Relative mRNA expression of cytokines and chemokines in 4T1 tumor tissue from breast
tumor mice model after four 0.5mg/kg injections of antiFAP-CD8h-PE38 or PE38.
Figure 5. Schematic of PEGylation reaction with pAcF incorporated protein.
Figure 6. Diagram of unnatural amino acid incorporation.
Figure 7. Unnatural amino acid incorporation sites (W281, A301, T355) on PE38 (PDB: 1IKQ).
Figure 8. pET28a-antiFAP-CD8h-PE38 plasmid map.
Figure 9. pEVOL-aaRS plasmid map.
Figure 10. Schematic of refolding of antiFAP-CD8h-PE38 by gradient dialysis.
Figure 11. The DNA and amino acid sequence of antiFAP-CD8h-PE38 gene with four mutations
sites.
Figure 12. SDS-PAGE analysis of purified wild-type and antiFAP-CD8h(C29AcF)-PE38 by Ni-
NTA column with high concentrations of imidazole.
Figure 13. SDS-PAGE analysis of purified mutant antiFAP-CD8h-PE38 by Ni-NTA column with
reduced Imidazole concentration.
Figure 14. SDS-PAGE analysis of purified antiFAP-CD8h-PE38(W281AcF) and antiFAP-
PE38(A301AcF) by Ni-NTA column eluted with low pH buffers.
V

Figure 15. Further purification by SEC of wild-type antiFAP-CD8h-PE38.
Figure 16. Further purification by SEC of antiFAP-CD8h(C29AcF)-PE38.
Figure 17. Further purification by SEC of antiFAP-CD8h-PE38(W281AcF).
Figure 18. Further purification by SEC of antiFAP-CD8h-PE38(A301AcF).
Figure 19. Further purification by SEC of antiFAP-CD8h-PE38(T355AcF).
Figure 20. Confirmation of PEGylated proteins.
Figure 21. Purification of PEGylated antiFAP-CD8h-PE38 by Superdex 200 10/300 column.

VI

List of Tables
Table 1. Designed primers for TAG mutation of four chosen sites.

VII

Abbreviations
aaRS, aminoacyl-tRNA-synthetase
E. coli, Escherichia coli
ECM, Extracellular Matrix
eEF2, eukaryotic Elongation Factor 2
FAP, Fibroblast Activation Protein
IPTG, Isopropyl β-D-1-thiogalactopyranoside
MMPs, Matrix Metalloproteinases
mPDA, m-Phenylenediamine
mRNA, Messenger RNA
NSAIDs, Nonsteroidal Anti-Inflammatory Drugs
pAcF, p-Acetylphenylalanine
PBS, Phosphate Buffered Saline
PE, Pseudomonas Exotoxin
PEG, Polyethylene Glycol
scFv, Single-Chain Variable Fragment
SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
TAFs, Tumor-Associated Fibroblasts
TGF-β, Transforming Growth Factor- β
VIII

tRNA, Transfer RNA
VEGF, Vascular Endothelial Growth Factor

IX

Abstract
Tumor-associated fibroblasts (TAFs)—a prominent cellular component in the tumor
microenvironment—play an important role in tumor growth and metastasis, which makes it a novel
target for tumor therapy. Previous studies have shown that inhibition of TAFs attenuated tumor
growth in mice. Recently, a recombinant antibody-toxin conjugate—antiFAP-CD8h-PE38—was
constructed by our collaborators Dr. Pin Wang and colleagues. This toxin targets TAFs by
recognizing fibroblast activation protein (FAP), an over-expressed biomarker on the surface of
TAFs. This immunotoxin worked effectively on inhibiting tumor growth and reducing
tumorigenesis-related chemokines and cytokines in vivo. However, the plasma half-life of
antiFAP-CD8h-PE38 in vivo is less than 30 minutes, which restricts its further application in the
clinic. In this study, a single linear 30 kDa Polyethylene Glycol (PEG) was site-specifically
conjugated to antiFAP-CD8h-PE38 at the incorporated unnatural amino acid p-
acetylphenylalanine (pAcF). The purpose of this conjugation is to increase the half-life of antiFAP-
CD8h-PE38. Four mutation sites in the antiFAP-CD8h-PE38 were chosen to incorporate pAcF
based on its crystal structure. The pAcF incorporated antiFAP-CD8h-PE38s were expressed in E.
coli and further purified by size exclusion chromatography (SEC). PEGylation of antiFAP-CD8h-
PE38 was conducted afterwards, and the PEGylated protein was confirmed and purified. It turned
out that the C29 and T355 incorporated antiFAP-CD8h-PE38 were more efficiently conjugated
with PEG compared to other mutation sites on the antiFAP-PE38, but the conditions for
PEGylation should be further optimized to increase the yield.
1

1. Introduction
1.1 Microenvironment of the tumor
Tumorigenesis is not only attributed to genetic mutation, but also to the corresponding
microenvironment. The tumor microenvironment tends to be referred to as the “soil” for the tumor
progression and includes the extracellular matrix (ECM), blood vessels, immune cells, fibroblasts,
and various cytokines (Tlsty and Coussens, 2006). The ECM-containing structural proteins,
specialized proteins, and proteoglycans provides a three dimensional structure to support the
surrounding cells. It has been shown that during tumor development, ECM expression and
degradation will be altered abnormally. Blood vessels—as the major constituent of the tumor
microenvironment—are responsible for providing oxygen and nutrients needed for tumor survival
and metastasis (Carmeliet and Jain, 2000). Formation of vessels results from the imbalance
between pro- and anti-angiogenic molecules released by tumor cells, endothelial cells, and ECM
(Carmeliet and Jain, 2000; Fukumura et al., 1998). Inhibition of pro-angiogenic molecules, like
vascular endothelial growth factor (VEGF), has been proven effective in the treatment of
metastatic breast cancer (Miller et al., 2007). The close association between chronic inflammation
and tumor has been also widely accepted. Evidence has shown that over 15% tumors were induced
by infection worldwide (Kuper et al., 2000). The chemokines secreted by the immune cells as well
as other genotoxins are likely to be involved in the regulation of tumor growth and metastasis
(Coussens and Werb, 2002). Long-term used nonsteroidal anti-inflammatory drugs (NSAIDs)
have been shown to reduce colon cancer risk up to 50% (Baron and Sandler, 2000).
1.2 Tumor-associated fibroblasts (TAFs) are a novel target for tumor therapy
Fibroblasts, as the major cells in the stroma, are recognized to play a more critical role in tumor
development and metastasis than previously expected. In the tumor, these cells are also referred to
2

