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Development of new approaches for antibody modification

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Development of new approaches for antibody modification

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Content i

Development of New Approaches for Antibody Modification

Yanran Lu
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTERS OF SCIENCE
(PHARMACEUTICAL SCIENCES)
May 2017
ii
Acknowledgements
I would like to thank my PI and thesis adviser throughout my time at USC, Dr. Yong (Tiger)
Zhang. He has always supported my research and the completion of this thesis. I have achieved
many research skills in both biochemistry and pharmaceutical sciences from him in these two years.
I would like to thank my thesis committee members, Dr. Curtis T. Okamoto and Dr. Wei-Chiang
Shen for their serving on my examination committee. Their knowledge and advice have been very
valuable and enriched my work.
I am thankful to Dr. Xiaonan Zhang and all my lab members; they have always been a great
support for my research and life throughout my time in USC.
Finally, I would like to thank my parents who are my whole world. I may not achieve my degree
and everything else without their love and support.
iii
Table of Content
Acknowledgements ................................................................................................................. ii
List of Figures ........................................................................................................................ v
List of Schemes ...................................................................................................................... vi
Abbreviations ....................................................................................................................... vii
Abstract ................................................................................................................................. ix
Chapter 1 .............................................................................................................................. 1
Introduction .................................................................................................................................... 1
Chapter 2 .............................................................................................................................. 9
Aims ................................................................................................................................................ 9
2.1 Aim 1: Molecular Cloning and Expression of Antibody-Actin Fusion Proteins ........................... 9
2.2 Aim 2: Expression and Purification of Photox Toxin and Its ADP-ribosyltransferase Activity .. 10
2.3 Aim 3: Chemical Synthesis of NAD
+
Analogues ....................................................................... 10
Chapter 3 ............................................................................................................................. 11
Experimental methods .................................................................................................................. 11
3.1 Molecular Cloning and Expression of Antibody-Actin Fusion Protein ....................................... 11
3.2 Generation of Photox Toxin and mART Activity Test ................................................................ 16
3.3 Chemical Synthesis of NAD
+
Analogues .................................................................................... 17
3.4 Data Analysis ............................................................................................................................... 27
Chapter 4 ............................................................................................................................. 29
Results ........................................................................................................................................... 29
iv
4.1 Molecular Cloning and Expression of Antibody-Actin Fusion Proteins ..................................... 29
4.2 Expression and Purification of Photox Toxin and Its ADP-ribosyltransferase Activity .............. 36
4.3 Chemically Synthesis of ‘Clickable’ Alkyne-Tagged NAD
+
Analogues ..................................... 42
Chapter 5 ............................................................................................................................. 53
Discussion & Conclusion & Future work ..................................................................................... 53
5.1 Aim 1: Generation of Antibody-Actin Fusion Antibody ............................................................. 53
5.2 Aim 2: Expression and Purification of Photox Toxin and ADP-ribosyltransferase Activity ....... 54
5.3 Aim 3: Chemically Synthesis of ‘Clickable’ Alkyne-Tagged NAD
+
Analogue .......................... 55
References ............................................................................................................................ 57

v
List of Figures
FIGURE 1: PCR PRODUCTS ANALYZED BY AGAROSE GEL ELECTROPHORESIS AND VISUALIZED BY
ETHIDIUM BROMIDE STAINING ................................................................................................................ 31
FIGURE 2: OVERLAP PCR PRODUCTS ANALYZED BY AGAROSE GEL ELECTROPHORESIS AND
VISUALIZED BY ETHIDIUM BROMIDE STAINING ................................................................................. 33
FIGURE 3: SDS-PAGE FOR HERCEPTIN-ACTIN FUSION PROTEIN ............................................................... 34
FIGURE 4: WESTERN BLOTTING OF HERCEPTIN-ACTIN FUSION PROTEIN. ............................................ 35
FIGURE 5: SDS-PAGE FOR PHOTOX SAMPLE PROTEIN ................................................................................. 38
FIGURE 6: HPLC ANALYSIS FOR MONO-ADP-RIBOSYLATION OF ACTIN BY PHOTOX (1-HOUR
REACTION). ..................................................................................................................................................... 40
FIGURE 7: HPLC ANALYSIS FOR MONO-ADP-RIBOSYLATION OF ACTIN BY PHOTOX (OVERNIGHT
REACTION) ...................................................................................................................................................... 41
FIGURE 8:
1
H NMR OF NICOTINAMIDE RIBOSIDE .......................................................................................... 45
FIGURE 9:
1
H NMR OF Β-NICOTINAMIDE MONONUCLEOTIDE ................................................................... 46
FIGURE 10:
1
H NMR OF 6-ALKYNE-ADENOSINE ............................................................................................. 47
FIGURE 11:
1
H NMR OF 6-ALKYNE-AMP MORPHOLIDATE ........................................................................... 48
FIGURE 12:
1
H NMR OF 8-ALKYNE-ADENOSINE WITH TMS ......................................................................... 49
FIGURE 13:
1
H NMR OF 8-ALKYNE-ADENOSINE ............................................................................................. 50
FIGURE 14: MS RESULT FOR 6-ALKYNE-NAD
+
................................................................................................ 51
FIGURE 15: HPLC FOR SEPARATION OF 6-ALYKNE-AMP ............................................................................. 52

vi
List of Schemes
SCHEME 1: SYNTHESIS OF Β-NICOTINAMIDE MONONUCLEOTIDE .......................................................... 19
SCHEME 2: SYNTHESIS OF 6-ALKYNE-NAD
+
. .................................................................................................. 21
SCHEME 3: SYNTHESIS OF 8-ALKYNE-NAD
+
................................................................................................... 24

vii
Abbreviations
ADP-ribose: Adenosine Diphosphate Ribose
ADC: Antibody-Drug Conjugate
AKT: Protein Kinase B
AML: Acute Myeloid Leukemia
Arg: Arginine
ARTs: ADP-Ribosyltransferases
ARTCs: ADP-Ribosyltransferase Cholera Toxin-Like
ARTDs: ADP-Riblosyltransferase Diphtheria Toxin-Like
ATP: Adenosine Triphosphate
BSA: Bovine Serum Albumin
CuAAC: Cu (I)-Catalyzed Azide-Alkyne Cycloaddition
DA
inv
Chemistry: Diels-Alder Reaction
DIFO: Difluorooctyne
DNA: Deoxyribonucleic Acid
FDA: Food and Drug Administration
HER-2/ErbB: Human Epidermal Growth Factor Receptor 2
HPLC: High Performance Liquid Chromatography
viii
IgG: Immunoglobulin G
mART: Mono-ADP-Ribosyltransferase
MAPK: Mitogen-Activated Protein Kinases
NAD
+
: Nicotinamide Adenine Dinucleotide
PARP: Poly-ADP-Ribose Polymerase
PCR: Polymerase Chain Reaction
PD: Pharmacodynamics
PI3K: Phosphoinositide 3-Kinase
PK: Pharmacokinetics
PTMs: Post Translational Modifications
SPAAC: Strain-Promoted Alkyne-Azide Cycloaddition
T-DM1: Trastuzumab Emtansine