as tumor-associated fibroblasts (TAFs). TAFs not only synthesize, decompose, and remodel the
ECM, but these fibroblasts also secrete chemokines and cytokines which promote tumor growth
and angiogenesis. (Bhowmick et al., 2004; Mueller and Fusenig, 2004). In normal tissue, tissue
homeostasis is modulated by the crosstalk between epithelial cells and stromal cells. The precursor
cells of the stroma secrete ECM and growth factors to induce the surrounding epithelial cells to
proliferate. As epithelial cells differentiate, they release cytokines to induce the differentiation of
precursor cells to stromal cells (Cunha et al., 1985). However, normal epithelial cells can become
malignant upon the accumulation of genetic mutations due to inflammation or wound. This cellular
transformation causes these tumor cells to secrete more growth factors to accommodate tumor
growth, resulting in dramatic alterations in the surrounding stroma. One major alteration in the
stroma is the transformation of normal fibroblasts to TAFs, and this transformation is stimulated
by tumor cell secretion of different cytokines, such as transforming growth factor-β (TGF-β). In
the process of transformation, the fibroblasts begin to overexpress fibroblast activation protein
(FAP) on the surface. (Zi et al., 2015). TAFs in turn express several growth factors and cytokines
that promote proliferation of pre-malignant and malignant cells and communication with immune
cells in autocrine and paracrine ways (Bhowmick et al., 2004). TAFs are also a primary source for
producing matrix metalloproteinases (MMPs), the important ECM-degrading enzymes which
promotes tumor invasion and metastasis (Bremnes et al., 2011). As described above, TAFs have
emerged as a novel target for tumor therapy. It is reported that in more than 90% of epithelial
tumors, FAP is excessively expressed on TAFs (Liu et al., 2012). Therefore, FAP can be a
promising biomarker for targeting TAFs. Indeed, previous studies have shown that depletion of
FAP genes attenuates tumor growth in mice (Santos et al., 2009) and increases uptake of
chemotherapy drugs in multidrug-resistant tumors (Loeffler et al., 2006).
3

1.3 AntiFAP-CD8h-PE38, a novel therapeutic against TAFs in breast cancer
Previously, investigators have searched for therapeutics against FAP, including small molecules
and large biologics. However, these small molecules did not exert complete inhibition of FAP
activity, thus causing minimal response in patients during clinical trials (Narra et al., 2007). One
potential reason for the ineffectiveness of these small molecules is that these molecules can
undergo cyclization at the tumor (Coutts et al., 1996). Small molecule inhibition is also dependent
on the enzymatic activities of FAP, but tumor growth can depend on FAP non-enzymatic activities
(Huang et al., 2011). As a result, these small-molecule therapeutics will have little to no effect at
the tumor site. Therefore, more effective and specific therapeutics against FAP should be
developed. Currently, large biologics have shown the advantages of specificity and efficacy
compared to small molecules. In our collaborator’s recent study, they have constructed a novel
recombinant immunotoxin antiFAP-CD8h-PE38 to suppress tumor progression in mouse breast
tumor models (Fang et al., 2016a). This recombinant immunotoxin contains two parts: antiFAP
and PE38 (Figure 1).

4

Figure 1. Diagram of construction of immunotoxin antiFAP-CD8h-PE38

5

AntiFAP is a single chain variable fragment (scFv) of the FAP monoclonal antibody which can
bind to the FAP specifically. The scFv, consisting of 250 amino acids, is a fusion protein
containing the variable regions of the heavy and light chains of the FAP monoclonal antibody
connected with a short flexible linker peptide of 25 amino acids. This scFv maintains the same
function as the original antibody in the recognition of antigen. PE38 is a 38kDa truncation of
Pseudomonas exotoxin (PE). PE acts as an inhibitor of eukaryotic elongation factor 2 (eEF2) in
protein translation, causing protein synthesis to cease (Yates and Merrill, 2004). There are three
domains in PE (Figure 2): domain I as the receptor binding domain, domain II as the translocation
domain which helps to translocate PE into the cytoplasm, and domain III as the catalytic domain
catalyzing the ADP-ribosylation of eEF2, inhibiting protein synthesis (Yates and Merrill, 2004;
Yates et al., 2005).

6

Figure 2. The crystal structure of PE (PDB:1IKQ). Domain I is shown in cyan (1-252) and
green (365-404). Domain II is in yellow (253-364), and domain III is in magenta (405-613).

7

PE38 consists of part of domain I (365-404), domain II and domain III. AntiFAP and PE38 are
connected by a short peptide CD8 hinge (CD8h) containing 48 amino acids, which might assist to
improve the expression of the scFv (Classon et al., 1992).
This construction has been shown to slow down tumor growth in vivo and change the tumor
microenvironment by downregulating the secretion of cytokines. 4T1 breast tumor buried female
BALB/c mice were used as the model to test the antitumor effect of antiFAP-CD8h-PE38. Mice
receiving four sequential 0.5mg/kg injections showed remarkable decrease in tumor volume
(Figure 3), and no difference in body weight and bone marrow toxicity was observed between the
control group and antiFAP-CD8h-PE38-treated group (Figure 4). Moreover, the expression of
cytokines TGF-β, MMP-9, and VEGF and chemokines CCL5, MCP-1, and SDF-1 in 4T1 tumor
tissue were reduced in the antiFAP-CD8h-PE38-treated group compared to the control (Fang et
al., 2016b).

8

A B C

Figure 3. Antitumor effect of antiFAP-CD8h-PE38 in mice. A) Tumor volume measured after
four injections of vehicle, PE38 or antiFAP-CD8h-PE38 in 4T1 breast tumor model. B) Body
weight measured from control group and antiFAP-CD8h-PE38 treated group in 4T1 breast tumor
model. C) The amount of live cells measured in bone marrow from control and antiFAP-CD8h-
PE38-treated group in 4T1 breast tumor model (Fang et al., 2016b).

9

Figure 4. Relative mRNA expression of cytokines and chemokines in 4T1 tumor tissue from
breast tumor mice model after four 0.5mg/kg injections of antiFAP-CD8h-PE38 or PE38
(Fang et al., 2016b).

10

1.4 PEGylation, an effective way to increase circulation half-life
Although this recombinant immunotoxin demonstrated a promising anti-tumor effect in vivo, its
half-life in blood circulation is less than 30 minutes, which restricts its further application in clinic.
The most common method to increase the half-life of a biologic at present is to attach the
polyethylene glycol (PEG) group on it. PEG, a linear polymer, is able to protect the protein which
it is conjugated to by spreading away the surrounding proteases and macrophages. Moreover,
adding a large molecular weight PEG on the protein helps to reduce kidney clearance, which is
also an important contributor to the short half-life of a protein in vivo. It is reported that the half-
life of PEGylated interferon α-2a for treatment of hepatitis C has increased 70-fold, and no
immunogenicity was observed compared to the non-PEGylated one in vivo (Bailon et al., 2001).
Thus, to increase the circulation half-life of antiFAP-CD8h-PE38, a single linear 30kDa PEG will
be conjugated to this immunotoxin.
Traditionally, the two common ways to conjugate a PEG to proteins is to activate the PEG with
a functional group that can react with either a primary amine group on lysine or a sulfhydryl group
on cysteine (Roberts et al., 2012). However, the drawbacks of these two routes are obvious. For
lysine conjugation, the protein bioactivity may be impaired due to the nonspecific conjugation of
PEG with the lysine located in the active site of protein. Moreover, the heterogeneity of the PEG-
protein conjugates is another concern for pharmaceutical manufacturing. The reason is that the
protein is likely to be conjugated with more than one PEG group, since the number of lysines can
make up about 10% of the total amount of amino acids in a protein. Additionally, the PEG
substitution positions on the protein may change every time. For the cysteine reaction, the
bioactivity of a protein may remain the same upon PEG conjugation, and heterogeneity is less
likely to happen since the number of cysteines in the protein is none or less than that of lysine.
11

Nevertheless, in order to achieve site-specific PEG conjugation in some cases, one or more
cysteines are introduced to the protein by genetic engineering (Tsutsumi et al., 2000). However,
the introduced cysteine can potentially form the wrong disulfide bonds which may affect the
protein bioactivity.
1.5 Increase the half-life of antiFAP-CD8h-PE38 by site-specific PEGylation using unnatural
amino acids
In order to increase the half-life of antiFAP-CD8h-PE38, maintain its bioactivity, and avoid
heterogeneity, an aminooxy-PEG will be site-specifically conjugated to an incorporated unnatural
amino acid, p-acetylphenylalanine (pAcF), at a certain location of antiFAP-CD8h-PE38 for this
study. The nucleophilic amine group of aminooxy-PEG attacks the ketone group of pAcF to form
a stable oxime bond (Figure 5). Moreover, an appropriate amount of meta-phenylenediamine
(mPDA) is added in the reaction as the catalyst, which is reported to improve the reaction rate in
the oxime ligation (Mahmoodi et al., 2015).

12

Figure 5. Schematic of PEGylation reaction with pAcF incorporated protein.

13

1.6 Genetic incorporation of unnatural amino acids
Protein translation is an intriguing process in nature. There are several components in protein
translation: messenger RNA (mRNA), ribosome, transfer RNA (tRNA), and 20 natural amino
acids. These 20 amino acids, preserved among all living organisms, serve elegantly as the building
blocks of all biological constructs. On the other hand, it is possible to introduce additional
complexity and function into proteins by incorporating chemically synthesized unnatural amino
acids with extra functional groups. In recent years, methods of incorporating unnatural amino acids
into proteins have developed rapidly, which provide an opportunity to probe protein structure and
function or attach new physical and chemical properties to proteins for therapeutic purposes.
However, there are limitations to some of these current techniques. For instance, solid-phase
peptide synthesis has the capability of synthesizing the protein with a maximum of about 60 amino
acids, which limits its application in producing larger proteins. The incorporation of unnatural
amino acids in a cell-free protein translation system in vitro is able to synthesize proteins with
more than 80 amino acids, but it is hard to charge the tRNA with its specific unnatural amino acid
through a chemical reaction, and the tRNA is used stoichiometrically in the system (Noren et al.,
1989). An alternative method suitable for larger scale manufacturing and for technical ease is
required. Then in vivo protein expression with unnatural amino acid incorporation was developed
by Dr. Schultz and colleagues (Figure 6). For instance, unnatural amino acids were incorporated
into recombinant proteins in a site-specific manner in Escherichia coli (E. coli) (Wang et al., 2001;
Xie and Schultz, 2005).
14

A

C

Figure 6. Diagram of unnatural amino acid incorporation. A) Aminoacylation on tRNA by
normal TyrRS (http://protein.gsc.riken.go.jp/sakamoto/research_en.html) B) Aminoacylation on
tRNA by selective TyrRS (http://protein.gsc.riken.go.jp/sakamoto/research_en.html) C)
Incorporation of UAA during translation (http://www.fjbio.com.tw/index.php?do=
prod&toppid=87&pid=169&id=274).

B
15

A unique stop codon TAG (amber codon) and congenial aminoacyl-tRNA-synthetase
(aaRS)/tRNA pair were used to incorporate an unnatural amino acid into proteins with high fidelity
and efficiency. The consideration of this method is that the new components added should be
orthogonal, not cross-reactive, to the endogenous components in E. coli. Thus, the specific
aaRS/tRNA pair from Methanococcus jannaschii (M. jannaschii) (Wang et al., 2000) was chosen
to incorporate unnatural amino acids in E. coli. The selective M. jannaschii TyrRS recognizes both
the corresponding tRNA, also referred to suppressor tRNA, and the unnatural amino acid, which
is specifically added onto the 3’ end of tRNA. The suppressor tRNA carrying the unnatural amino
acid is able to recognize the rare stop codon, UAG, on the mRNA and deliver the unnatural amino
acid at that site. Meanwhile, the releasing factor 1 (RF1) in E. coli, which also recognizes the stop
codon, can compete with the amber-suppressor tRNA and stop protein synthesis, thus generating
a truncated protein. However, the truncated protein can be separated from the full-length protein
by using Nickel-NTA affinity chromatography purification if there is a His6 tag at the C terminus
of the full-length protein. In a previous study, researchers have used this technique to incorporate
an unnatural amino acid pAcF at different sites of a human growth hormone (hGH) and to
specifically conjugate the pAcF to a PEG group in order to improve the clinical performance of
hGH (Cho et al., 2011).
1.7 Rationale of this project
For this thesis project, four potential mutation sites on antiFAP-CD8h-PE38 were chosen to
incorporate pAcF independently based on these following observations on the crystal structure: (1)
The incorporated pAcF should be fully exposed on the protein surface, which makes it more
accessible for PEGylation. (2) Moreover, the substitution of pAcF should not disturb the structure
and function of the original protein. Therefore, the 29
th
cysteine residue of the CD8 hinge and the
16

other three sites W281, A301, T355 on the domain II of PE38 were chosen to be potential
PEGylation sites (Figure 7).