ix
Abstract
Monoclonal antibodies possess exquisite specificity to their cognate antigens. By covalently
linking cytotoxic drugs to monoclonal antibodies, the resulting antibody-drug conjugates (ADCs)
represent a promising approach for targeted drug delivery. Numerous methods have been
developed for generating ADCs through chemically conjugating small-molecule drugs to surface
amino acid residues of monoclonal antibodies, which often leads to heterogenous products due to
non-specific chemical reactions. To make homogenous ADCs with site-specifically labelled drug
molecules, enzyme-catalyzed reactions could possibly offer a new solution. Using nicotinamide
adenine dinucleotide (NAD
+
) as a co-substrate, photox, a mono-ADP-ribosyltransferase from
Photorhabdus luminescens, catalyzes ADP-ribosylation of actin. It was envisioned that photox
could enzymatically add ADP-ribose groups with alkyne tags to antibodies fused with actin
polypeptides. The resulting antibody-actin fusions modified with alkyne-labelled ADP-ribose
could thus enable site-specific conjugation of cytotoxic drugs through well-established click
chemistry. Herein the photox enzyme was recombinantly expressed and purified from bacteria. By
genetically fusing human actin to an anti-HER2 monoclonal antibody, an antibody-actin fusion
protein was expressed from mammalian cells. Catalytic activities of purified photox were
examined with actin and the antibody-actin fusion. In addition, NAD
+
analogues with alkyne tags
were attempted for chemical synthesis. Through identification of active NAD
+
analogues for the
photox enzyme, antibody-actin fusions labeled with alkyne tags will likely be created for site-
specific conjugation of monoclonal antibodies.
1
Chapter 1
Introduction
Cancer management has been significantly improved over the past half century stemming from
advancement of chemotherapy. As well as chemotherapy, surgical removal, and radiation therapies,
targeted therapies and immunotherapies have emerging as major treatment options. (An, 2010).
Common mechanisms of chemotherapy involve in utilization of cytotoxic agents for inducing cell
deaths by disrupting microtubule function, DNA synthesis, and protein function. Severe adverse
effects derived from off-target effects still remain the major cause of treatment failures in
chemotherapy, despite the great success in treatment of cancer, in particular leukemia. Highly
potent cytotoxic agents are thus discouraged due to this fact.
Until recently, therapeutic antibody development was progressed slowly, although the study of
antibodies has been a popular area in both biology and medicine since early 1900s. The treatment
of infectious diseases by using serum from people who had recovered from the same diseases was
the first successful application of antibodies as therapeutics. César Milstein and Georges Köhler
both contributed to the methods for isolating mouse monoclonal antibodies from hybridoma cells,
a major progress in therapeutic antibody discovery and development. Then, not only have
monoclonal antibodies fueled breakthrough discoveries in basic research, but also developed as
live-saving medicines, effective clinical diagnostics, as well as useful agents for high throughput
drug screening. The US food and Drug Administration (FDA) approved the first therapeutic
monoclonal antibody muromonab-CD3 (OKT3) for reducing acute rejection in patients with organ
2
transplants (Thistlethwaite et al., 1987). Currently, more than 25 therapeutic antibodies have
served as a major drug modality in clinical use and over 100 monoclonal antibodies are in
development for excellent safety profiles, high binding affinity and specificity, and great efficacy
in disrupting protein-protein interactions (Reichert and Valge-Archer, 2007).
Highly potent cytotoxic agents conjugated with cell targeting moieties have been emerging as a
potentially important therapeutic strategy, given their substantial cytotoxicity for cancer treatment.
In particular, antibody-drug-conjugates (ADCs), resulted from the conjugation of humanized or
human monoclonal antibodies with cytotoxic small-molecule agents through chemical linkers,
have demonstrated to be an effective regimen for cancer therapy.
A number of monoclonal antibodies are characterized by remarkable anti-tumor activities, such
as anti-HER2, anti-EGFR, and anti-CD20, resulting from antigen-binding mediated
downregulation of signaling pathways. In comparison, most monoclonal antibodies themselves
even have little antitumor activities upon binding to their target antigens. Owing to their high
specificity towards the cognate antigens, monoclonal antibodies are usually considered as potential
cancer therapeutic agents despite their scant antitumor activity. In combination with distinct
effector molecules, including cytotoxic agents, bacterial or plant protein toxins (immunotoxins),
and radiopharmaceutical agents, monoclonal antibodies can exhibit potent antitumor activities
through induction of cell deaths following binding, internalization, and release of therapeutic
cargos.
Unlike conventional chemotherapeutics that display no specificity, ADCs show highly specific
activities and potency against antigen-expressing tumor cells (Sievers and Senter, 2013). Despite
the clear rationale of linking cytotoxic drugs to tumor-targeting antibodies for targeting therapies,
development of highly efficacious therapeutic ADCs still faces several technological challenges.
3
The early generation of ADCs featured with mouse-derived monoclonal antibodies using covalent
linkers with anticancer drugs, such as doxorubicin, vinblastine, and methotrexate. Little success
was achieved for those conjugates due to immunogenicity, scant potency caused by suboptimum
selection of targets, and insufficient selectivity (Chari, 1998). Those identified issues later on lead
to the technological improvements and renewed interests in developing ADCs (Chari, 1998). For
example, humanized or fully human antibodies have been used to largely prevent immunogenic
activity. Linkage of more potent drugs helps improve the potency. Comprehensive mechanistic
studies of targets and identified antibodies facilitate selection of candidates for enhanced
specificity and efficacy.
As a result, gemtuzumab ozogamicin was approved under an accelerated-approval process by
FDA in use of patients over the age of 60 who had relapsed CD33-positive acute myeloid leukemia
(AML)(Bross et al., 2001), or the patients who were not candidates for standard chemotherapy in
2000 (Bross et al., 2001). Trastuzumab emtansine was approved in 2013 for HER2-positive breast
cancer (Boyraz et al., 2013). It should be significantly notified that gemtuzumab ozogamicin was
later withdrawn from market in 2001 due to a clinical study showing increased patient deaths and
no obvious benefits over conventional cancer therapies. To date, more than 40 ADCs are in or
nearing to the clinical trials.
To develop ADCs, humanized or fully human monoclonal antibodies are the first choice in order
to achieve long circulating half-life in human blood stream, high targeting specificity, and minimal
immunogenicity (Tsuchikama et al., 2016). Upon administration into the blood stream, the
antibody components are responsible for recognizing and binding to cell-surface antigens that are
highly expressed on target cancer cells, which requires the antibody components have high
antigen-binding affinity and specificity. However, excellent antigen affinity can reduce efficiency
4
of solid tumor penetration (Polson et al., 2011). Moreover, it was suggested that efficacy of ADCs
is correlated with antigen density found on various types of cancer cells (Polson et al., 2011).
However, the relationship between overall efficacy of ADCs and the antigen expression levels
remains elusive.
On the other hand, it is still a long-term puzzle that how to efficiently deliver payload into target
cells. The actual uptake rates through ADCs-based delivery were estimated to be lower than 50%
(Teicher and Chari, 2011). In this case, payloads with picomolar cytotoxicity are required to show
and maximize treatment efficacy. In addition tumor cells, some of noncancerous cells may
internalize ADCs through different mechanisms, such as nonspecific picocytosis, and fragment
crystallizable region (Fc) receptor-mediated endocytosis (Lencer and Blumberg, 2005). The
payloads can also be released into circulating blood prior to reaching to target tissues. Thus, the
selection of payloads is another key for ADC development.
Besides the selections of optimal target-specific antibodies and potent payloads, the conjugation
methods and chemical linkers are also critical for the development of efficacious ADCs, which
fundamentally affect the overall design of ADC molecules and their physicochemical and
biological properties, such as pharmacokinetics (PK), pharmacodynamics (PD), and therapeutic
windows. Since the correlation between the linker stability and the adverse effects remaining
unclear (Drake and Rabuka, 2015), different linkers need be examined for their stability with
each combination of antigens, target tumors, and payloads.. To show excellent therapeutic benefits,
ADC linkers should be able to be rapidly cleaved for release of free payloads when the ADCs are
internalized by target tumor cells. Taken together, development of conjugation methods and linker
structures are crucial for design and discovery of potent ADCs. Chemical and enzymatic
5
conjugation are two popular methods for linking antibodies to the payloads. Utilization of
cleavable and non-cleavable linkers have been two common approaches.
A well-known reversible post-translational modification (PTM) is adenosine diphosphate
(ADP)-ribosylation which is catalyzed by ADP-ribosyltransferases (ARTs) to transfer single or
multiple ADP-ribose units from nicotinamide adenine dinucleotide (NAD
+
) to a specific amino
acid residue of proteins (Feijs et al., 2013). Mammalian ARTs share structurally conversed
catalytic domains and are categorized as ADP-ribosyltransferase cholera toxin-like (ARTCs)
(Glowacki et al., 2002), and diphtheria toxin-like (ARTDs) subfamilies, based on the homology
of the catalytic domains to bacterial cholera or diphtheria toxins (Feijs et al., 2013). ARTCs
enzymes are known to transfer single ADP-ribose to their substrates (Glowacki et al., 2002). On
the other hand, ARTDs are intracellular enzymes that can transfer either single ADP-ribose to an
acceptor amino acid residue, or attach multiple ADP-ribose moieties with the acceptor proteins
resulting in either linear or branched chains of poly-ADP-ribose (poly-ADP-ribosylation) (Kleine
et al., 2008). For instance, ARTD1 is shown to catalyze auto-poly-ADP-ribosylation as well as
poly-ADP-ribosylation of many other substrates, leading to the initial designation of PARP1 for
poly-ADP-ribose polymerase 1. Thus, this family of enzymes with relevant catalytic domains is
termed PARP family.
Produced by various highly pathogenic bacteria, a number of toxins share the enzymatic
functions to covalently modify host proteins by adding single ADP-ribose moiety from NAD
+
.
The attachments of ADP-ribose moieties can inhibit the natural functions of the target proteins
causing various diseases in human, such as diphtheria, pertussis, and cholera (Deng and Barbieri,
2008). Nine mono-ADP-ribosyltransferase (mARTs) have been identified to ADP-ribosylate actin
and disrupt actin polymerization. These enzymes are binary toxins that include a component
6
leading to binding/translocation, and another component with catalytic activity. The well-known
reaction mechanism for the actin-targeting mART toxins is formed by the cleavage of the
nicotinamide moiety from NAD
+
(Tsuge et al., 2008).
ADP-ribosylation can be directly visualized by using radiolabeled NAD
+
or functionalized
NAD
+
analogues. Bioorthogonal reactions based on the use of reporter-tagged NADs have been
developed for the investigation of ADP-ribosylation in vitro (Carter-O'Connell et al., 2014) and in
cells (Wallrodt et al., 2016). Fluorescently labeled NAD
+
analogues have been developed for the
real time visualization of PARylation in live cells (Buntz et al., 2016). Additionally, NAD
+