17

Figure 7. Unnatural amino acid incorporation sites (W281, A301, T355) on PE38 (PDB:
1IKQ).

18

The DNA codons encoding the chosen residues of the antiFAP-CD8h-PE38 gene (in the pET28a
plasmid) were mutated to TAG by site-directed mutagenesis. The two copies of pAcF aminoacyl-
tRNA-synthetase (aaRS) gene were inserted into the pEVOL plasmid, which also contains the
gene encoding corresponding amber-suppressor tRNA. The two plasmids were double-
transformed into E. coli BL21 (DE3) to express four different pAcF-incorporated antiFAP-CD8h-
PE38. Then Ni-NTA affinity chromatography was used to remove the truncated protein before
refolding the denatured protein by gradient dialysis. The refolded protein was further purified by
size exclusion chromatography (SEC) and afterwards, the purified pAcF incorporated antiFAP-
CD8h-PE38 was conjugated to aminooxy-PEG using the proper condition. Finally, the generation
of PEGylated antiFAP-CD8h-PE38 was confirmed by SDS-PAGE analysis, and the product was
further purified by cation exchange chromatography and SEC. The purified samples were then
stored in 50% glycerol for pharmacologic and pharmacokinetic studies in vivo.
2. Materials and methods
2.1 Equipment and Reagents
The UV-Vis spectrophotometer Nanodrop 2000 was purchased from Thermo Scientific
(Rockford, IL). T100 Thermal Cycler PCR machine was obtained from Bio-Rad (Hercules, CA).
HisTrap HP Ni-NTA column and Superdex 200 Increase 10/300 GL SEC column were purchased
from GE Healthcare (Chicago, IL). Amicon Ultra concentrators were purchased from Merk
Millipore (Temecula, CA). SnakeSkin pleated dialysis tubing was purchased from Pierce
(Rockford, IL). Bis-Tris SDS-PAGE gels were purchased from Invitrogen (Waltham, MA) and
GenScript (Pascataway, NJ). QIAprep Spin Miniprep Kit was purchased from QIAGEN (Valencia,
CA). QuikChange Site-Directed Mutagenesis Kit and XL-1 blue supercompetent E. coli were
purchased from Agilent Technologies (Santa Clara, CA). Quick PCR Purification Kit, DH5α
19

Subcloning competent cells and Pfx DNA polymerase were purchased from Invitrogen (Waltham,
MA). BL21(DE3) competent cells, T4 ligase, Bgl II, Sal I-HF, Nde I and Pst I-HF were purchased
from NEB (Ipswich, MA). Zymoclean Gel DNA Recovery Kit, Luria Broth (LB) powder and
IPTG were purchased from Genesee Scientific (San Diego, CA). pAcF powder was produced in
the lab. Urea and Protease inhibitor tablets were purchased from Thermo Scientific (Rockford, IL).
The 30 kDa Aminooxy-PEG powder was purchased from Sunbright (Tokyo, Japan). mPDA and
L-arabinose were purchased from Sigma-Aldrich (St. Louis, Missouri). Phosphate Buffered Saline
(PBS) powder was purchased from Gibco (Grand Island, NY).
The pET28a-antiFAP-CD8h-PE38 plasmid encoding antiFAP-CD8h-PE38 (Figure 8) was
obtained from our collaborator, Dr. Pin Wang’s lab. The pEVOL and pBK-AcFRS plasmids were
stored in our lab.
Detergent buffer (1% sodium deoxycholate, 1% NP-40, 50 mM Tris pH 8.0, 200 mM NaCl, 1
mM EDTA, 1 mM DTT), Wash buffer I (0.5% Trition X-100, 50 mM Tris pH 8.0, 100 mM NaCl,
0.1% azide, 1 mM EDTA, 1 mM DTT) and Wash buffer II (50 mM Tris pH 8.0, 100 mM NaCl,
0.1% azide, 1 mM EDTA, 1 mM DTT) are handmade in lab.
2.2 TAG mutations on antiFAP-CD8h-PE38 gene by site-directed mutagenesis
pET28a-antiFAP-CD8h-PE38 was transformed into DH5α competent E. coli for amplification
and then was extracted by QIAprep Spin Miniprep Kit according to the manufacturer’s protocol.
The quikchange site-mutagenesis PCR was performed to mutate the chosen sites on the CD8 hinge
(C29) and PE38 toxin (W281, A301, and T355) to the amber codon, TAG, separately. The parental
plasmid templates without the mutation were digested first by Dpn I enzyme for 1 hour at 37 ℃
before transformation of the remaining mutated plasmids into XL-1 blue supercompetent E. coli
20

by heat shock at 42 ℃ for 45 seconds. The four pET28a-antiFAP-CD8h-PE38 plasmids with
mutant TAG on different sites were purified by QIAprep Spin Miniprep Kit and verified by DNA
sequencing.

21

Figure 8. pET28a-antiFAP-CD8h-PE38 plasmid map

22

2.3 Construction of pEVOL plasmid containing pAcFRS/tRNA pair
The pAcFRS gene carrying Bgl II and Sal I restriction sites was amplified from the pBK plasmid
by PCR using Pfx DNA polymerase. The amplified gene products were purified by the Gel DNA
Recovery Kit, followed by double digestion of these gene products and the pEVOL vector by Bgl
II and Sal I-HF restriction enzymes at 37 ℃ for at least 4 hours (Figure 9). The digested gene
products were purified by the PCR Purification kit, and the backbones of the pEVOL vector were
purified by the Gel DNA Recovery Kit. Afterwards, the purified gene products and backbones
were ligated by T4 ligase at 16 ℃ for 2 hours to construct the pEVOL-pAcFRS I-tRNA. The
ligation product was transformed to DH5α competent E. coli by heat shock at 42 ℃ for 22 seconds,
and the cells were grown on LB agar plates (containing 30 µg/mL chloramphenicol) and incubated
at 37 ℃ overnight. The expected pEVOL-pAcFRS I-tRNA vector was extracted from the bacteria
solution of a single colony on the plate and confirmed by DNA sequencing. Once the first pAcFRS
gene was incorporated upstream of the constitutive glnS promoter in the pEVOL, a second copy
of the pAcFRS gene would be inserted upstream of the inducible araBAD promoter to increase the
yield of pAcFRS during protein synthesis. The procedure is the same as above mentioned. The
second pAcFRS gene carried the Nde I and Pst I restriction sites, and the purified gene products
and vectors were subjected to double digestion by Nde I and Pst I-HF restriction enzymes later.
Finally, the resulting pEVOL-pAcFRS I-pAcFRS II-tRNA vector was verified by DNA
sequencing.
23

Figure 9. pEVOL-aaRS plasmid map (Young et al., 2010).

24

2.4 Expression, purification and refolding of antiFAP-CD8h-PE38
The wild-type or mutant pET28a-antiFAP-CD8h-PE38 and pEVOL-pAcFRS I-pAcFRS II-
tRNA were double-transformed into BL21(DE3) E. coli via heat shock at 42 ℃ for 10 seconds.
The cells were grown on an LB agar plate with kanamycin (0.05 mg/mL) and chloramphenicol
(0.03 mg/mL) and incubated at 37 ℃ overnight.
A single colony on the plate was picked up and inoculated in a small scale (5 mL) LB medium
first and then transferred to a medium scale (about 20 mL) LB medium. Afterwards, the medium
scale bacteria were inoculated in 1 L LB medium containing kanamycin (0.05 mg/mL),
chloramphenicol (0.03 mg/mL) and pAcF (final concentration 1 mM) and grew in a 37 ℃ shaker
at 225 rpm. When the OD600 of the medium was over 0.6, 20% L-arabinose (final concentration
0.2%) and 1 M isopropyl-b-D-1-thiogalactopyranoside (IPTG, final concentration 1 mM) were
added to induce protein expression. For the wild-type antiFAP-CD8h-PE38 expression, pAcF and
L-arabinose were not added in the medium. The expression process continued at 37 ℃, 220 rpm
for 4 hours. Then cells were collected by centrifugation and lysed by lysis buffer (1 mM PMSF, 1
mM DTT, 10 mM MgCl2, 1 unit DNAase I, 0.001 mg/mL pepstatin A, 1 mg/mL lysozyme, 0.001
mg/mL leupeptin) at room temperature for 1 hour, followed by sonication. The antiFAP-CD8h-
PE38 (WT or mutated) inclusion bodies were separated and cleared of impurities by washing with
detergent buffer, wash buffer I and wash buffer II for several times and dissolved in denature buffer
containing 8 M urea. The presence of the hexa-histidine tag (His6 tag) at the C terminus enabled
the denatured full-length antiFAP-CD8h-PE38 to be purified by the Ni-NTA column. Afterwards,
the purified inactive antiFAP-CD8h-PE38 was refolded in a pH 8.0 buffer by gradient dialysis of
the urea from 8 M to 0 M at 4 ℃ (Figure 10).
25

Figure 10. Schematic of refolding of antiFAP-CD8h-PE38 by gradient dialysis.

26

2.5 Further purification of antiFAP-CD8h-PE38 by size exclusion chromatography (SEC)
After refolding, antiFAP-CD8h-PE38 was concentrated down to an appropriate volume (less
than 1 mL) by using the 10 kDa MWCO Amicon Ultra concentrator, and was further purified to
remove multimers or dimers formed during the refolding process by size exclusion
chromatography. The protein samples of wild-type and pAcF incorporated antiFAP-CD8h-PE38
were purified through the Superdex 200 10/300 gel filtration column and run at a speed of 0.5
mL/min at 4 ℃ using 1X PBS. The samples of fractions from each peak shown on chromatograms
were loaded on Bis-Tris SDS-PAGE gels and stained with GelCode blue stain reagent. The
purified monomer antiFAP-CD8h-PE38 fractions were then collected for PEGylation.
2.6 PEGylation of antiFAP-CD8h-PE38
The purified collections of wild-type and pAcF incorporated antiFAP-CD8h-PE38 were further
concentrated to 70-100 µM, and the 1x PBS buffer was exchanged to 0.1 M sodium acetate pH 4.2
for the PEGylation reaction. At present, a small-scale (30 µL) reaction was performed, and the
final concentration of the protein in the reaction was 50 µM. The 5 mM 30 kDa aminooxy-PEG
was added to the antiFAP-CD8h-PE38 in 5X, 10X or 20X molar excess, and meta-
phenylenediamine (mPDA) catalyst was also added 22-fold the concentration of PEG. The reaction
was conducted at room temperature for 24 hours with gentle rotation. Then the PEGylation
efficiency was confirmed by running the reaction samples on Bis-Tris SDS-PAGE gels followed
by staining with GelCode blue stain reagent.
2.7 Purification of PEGylated antiFAP-CD8h-PE38
Afterwards, the PEGylated antiFAP-CD8h-PE38 was separated by Superdex 200 10/300 column
using a flow rate of 0.5 mL/min and 1xPBS running buffer at 4 ℃. The sample fractions of each
27

peak were loaded on the Bis-Tris gels to identify the PEGylated antiFAP-CD8h-PE38. The
purified PEGylated antiFAP-CD8h-PE38 was collected and stored at -20 ℃ with 50% glycerol.
3. Results
3.1 Four mutation sites on antiFAP-CD8h-PE38 gene and mutation primers
Four mutation sites were chosen on the antiFAP-CD8h-PE38 gene (Figure 11) and PCR primers
were designed for the TAG mutation (Table 1).

28

Figure 11. The DNA and amino acid sequence of antiFAP-CD8h-PE38 gene with four
mutations sites. A) The mutated C29AcF is the 29
th
cysteine of CD8 hinge which contains 48
amino acids. B) The three mutation sites (W281, A301, T355) on PE38.

B
A
29

Table 1. Designed primers for TAG mutation of four chosen sites.
Primers
for
C29
Mutation
CD8hinge
C29AcF-
Forward
primer:
5’-GCGCCCAGAGGCGTAGCGGCCAGCGGCGGG-3’

CD8hinge
C29AcF-
Reverse
primer:
5’-CCCGCCGCTGGCCGCTACGCCTCTGGGCGC-3’

Primers
for
W281
Mutation
PE38-
W281AcF-
Forward
primer:
5’-GCCAGCCGCGCGGCTAGGAACAACTGGAGCAG-3’

PE38-
W281AcF-
Reverse
primer:
5’-CTGCTCCAGTTGTTCCTAGCCGCGCGGCTGGC-3’

Primers
for
A301
Mutation
PE38-
A301AcF-
Forward
primer:
5’-CGCCCTCTACCTGGCGTAGCGGCTGTCGTGGAAC-3’

PE38-
A301AcF-
Reverse
primer:
5’-GTTCCACGACAGCCGCTACGCCAGGTAGAGGGCG-3’

Primers
for
T355
Mutation
PE38-
T355AcF-
Forward
primer:
5’-
CGCTTCGTCCGGCAGGGCTAGGGCAACGACGAGGCCC-
3’