analogues in association with fluorescence lifetime imaging served as chemical tools allows
protein-specific detection and the study of PARylation in cells after exposure to genotoxic damage
(Buntz et al., 2016). Recently, “clickable” alkyne-tagged NADs (Carter-O'Connell et al., 2014)
have been reported to be used for the research on PARylation. Some new functionalized NAD
+

analogues based on bioorthogeonal chemistry have been developed for the intracellular imaging
of PAR. By varying the modification positions and the types of chemical groups, several NAD
+

analogues were generated for both copper (I)-mediated and copper-free click reactions (Wallrodt
et al., 2016). It was reported that clickable NAD analogues with terminal alkyne groups allow the
combination of fluorescent or affinity tags with the substrate proteins (Jiang et al., 2010) for
visualization and proteomic analysis.
Breast cancer, one of the major public health issue, is the leading type of cancer in women.
Nearly 20-50% of breast cancers in human are with an amplification or overexpression of HER2
(also known as ErbB2) (Owens et al., 2004). The 185-kDa transmembrane receptor protein, HER2,
a member of the epidermal growth factor receptor family, plays an important role in cell
differentiation, proliferation and survival (Yarden and Sliwkowski, 2001). The amplification and
7
upregulation of this gene are associated with the pathogenesis of aggressive tumors with poor
prognosis.
Trastuzumab, a monoclonal antibody (brand name Herceptin, used hereafter), targets the
extracellular domain of HER2. It was approved as a first-line treatment in combination with
paclitaxel for HER2-positive metastatic breast cancer (Slamon et al., 2001). Several action
mechanisms for this therapeutic antibody were proposed, including inhibition of PI3K/AKT and
MAPK signaling pathways (Junttila et al., 2009), prevention of HER2 ectodomain cleavage
(Molina et al., 2001), antibody dependent cell-mediated cytotoxicity (ADCC), and induction of
apoptosis (Lewis et al., 1993). HER2-targeted therapy has been recommended in the patients with
early and metastatic HER2-positive breast cancer. Trastuzumab emtansine (T-DM1), a novel ADC,
is composed of trastuzumab targeting to HER2, a stable thioether liker, and the potent cytotoxic
agent DM1 (derivative of maytanisine). In combination with other chemotherapies, T-DM1
revealed excellent clinical activity and favorable safety profiles in patients with HER2-positive
metastatic breast cancer (Boyraz et al., 2013).
Photorhabdus luminescens is a pathogenic bacterium producing many toxic proteins. An
extensive variety of toxins and hydrolytic enzymes are encoded by P.luminescens genome, many
of these toxins and enzymes are being studied as potential virulence factors (Visschedyk et al.,
2010). P.luminescens can produce toxins complexes, high molecular weight, multi-subunit,
insecticidal toxins (Waterfield et al., 2001). P.luminescens has gained considerable interests in the
area of biopesticides with increasing resistance against conventional pesticides (Waterfield et al.,
2001). ADP-ribosyltransferase enzymes have been extensively studied since Clostridium
botulinum was first reported in 1986 (McGuffin et al., 2004). Previous study demonstrated that
photox is a novel actin-targeting mono-ADP-ribosyltransferase from photorhabdus luminescens
8
(Visschedyk et al., 2010). By sharing high homology in catalytic regions and a similar core
catalytic fold with identified mono-ADP-ribosylfransferase, this 46-kDa protein was found to
ADP-ribosylate many different actin catalogues, such as monomeric α-skeletal actin and
nonmuscle β- and γ-actin (Visschedyk et al., 2010).
Click chemistry is considered as a type of biocompatible reaction that provides efficient methods
for coupling of substrates and specific biomolecules with high yields under mild conditions. By
ligating biomolecules to a reporter groups, click chemistry has been found in an increased number
of applications in pharmacological and biomimetic fields. Serving as an extension and
complementary technology for drug discovery, click chemistry also allows generation of new
structures for pharmacophores, as well as covalent links between two different biomolecules for
achieving desired interactions.
The first class of click reaction with high efficiency in biological conditions but potential
cytotoxicity is the copper-catalyzed reaction of an azide with an alkyne group to form a 5-
membered heteroatom ring: a Cu (I)- catalyzed Azide-Alkyne cycloaddition (CuAAC) (Kolb and
Sharpless, 2003). To minimize possible cytotoxicity of CuAAC reactions, the alkyne group can
be introduced in a strained difluorooctyne (DIFO) for strain promoted cycloaddtion (Agard et al.,
2006). This reaction has been applied to probe azides in living systems with somewhat slower
reaction rates than those of the CuAAC. Due to the two appropriate characteristics, bio-
orthogonality and mild and relatively low toxic conditions, click chemistry are becoming
promising approaches for assembling both small and large biomolecules.
9
Chapter 2
Aims
The goals of this thesis are to generate new antibody-drug conjugate scaffolds that can minimize
systematic effects of cytotoxic drugs. Through click chemistry, cytotoxic drugs can be covalently
linked to tumor-targeting antibodies. Enzymatic conjugation holds the potential for site-specific
conjugation of antibody with distinct payloads, which can likely increase their therapeutic indexes.
This will be accomplished by testing the hypothesis that the engineered antibody-polypeptide
fusion proteins can be recognized by photox enzyme for efficient site-specific transfer of modified
ADP-ribose moiety from NAD
+
analogues. Towards this end, we have been trying to synthesize
alkyne-labeled NAD
+
analogues for site-specific modification of antibody-polypeptide fusion
proteins, which would enable attachments of payloads through click chemistry. Herceptin as a
model monoclonal antibody, will be utilized for generation of antibody fusion proteins. Three
aims were set forth to achieve the goals for facilitating the development of antibody fusion proteins
with alkyne tags.
2.1 Aim 1: Molecular Cloning and Expression of Antibody-Actin Fusion
Proteins
Through genetic engineering, gene encoding human α-actin was fused to C-terminus of
Herceptin heavy chain. The resulting construct was tested for mammalian expression of antibody-
actin fusion as potential substrates of photox.
10
2.2 Aim 2: Expression and Purification of Photox Toxin and Its ADP-
ribosyltransferase Activity
Photox enzyme was recombinantly expressed in E.coli. Overexpressed photox was purified
through affinity chromatography and examined for enzymatic activity.
2.3 Aim 3: Chemical Synthesis of NAD
+
Analogues
Chemical synthesis of NAD
+
analogues modified with alkynes in two different positions were
attempted according to previously published procedures. By using these reported NAD
+
analogues
(Jiang et al., 2010), the ADP-ribosylation activity with photox and antibody-actin fusion will be
evaluated. Collectively, Herceptin-actin fusion proteins would be first generated to serve as the
targeting component. Through Photox-mediated ADP-ribosylation with NAD
+
analogues,
antibody surface handles would be created for attaching stable chemical linkers with distinct
payloads.

11
Chapter 3
Experimental Methods
3.1 Molecular Cloning and Expression of Antibody-Actin Fusion Protein
3.1.1 Polymerase chain reaction (PCR) and overlap polymerase chain reaction
3.1.1.1 Polymerase chain reaction (PCR)
Genes encoding human α-actin and Herceptin heavy chain were utilized as templates for PCR
reactions. Their concentrations and purity were verified through UV absorbance and DNA agarose
gel electrophoresis, respectively before mixing with primers for amplification using AccuPrime™
Pfx DNA polymerase.
The following sets of primers were used:
Herceptin heavy chain: forward: 5′- CACGAATTCGGAGGTGCAGC-3′,
Reverse:5′- CCGCCACCAGAGCCACCTCCCCCTTTACCCGGAGACAGGGAGAG-3′
Human α-actin: forward: 5′-
AGGTGGCTCTGGTGGCGGAGGGAGCATGTGCGACGAAGACGAGACC-3’
Reverse:5’- CCAGCTAGCACTTATCACTAGAAGCATTTGCGGTGGAC-3’
The reactions were performed by following procedures:
1) Adding the following components to an autoclaved PCR tube at room temperature:
12
10X AccuPrime™ Pfx reaction mix (5 µL, final concentration: 1X), primer mix (10 µM
each, 1.5 µL each, final concentration: 0.3 µM), template DNA (100 ng, ≥1 µL, final
concentration is as required), AccuPrime™ Pfx DNA polymerase (0.5 µL, final
concentration: 1.25 units), and autocleaved distilled water (to 50 µL).
2) Mixing contents of the tubes, capping the tubes, and centrifuging briefly to collect the
contents.
3) Denaturing the template for 2 min at 95°C.
4) Performing 35 cycles of PCR amplification as follows (Three-step cycling):
5) Denature: 95°C for 15 s
6) Anneal: 57°C for 30 s
7) Extend: 68°C for 2 min
8) Maintaining the reaction at 4°C after cycling
9) Analyzing the products by agarose gel electrophoresis and visualizing by ethidium
bromide staining.
10) Cleaning up the fragments by using Zymoclean Gel DNA Recovery Kit (Genesee
Scientific, San Diego, CA)
3.1.1.2 Overlap extension polymerase chain reaction
The samples were prepared in the same manner as in polymerase chain reaction. To perform
overlap extension PCR reactions, the PCR products of both Herceptin heavy chain and actin were
mixed without any primers and amplified by using annealing temperature of 57°C for 15 cycles.
The reaction system was then added with Herceptin forward primer and actin reverse primer for
further amplification by using annealing temperature of 57°C for another 25 cycles. On the basis
13
of DNA agarose gel analysis, DNA fragments with correct sizes were purified by gel extraction
using Zymoclean Gel DNA Recovery Kit (Genesee Scientific, San Diego, CA)
3.1.1.3 Double digestion, ligation, and transformation
The gene of Herceptin-actin fusion protein was cloned into pFUSE vector by following procedures:
1) Digesting the both DNA fragments and pFUSE vector by restriction enzymes, EcoRI and
Nhel (New England Biolabs, Ipswich, MA). The designed DNA fragments contained
EcoRI and Nhel restriction enzymes. Setting up the reaction in a microcentrifuge tube on
ice:
Restriction enzymes 10 units each, DNA (1 µg), 10 × NEB Buffer (5 µL, 1 ×), the total
reaction volume was 50 µL. the reactions were incubated for 1 hour at 37°C.
2) Setting up the following ligation reaction by using T4 DNA Ligase (New England Biolabs,
Ipswich, MA) in a microcentrifuge tube at room temperature:
T4 DNA Ligase Buffer (10 ×) (2 µL), pFUSE-Fc vector DNA (0.070 pmol), Insert DNA
(0.010 pmol), T4 DNA ligase (1 µL), and adding nuclease-free water to 20 µL, the total
volume of the reaction was 20 µL.
3) Incubating the reaction mixture at room temperature for 3 hours
4) Directly transforming 2 µL into DH10B electrocompetent cells
5) After 60-min incubation at 37°C post transformation, plating the competent cells onto low
salt-LB plates containing zeocin to grow overnight at 37°C
6) Obtaining Herceptin-actin fusion protein plasmid by ZR Plasmid Miniprep-Classic Kit
(Genesee Scientific, San Diego, CA).

14
3.1.2 Expression of antibody-actin fusion proteins
To express the resulting fusion protein, the Herceptin-heavy-chain-actin plasmid and Herceptin
light chain plasmid were cotransfection into the Expi293F™ cells using Gibco™ Expifectamine™
293 transfuction Kits (Thermo Fisher Scientific, Houston, TX). The general transfection guidelines
are following with the online protocol:
1) Adding 7.5×10
'
cells in 25.5 mL of Expi293F™ expression medium to sterile, disposable
Erlenmeyer shaker flask
2) Diluting total 30 µg of plasmid DNA (the ratio of Herceptin-heavy-chain-actin plasmid
DNA and Herceptin light chain plasmid DNA: 2:1) in Opti-MEM™ I Reduced Serum
Medium to a total volume of 1.5 mL and mixing gently.
3) Diluting 80 µL of ExpiFectamine™ 293 Reagent in Opti-MEM™ I medium to 1.5 mL and
incubating for 5 min at room temperature.
4) Adding diluted plasmid DNA to diluted ExpiFectamine™ 293 Reagent, incubating DNA-
ExpiFectamine™ 293 Reagent mixture for 25 min at room temperature.
5) Incubating the cells in a 37°C incubator after adding DNA- ExpiFectamine™ 293 Reagent
mixture for approximately 16-18 hours.
6) After 16-18 hours’ post-transfection, 150 µl of ExpiFectamine™ 293 transfection enhancer
1 and 1.5ml of ExpiFectamine™ 293 transfection enhancer 2 were added to flasks.
7) Media were harvested at day 3 and day 6 from the day of transfection.
3.1.3 Purification of Herceptin-actin fusion proteins
The harvested media from ExpiFectamine™ 293 cells were purified by using protein G Resin
(GeneScript, Piscataway, NJ). The purification procedure as follows:
15
1) Applying 15 ml autocleaved distilled water onto the column and draining the flow-
through
2) Applying 15 ml 1×Phosphate-buffered saline buffer onto column and draining the flow-
through
3) Applying the harvested media onto the column and draining the flow-through, collecting
the flow-through for reapplying onto the column
4) Washing the column with 30 ml 1×Phosphate-buffered saline buffer and draining the
buffer
5) Eluting the immunoglobulins with 15 ml elution buffer with 0.1 M glycine, pH 2.5,
collecting the eluate and immediately neutralizing to pH 7.4 with 1 M Tris-HCl, pH 8.5
(1/10 volume of total eluate)
6) Dialyzing protein into 1×Phosphate-buffered saline buffer and concentrating to 0.5-1.0
mg ml
-1