PE38-
T355AcF-
Reverse
primer:
5’-
GGGCCTCGTCGTTGCCCTAGCCCTGCCGGACGAAGCG-
3’

30

3.2 Expression and purification of wild-type and pAcF incorporated antiFAP-CD8h-PE38
Wild-type and pAcF incorporated antiFAP-CD8h-PE38 were expressed in E. coli before the
inclusion bodies for these proteins were washed and dissolved in 8 M urea buffer. The denatured
wild-type and antiFAP-CD8h(C29AcF)-PE38 were purified by the Ni-NTA column with binding
buffer (25 mM imidazole), wash buffer (40 mM imidazole) and elution buffer (500 mM imidazole).
Samples of each fraction were loaded on the Bis-Tris gels to test the purity of antiFAP-CD8h-
PE38 (Figure 12). Some of the full-length antiFAP-CD8h-PE38 were eluted during the wash step,
especially for antiFAP-CD8h(C29AcF)-PE38. The potential reason may be that imidazole
concentrations in the binding buffer, wash buffer and elution buffer were a little too high for
antiFAP-CD8h(C29AcF)-PE38 purification.

31

Figure 12. SDS-PAGE analysis of purified wild-type and antiFAP-CD8h(C29AcF)-PE38 by
Ni-NTA column with high concentrations of Imidazole. BC, before column; FT, flow-
through; W, wash; E, elution.

32

Therefore, in order to prevent protein loss during the wash steps, the concentration of imidazole
in all buffers were further reduced to 20 mM in the binding buffer, 30 mM in the wash buffer, and
300 mM in the elution buffer (Figure 13). Reducing the imidazole concentration in Ni-NTA
purification was more effective to separate truncated proteins and reduce elution of full-length
protein in wash step for antiFAP-CD8h(C29AcF)-PE38 and antiFAP-CD8h-PE38(T355AcF), but
not for the antiFAP-CD8h-PE38(A301AcF) and antiFAP-CD8h-PE38(W281AcF) types. Almost
all the antiFAP-PE38(W281AcF) full-length protein and truncated one were eluted out at the same
time in the wash step.

33

Figure 13. SDS-PAGE analysis of purified mutant antiFAP-CD8h-PE38 by Ni-NTA column
with reduced imidazole concentration. BC, before column; FT, flow-through; W, wash; E,
elution. A) SDS-PAGE gel of purified antiFAP-CD8h(C29AcF)-PE38 and antiFAP-
PE38(T355AcF). B) SDS-PAGE gel of purified antiFAP-CD8h-PE38(W281AcF). C) SDS-PAGE
gel of purified antiFAP-CD8h-PE38(A301AcF).
B
C
A
34

Thus, to purify the full-length protein of antiFAP-CD8h-PE38(A301AcF) and antiFAP-CD8h-
PE38(W281AcF) , the low pH elution method (pH 6.3 wash buffer, pH 5.9 elution buffer I, pH 4.5
elution buffer II) was performed on the Ni-NTA column (Figure 14). Changing the pH of the wash
and elution buffers was more effective than imidazole elution to separate antiFAP-CD8h-
PE38(A301AcF). In the wash step, a large amount of truncated protein was eluted and there was
little truncated protein in elution II (pH 4.5). For antiFAP-CD8h-PE38(W281AcF) purification,
the low pH elution method eluted a large amount of truncated protein in the wash step without the
full-length protein, but the truncated one still existed in the full-length protein solution. The low
pH buffer elution was also not effective to purify antiFAP-CD8h-PE38(W281AcF). Elution II of
antiFAP-CD8h-PE38(A301AcF) and Elution I and II of antiFAP-CD8h-PE38(W281AcF) were
collected and refolded next.

35

Figure 14. SDS-PAGE analysis of purified antiFAP-CD8h-PE38(W281AcF) and antiFAP-
PE38(A301AcF) by Ni-NTA column eluted with low pH buffers. BC, before column; FT,
flow-through; W, wash; E, elution. A) SDS-PAGE gel of purified antiFAP-CD8h-
PE38(W281AcF). B) SDS-PAGE gel of purified antiFAP-CD8h-PE38(A301AcF).

A
B
36

3.3 Further purification of antiFAP-CD8h-PE38 by SEC
The eluted protein from Ni-NTA column was then refolded by the same gradient dialysis method.
The refolded proteins were further purified by SEC. Wild-type antiFAP-CD8h-PE38 was first
purified by Superdex 200 10/300 column with an approximate 24 mL column volume (Vc) at a
flow rate of 0.5 mL/min in 1xPBS buffer at 4 ℃. Since the elution volume of the concentrated
protein exceeded the recommended sample volume (0.1-1% of Vc), the Superdex 200 16/600
column with Vc of 120 mL was used to purify the other four pAcF incorporated antiFAP-CD8h-
PE38s for higher resolution. The sample fractions from each main peak were run on the Bis-Tris
gels to identify the purified antiFAP-CD8h-PE38. For all the antiFAP-CD8h-PE38s, the first peak
on each chromatogram should represent the multimers or dimers formed wrongly during protein
renaturation based on various bands above 98 kDa on the SDS-PAGE gel. The second peak of
each chromatogram was confirmed to be the purified antiFAP-CD8h-PE38 based on molecular
weight (70,000 kDa) from the gel. And the third peak showed in the chromatograms for A301,
W281 and T355 is likely protein degradation products. Moreover, from all the gel results, protein
degradation existed in all antiFAP-CD8h-PE38s to a certain extent. Therefore, protease inhibitor
tablet was added into all these samples to prevent further degradation.
For the purification of wild-type antiFAP-CD8h-PE38, there were two peaks present in the
chromatogram (Figure 15). Fractions 24-27 of peak 2 were collected and concentrated for
subsequent PEGylation.
37

Figure 15. Further purification by SEC of wild-type antiFAP-CD8h-PE38. B.C., before
column. A) SEC chromatogram of wild-type antiFAP-CD8h-PE38 from Superdex 200 10/300
column. B) SDS-PAGE gel of purified wild-type antiFAP-CD8h-PE38.

A
B
38

For the purification of antiFAP-CD8h(C29AcF)-PE38, there were two peaks showed in the
chromatogram (Figure 16). Fractions 13-17 of peak 2 were collected and concentrated for
PEGylaton.

39

Figure 16. Further purification by SEC of antiFAP-CD8h(C29AcF)-PE38. b.c., before
column. A) SEC chromatogram of antiFAP-CD8h(C29AcF)-PE38 from Superdex 200 16/600
column. B) SDS-PAGE gel of purified antiFAP-CD8h(C29AcF)-PE38.