7) Analyzing the products by protein SDS-PAGE
3.1.4 Western blotting
Once the concentration of recombinant protein was determined, samples with NuPAGE
®
LDS
Sample Buffer (4X) (Invitrogen, Carlsbad, CA) were separated by 10% sodium dodecyl sulfate-
polyarylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes.
Membranes were subsequently blotted in 1% BSA (VWR international, Radnor, PA) and then
incubated with Goat anti-Human Kappa Light Chain Secondary Antibody, HRP (1:20000, Thermo
Fisher Scientific Houston, TX). After adding the HRP chemiluminescent substrates (Thermo
16
Fisher Scientific, Houston, TX), Membranes were then imaged using ChemiDoc Touch Gel
Imaging System (Bio-Rad, Irvine, CA).
3.2 Generation of Photox Toxin and mART Activity Test
3.2.1 Overexpression and purification of photox toxin
The photox gene was overexpressed in E. coli cells following the reported literature (Visschedyk,
2010). pET-28b vector tagged with N-terminal His
6
and Photox gene was used for expression in
E.coli. BL21 (DE3) competent cells were transformed with plasmid and plated onto LB plates
containing kanamycin to grow overnight at 37°C. Cells were grown at 37°C in 2-liter LB media
containing 100 µg mL
-1
of kanamycin to OD
600
value of ∼0.6 before induction with 1 mM isopropyl
1-thio-β-D-galactopyranoside (IPTG) at 25°C. Twelve hours post-induction cells were harvested
by centrifugation at 5000 × g for 1 hour. Cells were resuspended in lysis buffer (20 mM Tris-HCl,
pH 7.5, 50 mM NaCl) and the cells were lysed using a French press for three times. Supernatant
was obtained by lysates were centrifuged for 30 min at 14,000 × g. The photox toxin was purified
by Hispur Ni-NTA Superflow Agarose (Thermo Fisher Scientific, Houston, TX). The general
purification protocol as follows:
1) Packing column, equilibrating the column using equilibration buffer (20 mM Tris-HCl
pH 7.5, 200 mM NaCl, 20 mM imidazole)
2) Applying samples onto the column, draining the flow-through and collecting the flow-
through for reapplying onto the column
3) Washing the column with 30 mL wash buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl,
30 mM imidazole) and draining the buffer
17
4) Eluting the protein with 15 mL elution buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl,
400 mM imidazole)
5) Dialyzing protein into 20 mM Tris-HCl pH 7.5, 200 mM NaCl and concentrating to 0.5-
1.0 mg mL
-1
for further analyzing and biological assay
6) Analyzing the purified products by protein SDS-PAGE
3.2.2 Mono-ADP-ribosylation of actin by photox
The nonmuscle β- and γ-actin mixture was commercially available (Cytoskeleton, Denver, CO),
5 µM of photox was mixed with 5 µM nonmuscle β- and γ-actin mixture after equilibration of all
samples to ambient temperature. The generation of nicotinamide was analyzed by High
Performance Liquid Chromatography (HPLC). All reactions were performed in 10 mM Tris-HCl,
pH 8.0, 0.2 mM ATP, 0.2 mM CaCl
2
, 0.2 mM 2-mercaptoethanol. All samples were analyzed by
HPLC system after 1-hour reaction without light.
3.3 Chemical synthesis of NAD
+
analogues
General synthesis methods were following the reported methods (Wang, 2014). Reagents were
obtained from Aldrich, Acros, and Oakwood in the highest purity available and used as supplied.
1
H NMR was performed on Varian 400 spectrometers.
HPLC experiments were accomplished on Waters 2695 Separation module & Waters 2487
detector with column (150 × 21.2 𝑚𝑚) monitoring at 260 nm. Solvent for HPLC were water with
0.1% formic acid (Solvent 1) and acetonitrile with 0.1% formic acid (Solvent 2). Compounds were
eluted at a flow rate of 3 mL min
-1
with 0% solvent 2 for 4 min, then a linear gradient of 0% to X%
18
(X depends on different compounds) solvent 2 over 24 min, and finally 100% solvent for 5min
before equilibrating the column back to 0% solvent 2 over 5 min.

19
3.3.1 β-Nicotinamide mononucleotide

Scheme 1: β-Nicotinamide mononucleotide a) trimethyl phosphate, phosphoryl chloride, 0°C, 12
hours

20
Nicotinamide riboside (255 mg, 1.0 mmol) was added with the mixture solution of trimethyl
phosphate (1 mL) and phosphoryl chloride (1.8 mL, 7.0 mmol, 7 equiv) with a stirring at 0°C. The
reaction was allowed to proceed for 12 hours at 0°C. Then cold water (5 mL) was added to the
reaction mixture, then dried in vacuo. Methanol was added to the residue and washed with ethyl
ether to collect precipitate. When the mother liquor was mixed with the fresh cold ethyl ether,
more participate formed, then the participate was collected by centrifugation. The participate
combined with previous one was dissolved in cold water (10 mL) and purified by HPLC. The
collected fractions were concentrated and checked by LC-MS and those that contained the desired
product were lyophilized to give the product.

21
3.3.2 6-alkyne-NAD
+

Scheme 2: 6-alkyne-NAD
+
: a) calcium carbonate, propargylzmine, absolute ethanol, refluxed, 12
hours; b) trimethyl phosphate, phosphoryl chloride, 0°C, 12 hours; c) β-NMN, CDI, triethylamine,
DMF, rt, 176 hours.

22
3.3.2.1 Synthesis of 6-alkyne-adenosine
6-chloropurine nucleoside (1g, 3,5 mmol), calcium carbonate (0.7g, 7mmol, 2 equiv) and
propargylzmine (1.2 mL, 17.5 mmol 5 equiv) were added to absolute ethanol (30 mL). The reaction
mixture was refluxed under argon for 12 hours. Then the calcium salts were removed by filtration
from the reaction mixture, and the filtrate was kept at -20°C for 1 hour. The white precipitate was
collected by centrifugation and washed with ethyl ether. When the mother liquor was mixed with
the fresh cold ethyl ether, more participate formed. The precipitate was combined with previous
one, then dried in vacuo to get the desired product.
3.3.2.2 Synthesis of 6-alkyne-AMP
6-alkyne-adenosine (305 mg, 1.0 mmol) was added with a solution mixture of trimethyl
phosphate (1 mL) and phosphoryl chloride (1.8 mL, 7.0 mmol, 7 equiv) with a stirring at 0°C. The
reaction was allowed to proceed for 12 hours at 0°C. Then cold water (5 ml) was added to the
reaction mixture, then dried in vacuo. Methanol was added to the residue and washed with ethyl
ether to collect precipitate. When the mother liquor was mixed with the fresh cold ethyl ether,
more participate formed, then the participate was collected by centrifugation. The participated
combined with previous one was dissolved in cold water (10 mL) and purified by HPLC. The
collected fractions were concentrated and checked by LC-MS and those that contained the desired
product were lyophilized to give the product. The HPLC method: compounds were eluted at a flow
rate of 3 ml min
-1
with 60% solvent 2 for 4 min, then with a linear gradient of 60% to 70% solvent
2 in 2 min, then remaining 70% solvent 2 for 6min, then with a linear gradient of 70% to 100%
solvent 2 in 4min, then remaining 100% solvent 2 for 2min, and finally 50% solvent 2 for 5min
before equilibrating the column back to 60% solvent 2 over 5 min.
23
3.3.2.3 Synthesis of 6-alkyne NAD
+
The crude 6-alkyne-AMP purified by HPLC was dissolved in anhydrous DMSO (1.5 mL) and
the following were added in sequences: Triphenylphosphine (656 mg, 2.5 mmol, 5 equiv),
morpholine (369 µL, 4.2 mmol, 8.5 equiv), and 2.2’-dipyridyldisulfide (551 mg, 2.5 mmol, 5
equiv). The reaction mixture was allowed to stir at room temperature for 3 hours. The participate
was formed by adding Sodium iodide (0.2 M) in acetonitrile dropwise into the reaction. The
participate was collected by centrifugation and washed with cold acetonitrile. Then the participate
was dissolved in cold methanol and dried under vacuum. This crude product was dissolved in a
solution of Manganese(II) chloride (500 µL, 0.2 M in formamide) with β-Nicotinamide
mononucleotide (20 mg, 0.055 mmol), and Magnesium sulfate (12 mg, 0.100 mmol). The reaction
mixture was allowed to stir at room temperature for 48 hours. After that, the reaction was
concentrated and then added with cold water, and be proposed to be purified by HPLC.

24
3.3.3 8-alkyne-NAD
+

Scheme 3: 8-alkyne-NAD
+
: a) 1. CuI, trimethylsilylacetylene, Pd (PPh
3
) Cl
2
, Et
3
N, DMF, rt, 18
hours; 2. NH
3
/MeOH, rt, 2 hours; b) trimethyl phosphate, phosphoryl chloride, 0°C, 12 hours; c)
β-NMN, CDI, triethylamine, DMF, rt, 176 hours.