A
B
40

For the purification of antiFAP-CD8h-PE38(W281AcF), there were three peaks present in the
chromatogram (Figure 17). The second peak, which was supposed to contain the purified antiFAP-
CD8h-PE38(W281AcF), was found to additionally contain a 38 kDa impurity as depicted on an
SDS-PAGE gel. This impurity may represent the degradation of antiFAP-CD8h-PE38. The
fractions 14-17 of the second peak were still collected and concentrated for the next PEGylation.
The degradation may be removed by further purification of PEGylated antiFAP-CD8h-PE38 in
the next step.

41

Figure 17. Further purification by SEC of antiFAP-CD8h-PE38(W281AcF). B.C., before
column. A) SEC chromatogram of antiFAP-CD8h-PE38(W281AcF) from Superdex 200 16/600
column. B) SDS-PAGE gel of purified antiFAP-CD8h-PE38(W281AcF).

A
B
42

For the purification of antiFAP-CD8h-PE38(A301AcF), there were three peaks present in the
chromatogram (Figure 18). Compared to antiFAP-CD8h-PE38(W281AcF), the antiFAP-CD8h-
PE38(A301AcF) fractions in the second peak were able to be separated from the multimers and
degradation from the third peak. Fractions 15-18 of second peak were collected and concentrated
for subsequent PEGylation.

43

Figure 18. Further purification by SEC of antiFAP-CD8h-PE38(A301AcF). B.C., before
column. A) SEC chromatogram of antiFAP-CD8h-PE38(A301AcF) from Superdex 200 16/600
column. B) SDS-PAGE gel of purified antiFAP-CD8h-PE38(A301AcF).

B
A
44

For the purification of antiFAP-CD8h-PE38(T355AcF), there were three peaks present in the
chromatogram (Figure 19). The purification results were similar to the antiFAP-CD8h-
PE38(A301AcF) mutant protein in which the full-length protein and impurities were successfully
separated by gel filtration. Fractions 14-18 of the second peak were collected and concentrated for
PEGylation.

45

Figure 19. Further purification by SEC of antiFAP-CD8h-PE38(T355AcF). A) SEC
chromatogram of antiFAP-CD8h-PE38(T355AcF) from Superdex 200 16/600 column. B) SDS-
PAGE gel of purified antiFAP-CD8h-PE38(T355AcF).

B
A
46

3.4 PEGylation of antiFAP-CD8h-PE38
The purified pAcF-incorporated antiFAP-CD8h-PE38s were concentrated down to 70-100 µM,
and the 1xPBS buffer was exchanged to a pH 4.2 reaction buffer. The conjugation reaction was
conducted for 24 hours at room temperature. After the reaction, the samples of each reaction were
loaded onto SDS-PAGE gel to examine the formation of PEGylated protein (Figure 20).
The band around 100 kDa present on the SDS-PAGE gel is the PEGylated antiFAP-CD8h-PE38
due to the increased molecular weight of 30 kDa PEG, while the band at 70 kDa represents
unreacted protein. The antiFAP-CD8h(C29AcF)-PE38 and antiFAP-CD8h-PE38(T355AcF)
mutant proteins were able to conjugate with PEG, whereas the antiFAP-CD8h-PE38(A301AcF)
and antiFAP-CD8h-PE38(W281AcF) proteins were unconjugated. The potential reason is that the
C29 site of the CD8 hinge and the T355 site of PE38 are more exposed to the solvent than W281
and A301. Moreover, the band representing PEGylated antiFAP-CD8h(C29AcF)-PE38 is darker
than that of PEGylated antiFAP-CD8h-PE38(T355AcF) even though the PEG concentration is
only 5x molar excess, suggesting that the PEGylation efficiency of antiFAP-CD8h(C29AcF)-PE38
was higher. It may indicate that pAcF at T355 site is less exposed to the surface of protein.
Although the PEGylation worked for the antiFAP-CD8h(C29AcF)-PE38 and antiFAP-CD8h-
PE38(T355AcF) mutant proteins, there was still large amount of unreacted protein. Additionally,
during PEGylation, protein precipitation was observed for all the pAcF incorporated antiFAP-
CD8h-PE38s, and the formation of these precipitates may contribute to the reduced PEGylation
efficiency. The conditions mentioned above have to be further optimized in the future to increase
protein PEGylation yield and reduce protein precipitation.

47

Figure 20. Confirmation of PEGylated protein. A) SDS-PAGE gel for confirmation of
PEGylated antiFAP-CD8h(C29AcF)-PE38 with 5X molar excess of PEG in a 30 µl reaction. B)
SDS-PAGE gel for confirmation of PEGylated antiFAP-CD8h-PE38(W281AcF) with 10X or 20X
molar excess of PEG in a 30 µl reaction. C) SDS-PAGE gel for confirmation of PEGylated
antiFAP-CD8h-PE38(T355AcF) with 10X or 20X molar excess of PEG in a 30 µl reaction. D)
A
B
C
D
48

SDS-PAGE gel for confirmation of PEGylated antiFAP-CD8h-PE38(A301AcF) with 10X or 20X
molar excess of PEG in a 30 µl reaction.

49

3.5 Purification of PEGylated antiFAP-CD8h-PE38
The PEGylated antiFAP-CD8h-PE38 protein was further separated from the unreacted protein
by the Superdex 200 10/300 column with 0.5 mL/min flow rate and 1xPBS running buffer at 4 ℃.
Samples from each main peak were loaded on an SDS-PAGE gel to test the purity of PEGylated
antiFAP-CD8h-PE38 (Figure 21). The PEGylated antiFAP-CD8h-PE38 was separated from
unreacted protein. There was only one band (~100 kDa) present on the SDS-PAGE gel for all the
fractions for the first peak, suggesting that it is the PEGylated antiFAP-CD8h-PE38. The lower
bands, representing the unreacted antiFAP-CD8h-PE38 at about 70 kDa, were only present in the
second peak fractions. For the PEGylated antiFAP-CD8h(C29AcF)-PE38, fractions 17-19 of the
first peak were collected and concentrated. For the PEGylated antiFAP-CD8h-PE38(T355AcF),
fractions 18-21 of first peak were collected and concentrated. Both the PEGylated antiFAP-CD8h-
PE38 were stored at -20 ℃ with 50% glycerol. Afterwards, the samples will be sent to our
collaborator for pharmacological and pharmacokinetic studies.