25
3.3.3.1 Synthesis of 8-alkyne-adenosine
Copper (I) iodide (19 mg, 0.1 mmol, 0.2 equiv) were dissolved in dried dimethylformamide.
under an argon atmosphere, trimethylsilyl-acetylene (105 µL, 0.750mmol, 1.5 equiv), dichlorobis
(tripphenylphosphine) palladium (II) (35.1 mg, 0.500 mmol. 0.1 equiv) and triethylamine (210 µL,
1.5 mmol, 3.0 equiv) were added. The reaction was allowed to stir under the argon without light
at room temperature for 18 hours. Then ethyl acetate (20 mL) was added to the reaction mixture.
The organic phase was washed with 1M hydrochloric acid and saturated sodium bicarbonate, and
then dried by sodium sulfate. The solvent was removed under vacuum. The crude product was
purified by column chromatography on silica gel (methylene chloride: methanol = 10:1). The
obtaining product was added in dried methanol (10 mL) which was saturated with ammonia at
room temperature. The reaction was allowed to stir at room temperature for 2 hours. After that
the solvent was removed under vacuum at 20°C. Then the residue was added with dried methanol
and washed by ethyl ether to collect precipitate. When the mother liquor was mixed with the fresh
cold ethyl ether, more participate formed, then the participate was collected by centrifugation. The
participated combined with previous one was the desired product.
3.3.3.2 Synthesis of 8-alkyne-AMP
2-ethynyladenosine (29.0 mg, 0.100 mmol) and 1,8-bis (dimethylamino) napthale (32 mg, 0.150
mmol, 1.5 equiv) were dissolved in 1 mL trimethyl phosphate and then cooled to 0°C. Below this
temperature freshly distilled phosphoryl chloride was added slowly to the solution. The reaction
mixture was allowed to stir for 6 hours at 0°C. the reaction was quenched by 0.1 M TEAB buffer.
The mixture was stirred for 10 min and then twice extracted with ethyl acetate. The aqueous phase
was dried at 20°C under vacuum. The residue was added with cold methanol and washed by ethyl
26
ether to collect precipitate. When the mother liquor was mixed with the fresh cold ethyl ether,
more participate formed, then the participate was collected by centrifugation. All the participate
was dissolved in cold water (10 mL) and purified by HPLC. The crude product was proposed to
be obtained. The HPLC method: compounds were eluted at a flow rate of 3 mL min
-1
with 60%
solvent 2 for 4 min, then with a linear gradient of 60% to 70% solvent 2 in 2 min, then remaining
70% solvent B for 6min, then with a linear gradient of 70% to 100% solvent 2 in 4min, then
remaining 100% solvent 2 for 2min, and finally 50% solvent 2 for 5min before equilibrating the
column back to 60% solvent 2 over 5 min.
3.3.3.3 Synthesis of 8-alkyne-NAD
+

Method 1: β-Nicotinamide mononucleotide (β-NMN) (10.0 mg, 0.030 mmol, 1.5 equiv) was
added into dried dimethylformamide (1 mL). 1,1’-carbonyldiimidazole (CDI) (16.2mg,
0.100mmol, 5.0 equiv) and triethylamine (4.45 µL, 0.032 mmol, 1.6 equiv) were added. The
reaction was allowed to stir at room temperature for 18 hours. And then the reaction was quenched
with 200 µl dried methanol. The solvent was dried under vacuum. The crude 8-alkyne –AMP was
dissolved in 1 ml dried dimethylformamide and added to the activated β-NMN. The mixture
reaction was stirred at room temperature at room temperature. after 7 days the starting materials
were not seen the reaction was added with cold water (10 mL) and proposed to be purified by
HPLC.
Method 2: To a solution containing β-Nicotinamide mononucleotide (β-NMN) (10.0 mg, 0.030
mmol, 0.67 equiv) in dioxane (200 µL), dimethyl formamide (75 µL), and hexamethyl
phosphoamide (75 µL) were added with diphenyl phosphochloridate (12.5 µL, 0.060 mmol, 1.5
equiv), tri-n-butylamine (18 µL, 0.075 mmol, 1.7 equiv), and benzyltributylammonium chloride
27
(9.4 mg, 0.030 mmol, 0.67 equiv). The mixture was allowed to vigorously stir at room temperature
for 1 hour under argon. Some participate was formed by adding ethyl ether (500 µL) to the reaction
mixture. The participate was collected by centrifugation, washed with ethyl ether, and dried in
vacuo. The residue was dissolved in dioxane (150 µL). the crude 8-alkyne-AMP in anhydrous
dimethyl formamide (200 µL) was added to the activated β-Nicotinamide mononucleotide solution,
and anhydrous pyridine (200 µL) was added to the mixture immediately. The reaction mixture was
allowed to stir for 18 hours at room temperature. After removal the solvent by vacuum, the residue
was added with cold water (5 mL) and proposed to be purified by HPLC.
Method 3: the crude 8-alkyne-AMP was dissolved in anhydrous DMSO (2 mL) and the
following were added with following reagents: triphenylphosphine (656 mg, 2.5 mmol, 5 equiv),
morpholine (369 µL, 4.2 mmol, 8.5 equiv), and 2.2’-dipyridyldisulfide (551 mg, 2.5 mmol, 5
equiv). the reaction mixture was processed to stir at room temperature for 3 hours. The participate
was formed by adding Sodium iodide (0.2 M) in acetonitrile dropwise into the reaction. The
participate was collected by centrifugation and washed with cold acetonitrile. Then the participate
was dissolved in cold methanol and dried under vacuum. This crude product was dissolved in a
solution of Manganese(II) chloride (500 µL, 0.2 M in formamide) with β-Nicotinamide
mononucleotide (20 mg, 0.055 mmol), and Magnesium sulfate (12 mg, 0.100 mmol). The reaction
mixture was allowed to stir at room temperature for 48 hours. After that, the reaction was
concentrated and then added with cold water, and be proposed to be purified by HPLC.
3.4 Data analysis
PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide
staining. Western Blots were analyzed by using Image Lab (Bio-Rad, Irvine, CA).
1
H NMR was
28
performed on Varian 400 spectrometers, NMR data were analyzed by using Nuts software (Acorn
NMR Inc. Livermore, CA).

29
Chapter 4
Results
4.1 Monoclonal Cloning and Expression of Antibody-Actin Fusion
Proteins
4.1.1 Rationale
Preferentially, ADCs should deliver the cytotoxic molecules to tumor cells with the target
antigen rather than to healthy tissue in order to achieve high efficacy. Several key areas of research
contribute to the development of ADCs, including the choice of an appropriate antigen target,
development of potent payload, and stable linkers with the releasing strategies of the cytotoxic
payload upon internalization, and conjugation technology.
Rather than normal tissues, some metastatic breast cancer tumors can highly overexpress the
HER2 antigen (Lewis Phillips et al., 2008). Trastuzumab-based therapy is attributed to aberrant
activation of signaling pathways, including in the phosphatidylinositol 3-kinase pathway (Nagata
et al., 2004), activation of compensatory signaling through up-regulation of the insulin-like growth
factor-I receptors (Nahta et al., 2005), or ErbB/HER ligands (Helft et al., 2004), or generation of
a constitutively active truncated from of HER2, designated p95HER2 (Anido et al., 2006).
Previous studies have been showed that the structure of a Michaelis complex with iota toxin,
actin, and a non-hydrolyzable NAD
+
analogue (Tsuge et al., 2008). It is proposed that the common
reaction mechanism for the actin-targeting mART toxins is formed following the cleavage of the
30
nicotinamide moiety from NAD
+
(Tsuge et al., 2008). Photox toxin is indeed an ADP-
ribosyltransferase, making it served as the member of the actin-targeting mART family.
Fusion proteins are engineered biomolecules including parts from two or more genes
synthesized as a single multi-functional construct. Bi-specific fusion proteins have been served as
biopharmaceuticals, with an active drug domain fused to a carrier domain, allowing for proper
transport (Chen et al., 2013). Linker can be classified into three groups: flexible, rigid, and
cleavable (Chen et al., 2013). The most common is the (Gly4Ser)
n
linker (Gly-Gly-Gly-Gly-Ser)
n
,
and n indicates the number of repeats of the motif. It is the flexible linker with generally composed
of small, non-polar or polar residues. The additional polar residue, serine can reduce linker-protein
interactions and preserve protein functions (Chen et al., 2013).
In aim 1 studies, the DNA sequence of actin fused to the C-terminus of the Herceptin heavy
chain was designed to contain a (Gly4Ser)
2
linker. This resulting heavy chain fusion construct was
then co-transfected with the expression construct of Herceptin light chain into the Expi293F™
cells. The proposed secreted Herceptin-actin fusion protein was obtained by collecting the
Expi293F™ expression medium and purified using protein G Resin.
4.1.2 Results
4.1.2.1 Molecular Cloning of Antibody-Actin Fusion Proteins
After the PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium
bromide staining, there were two clean bands at nearly 1.4 kilobases and 1.3 kilobases, respectively.
The results confirmed with the design of DNA template for Herceptin heavy chain fragment
(including the forward and reserve primers, 1383 bases), and the design of DNA template for actin
(including the forward and reserve primers, 1161 bases).
31

Figure 1: PCR products were analyzed by agarose gel electrophoresis and visualized by
ethidium bromide staining: lane 1: Quick-Load
®
Purple 2-Log DNA Ladder (0.1-10.0kb); lane
2: 50 µl Herceptin heavy chain PCR reaction with 10 µl Gel Loading Dye, Purple (6×), no SDS
loading buffer; lane 3: 50 µl actin PCR reaction with 10 µl Gel Loading Dye, Purple (6×), no SDS
loading buffer. Gel electrophoresis running time: 55 min.

32
After the generated DNA templates were quantified and purity was verified, these DNA
templates were then conducted for overlap extension PCR. After the PCR products analyzed by
agarose gel electrophoresis and visualized by ethidium bromide staining, there was a clean band
between 3.0 kilobases and 2.0 kilobases. The shown result was confirmed with the whole length
of designed DNA sequences (including the Herceptin heavy chain forward primer and actin reverse
primer, 2526 bases).
The DNA insert was cloned into the pFUSE vector through T4 DNA ligase. Plasmid was
obtained by ZR Plasmid Miniprep-Classic Kit. The constructs were confirmed by DNA sequencing.
4.1.2.2 Expression Herceptin-actin fusion antibody
After purification by protein G Resin, the concentrated protein samples were analyzed by SDS-
PAGE and visualized by coomassie brilliant blue staining. However, there were not obvious bands
with correct size shown on the gel due to the low concentration that was 0.083 mg/ml by using
NanoDrop 2000C Spectrophotometer (Thermo Scientific, Houston, TX).
Thus, the western blotting was conducted to determine whether the sample protein was the
desired fusion protein with highly sensitivity. There were some bands at 190 kDa, 25 kDa, and
between 58 kDa and 46 kDa.

33

Figure 2: Overlap PCR products analyzed by agarose gel electrophoresis and visualized by
ethidium bromide staining: lane 1: Quick-Load
®
Purple 2-Log DNA Ladder (0.1-10.0kb); lane
2: 50 µl overlap PCR reaction with 10 µl Gel Loading Dye, Purple (6×), no SDS loading buffer;
gel electrophoresis running time: 55 min.

34
Figure 3:SDS-PAGE for Herceptin-actin fusion protein: lane 1: 5 µl Blue Protein Standard,
Broad Range; lane 2: 1.5 µg sample protein (denatured) with 10 mM DTT and NuPAGE
®
LDS
Sample Buffer (4X), boiled for 10 min; lane 3: 1.5 µg sample protein (not denatured) and
NuPAGE
®
LDS Sample Buffer (4X); lane 4: 20 µl cell lysis with 10 mM DTT and NuPAGE
®
LDS Sample Buffer (4X), boiled for 10 min; lane 5: 20 µl cell lysis with NuPAGE
®
LDS Sample
Buffer (4X); lane 6: 20 µl cell medium from Day 3 with 10 mM DTT and NuPAGE
®
LDS
Sample Buffer (4X), boiled for 10 min; lane 7: 20 µl medium from Day 3 with NuPAGE
®
LDS
Sample Buffer (4X); lane 8: 20 µl medium from Day 6 with 10 mM DTT and NuPAGE
®
LDS
Sample Buffer (4X), boiled for 10 min; lane 9: 20 µl medium from Day 6 with NuPAGE
®
LDS
Sample Buffer (4X); lane 10: 20 µl flow through with 10 mM DTT and NuPAGE
®
LDS Sample
Buffer (4X), boiled for 10 min; lane 11: 20 µl flow through with 10 mM DTT and NuPAGE
®

LDS Sample Buffer (4X), boiled for 10 min; lane 12: 5 µl Blue Protein Standard, Broad Range;
SDS-PAGE running time: 55min.

35
Figure 4:Western Blotting of Herceptin-actin fusion protein: lane 1: 5 µl Blue Protein
Standard, Broad Range; lane 2: 1.5 µg sample protein (denatured) with 10 mM DTT and
NuPAGE
®
LDS Sample Buffer (4X), boiled for 10 min; lane 3: 1.5 µg sample protein (not
denatured) and NuPAGE
®
LDS Sample Buffer (4X); lane 4: 20 µl cell lysis with 10 mM DTT
and NuPAGE
®
LDS Sample Buffer (4X), boiled for 10 min; lane 5: 20 µl cell lysis with
NuPAGE
®
LDS Sample Buffer (4X); lane 6: 20 µl cell medium from Day 3 with 10 mM DTT
and NuPAGE
®
LDS Sample Buffer (4X), boiled for 10 min; lane 7: 20 µl medium from Day 3
with NuPAGE
®
LDS Sample Buffer (4X); lane 8: 20 µl medium from Day 6 with 10 mM DTT
and NuPAGE
®
LDS Sample Buffer (4X), boiled for 10 min; lane 9: 20 µl medium from Day 6
with NuPAGE
®
LDS Sample Buffer (4X); lane 10: 20 µl flow through with 10 mM DTT and
NuPAGE
®
LDS Sample Buffer (4X), boiled for 10 min; lane 11: 20 µl flow through with 10 mM
DTT and NuPAGE
®
LDS Sample Buffer (4X), boiled for 10 min; lane 12: 5 µl Blue Protein
Standard, Broad Range; SDS-PAGE running time: 55min.

36
4.2 Expression and Purification of Photox Toxin and Its ADP-
ribosyltransferase Activity
4.2.1 Rationale
Produced by P. luminescens , Photox, a novel putative virulence factor, is one of the member of
mART family (Visschedyk et al., 2010). Photox targets Arg
177
of actin through ADP-
ribosyltransferase activity with relatively high enzymatic activity (k
cat
, 1680± 75 min
-1
) compared
with known actin-targeting mARTs (Visschedyk et al., 2010).
Although most of mARTs are known to process the NAD
+
-glycohydrolase activity to hydrolyze
the NAD
+
substrates. It has been reported that both C21 (Schleberger et al., 2006) and iota (Otto
et al., 2000) toxin shows the NAD
+
-glycohydrolase activity. However, both wild-type and mutant
photox enzymes do not show detectable the NAD
+
-glycohydrolase activity (Visschedyk et al.,
2010). This residual activity is also not processed by SpvB highlighting this similarity of these two
toxins (Sakurai et al., 2003).
Photox can catalyze the enzymatic function of covalent modifying the host protein, actin,
through transfer of an ADP-ribose moiety from NAD
+
, and releasing free nicotinamide. The
photox was tested for in vitro mART activity by incubating Photox with normal NAD
+
, and
mixture of the nonmuscle β- and γ-actin. The free nicotinamide, proposed to be generated by ADP-
ribosylation, were detected by HPLC system with multiple control groups.
In Aim 2 studies, the photox gene was overexpressed in E.coli. The protein was tried to be
purified from supernatant of the cell lysis rather than the inclusion bodies from the reported studies.
37
4.2.2 Results
4.2.2.1 Overpression and purification of photox
After purified by Hispur Ni-NTA Superflow Agarose, the concentrated protein samples were
analyzed by SDS-PAGE and visualized by coomassie brilliant blue staining. The level of purity
and the size of the protein on the basis of SDS-PAGE gel indicated that the protein was isolated
from the supernatant. The purified protein was shown to have a His-tag by using Western blotting
with a monoclonal antibody against the ploy-His tag (data not shown).
The yield of purified photox protein was ~ 1 mg liter
-1
which was lower than the reported yield
of ~ 10 mg liter
-1
of culture.