50

A
B
C
51

Figure 21. Purification of PEGylated antiFAP-CD8h-PE38 by Superdex 200 10/300 column.
A) SEC chromatogram of purified PEGylated antiFAP-CD8h(C29AcF)-PE38. B) SDS-PAGE gel
of purified PEGylated antiFAP-CD8h(C29AcF)-PE38. C) SEC chromatogram of purified
PEGylated antiFAP-CD8h-PE38(T355AcF). D) SDS-PAGE gel of purified PEGylated antiFAP-
CD8h-PE38(T355AcF).

D
52

4. Discussion
The tumor microenvironment has been recognized increasingly to play an essential role in
tumorigenesis and progression. TAFs— the predominant cells in the tumor microenvironment—
have attracted considerable attention recently. Not only do these TAFs promote tumor growth and
angiogenesis by secreting cytokines and chemokines, but these malignant cells also contribute to
tumor metastasis through degrading and remodeling the ECM. Thus, TAFs are seen as a promising
target for tumor therapy. Studies have shown that inhibition of TAF is effective on hindering tumor
growth. Previously, our collaborator has developed a novel recombinant immunotoxin, antiFAP-
CD8h-PE38, which can target TAFs by recognizing FAP, an exceedingly overexpressed biomarker
on the surface of TAFs. This immunotoxin was tested to effectively attenuate tumor growth and
reduce the secretion of tumorigenesis-related chemokines and cytokines in vivo. However, the half-
life of antiFAP-CD8h-PE38 in blood circulation is 13 minutes, which significantly restricts its
further application in the clinic. Therefore, to increase the half-life of antiFAP-CD8h-PE38 in vivo,
we aim to attach a single linear 30 kDa PEG to the immunotoxin.
This study focuses on incorporating the unnatural amino acid pAcF into antiFAP-CD8h-PE38
for site-specific conjugation with aminooxy-PEG. This PEGylation strategy helps maintain the
bioactivity of the protein and prevent heterogeneity of PEGylation. Four sites were chosen to
incorporate pAcFs on the protein: one is on the C29 of the CD8 hinge between antiFAP and PE38,
the other three sites—W281, A301 and T355—were on PE38. The wild type and four mutant
antiFAP-CD8h-PE38s were expressed in E. coli and then purified by both the Ni-NTA and
Superdex 200 columns. Afterwards, the purified proteins were conjugated to PEG before further
purification and separation of the PEGylated products. It was found that the antiFAP-
CD8h(C29AcF)-PE38 and antiFAP-CD8h-PE38(T355AcF) were conjugated with PEG
53

successfully, but not the antiFAP-CD8h-PE38(A301AcF) and antiFAP-CD8h-PE38(W281AcF).
We speculated that the pAcF incorporated at C29 and T355 sites are more exposed to the protein
surface whereas pAcF at W281 and A301 may be blocked by the surrounding amino acids.
However, the efficiency of the PEGylation for C29 and T355 mutant proteins were not high enough
to obtain a decent yield. One potential reason is that the pAcF incorporated at the C29 and T355
sites were not completely exposed to the surface. Moreover, it is known that the ketone group on
pAcF is less reactive than an aldehyde in the nucleophilic reaction. The other reason is that protein
precipitation occurred in all the PEGylation reactions, which reduces the final yield of PEGylated
products. The precipitation may be accredited to the water absorption from protein molecules by
PEG whose ethylene oxide unit can bind 2 to 3 water molecules in solution (Polson, 1977).
Therefore, PEG in the reaction should be limited to a certain concentration, at which the yield is
enhanced but precipitation is reduced. In the future, the conditions for this reaction will be further
optimized.
Another important issue which arose in this project is protein degradation. Proteins were
degraded to a certain extent after purification by the Superdex column. Thus, the protease inhibitor
cocktail tablet was added into protein solutions to prevent degradation.
Once the conditions of PEGylation are optimized, the PEGylated antiFAP-CD8h(C29AcF)-
PE38 and PEGylated antiFAP-PE38(T355AcF) will be accumulated and sent to our collaborator
for the next pharmacological and pharmacokinetics studies.

54

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Abstract (if available)

Abstract Tumor-associated fibroblasts (TAFs)—a prominent cellular component in the tumor microenvironment—play an important role in tumor growth and metastasis, which makes it a novel target for tumor therapy. Previous studies have shown that inhibition of TAFs attenuated tumor growth in mice. Recently, a recombinant antibody-toxin conjugate—antiFAP-CD8h-PE38—was constructed by our collaborators Dr. Pin Wang and colleagues. This toxin targets TAFs by recognizing fibroblast activation protein (FAP), an over-expressed biomarker on the surface of TAFs. This immunotoxin worked effectively on inhibiting tumor growth and reducing tumorigenesis-related chemokines and cytokines in vivo. However, the plasma half-life of antiFAP-CD8h-PE38 in vivo is less than 30 minutes, which restricts its further application in the clinic. In this study, a single linear 30 kDa Polyethylene Glycol (PEG) was site-specifically conjugated to antiFAP-CD8h-PE38 at the incorporated unnatural amino acid p-acetylphenylalanine (pAcF). The purpose of this conjugation is to increase the half-life of antiFAP-CD8h-PE38. Four mutation sites in the antiFAP-CD8h-PE38 were chosen to incorporate pAcF based on its crystal structure. The pAcF incorporated antiFAP-CD8h-PE38s were expressed in E. coli and further purified by size exclusion chromatography (SEC). PEGylation of antiFAP-CD8h-PE38 was conducted afterwards, and the PEGylated protein was confirmed and purified. It turned out that the C29 and T355 incorporated antiFAP-CD8h-PE38 were more efficiently conjugated with PEG compared to other mutation sites on the antiFAP-PE38, but the conditions for PEGylation should be further optimized to increase the yield.

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Asset Metadata

Creator Zhao, Qianqian (author)

Core Title Site-specific PEGylation of recombinant immunotoxin antiFAP-CD8h-PE38 with an expanded genetic code

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/15/2016

Defense Date 07/15/2016

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

Tag fibroblast activation protein,immunotoxin,OAI-PMH Harvest,PEGylation,tumor-associated fibroblast,unnatural amino acid

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Xie, Jianming (committee chair), Okamoto, Curtis T. (committee member), Wang, Pin (committee member)

Creator Email zhaoqian@usc.edu,zhaoqianqian1116@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-270036

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Legacy Identifier etd-ZhaoQianqi-4551.pdf

Dmrecord 270036

Document Type Thesis

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Rights Zhao, Qianqian

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

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

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