38

Figure 5:SDS-PAGE for Photox sample protein: lane 1: 5 µl Blue Protein Standard, Broad
Range; lane 2: 5 µg Photox sample protein (denatured) with 10 mM DTT and NuPAGE
®
LDS
Sample Buffer (4X), boiled for 10 min; lane 3: 5 µg Photox sample protein with NuPAGE
®
LDS
Sample Buffer (4X); lane 4: 20 µl soluble part of inclusion bodies in dialysis buffer with 10 mM
DTT and NuPAGE
®
LDS Sample Buffer (4X), boiled for 10 min; lane 5: 20 µl soluble part of
inclusion bodies in dialysis buffer with NuPAGE
®
LDS Sample Buffer (4X); lane 1: 5 µl Blue
Protein Standard, Broad Range.

39
4.2.2.2 Mono-ADP-ribosylation of Actin by Photox
After photox incubating with normal NAD
+
and the mixture of nonmuscle β- and γ-actin in
appropriate buffer for 1 hour without light, the samples were detected by the HPLC system
(Method: 0.1% formic acid (Solvent 1) and methanol with 0.1% formic acid (Solvent 2). samples
were eluted at a flow rate of 2 ml min
-1
with 0% solvent 2 for 2 min, then a linear gradient of 0%
to 50% solvent 2 over 12 min, and finally 100% solvent 2 for 5min before equilibrating the column
back to 0% solvent 2 over 5 min). At the same time, multiple control groups were also detected by
the HPLC with the same method. However, the HPLC did not show the new generation of free
nicotinamide, remaining pretty amount of normal NAD
+
.
Due to the undetectable generation of free nicotinamide, the reaction samples were detected by
the same HPLC system methods after overnight reaction. However, it still did not show the new
generation of free nicotinamide with remaining normal NAD
+
.

40
Figure 6:HPLC analysis for Mono-ADP-ribosylation of Actin by Photox (1-hour reaction):
A: 5 µM Photox incubating with 5 µM actin and 200 µM NAD
+
performed in 10 mM Tris-HCl,
pH 8.0, 0.2 mM ATP, 0.2 mM calcium chloride, 0.2 mM 2-mercaptoethanol; B: only 5 µM actin
and 200 µM NAD
+
performed in 10 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2 mM calcium
chloride, 0.2 mM 2-mercaptoethanol; C: only 200 µM nicotunamide performed in 10 mM Tris-
HCl, pH 8.0, 0.2 mM ATP, 0.2 mM calcium chloride, 0.2 mM 2-mercaptoethanol; D: 200 µM
NAD
+
performed in 10 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2 mM calcium chloride, 0.2 mM
2-mercaptoethanol; E: 100 µM ATP performed in 10 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2
mM calcium chloride, 0.2 mM 2-mercaptoethanol F: 100 µM adenosine diphosphate ribose
performed in 10 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2 mM calcium chloride, 0.2 mM 2-
mercaptoethanol.

41
Figure 7: HPLC analysis for Mono-ADP-ribosylation of Actin by Photox (overnight
reaction): A: 5 µM Photox incubating with 5 µM actin and 200 µM NAD
+
performed in 10 mM
Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2 mM calcium chloride, 0.2 mM 2-mercaptoethanol; B: only 5
µM actin and 200 µM NAD
+
performed in 10 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2 mM
calcium chloride, 0.2 mM 2-mercaptoethanol; C: only 200 µM nicotunamide performed in 10
mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2 mM calcium chloride, 0.2 mM 2-mercaptoethanol; D:
200 µM NAD
+
performed in 10 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2 mM calcium chloride,
0.2 mM 2-mercaptoethanol; E: 100 µM ATP performed in 10 mM Tris-HCl, pH 8.0, 0.2 mM
ATP, 0.2 mM calcium chloride, 0.2 mM 2- mercaptoethanol F: 100 µM adenosine diphosphate
ribose performed 10 mM Tris-HCl, pH 8.0, 0.2 mM ATP, 0.2 mM calcium chloride, 0.2 mM 2-
mercaptoethanol.

42
4.3 Chemically Synthesis of ‘Clickable’ Alkyne-Tagged NADs
Analogues
4.3.1 Rationale
It has been reported that development of functionalized NAD
+
analogues for research on
PARylation processes. There are some bioorthogonal reporters and NAD
+
aides at different
positions as well as the NAD
+
with terminal alkenes that can label PAR through either SPAAC or
DA
inv
chemistry (Wallrodt et al., 2016). Recently, the “clickable” NAD
+
analogues based on
bioorthogonal chemistry served as chemical tools for the intracellular imaging of PAR (Wallrodt
et al., 2016).
In order to explore the best substrate properties, the “clickable” NAD
+
analogues with alkyne
group at varied positions were generated, thereby allowing copper(I)-catalyzed azide-alkyne
cycloaddition reactions. Both 6-alkyne-NAD
+
and 8- alkyne-NAD
+
have been reported to be used
by ARTD 1 to form modified polymers.
4.3.2 Results
The structures of the intermediates were confirmed by
1
H NMR.
4.3.2.1 β-Nicotinamide mononucleotide
4.3.2.1.1 Nicotinamide riboside
Nicotinamide riboside was re-purified from the previous storage in our lab. It yielded a white
solid (255 mg, 1.0 mmol).
1
H NMR (400MHz, D
2
O) δ 9.6 (s, 1H), 9.26 (d, J = 6.4 Hz, 1H), 8.97
43
(d, J = 8 Hz, 1H), 8.27(t, J = 6.8 Hz, 1H), 6.24(d, J = 4 Hz, 1H), 4.51-4.47 (m, 2H), 4.35 (t, J =
4.8 Hz, 1H), 4.04(dd, J = 13.2, 10 Hz, 1H), 3.89 (dd, J = 13.2, 10Hz, 1H).
4.3.2.1.2 β-Nicotinamide mononucleotide
The reaction yielded a white solid (170mg, 51%).
1
H NMR (400MHz, D
2
O) δ 9.46 (s, 1H),
9.28(d, J = 6.4 Hz, 1H), 8.97 (d, J = 8 Hz, 1H), 8.29(t, J = 6.8 Hz, 1H), 6.21(d, J = 4 Hz, 1H),
4.82(s, 1H), 4.74(t, J = 4.8 Hz, 1H), 4.49 (dd, J = 13.2, 10 Hz, 1H), 4.33(dd, J = 13.2, 10Hz, 1H).
4.3.2.2 6-alkyne-NAD
+

4.3.2.2.1 6-alkyne-adenosine
The reaction yielded a white solid (600 mg, 56%).
1
H NMR (400MHz, CD
3
OD) δ 8.29-8.28 (m,
2H), 5.98 (d, J = 6 Hz, 1H), 4.75 (t, J = 5.2 Hz, 1H), 4.41 (s, 2H), 4.33-4.32 (m, 1H), 4.18-4.17
(m, 1H), 3.88 (dd, J = 12.4, 2.4 Hz, 1H), 3.75 (dd, J = 12.4, 2.4 Hz, 1H), 2.62-2.60 (m, 1H).
4.3.2.2.2 6-alkyne-AMP
After further purification by using HPLC, t
R
= 10.91 min (collection time: 10.50-12.00 min) the
reaction was carried out with this product to synthesize 6-alkyne-NAD
+
without analyzing by
NMR
4.3.2.2.3 6-alkyne-NAD
+
4.3.2.2.3.1 6-alkyne-AMP morpholidate
The reaction yielded a white solid (37 mg, 0.050 mmol).
1
H NMR (400MHz, D
2
O) δ 8.39 (s,
1H), 8.27 (s,1H), 6.09 (d, J = 5.2 Hz, 1H), 4.52 (t, J = 4.4 Hz, 1H), 4.32 (s, 3H), 4.06-3.94 (m, 2H),
3.34 (s, 4H), 2.87 (7, J = 4 Hz, 4H), 2.61 (t, J = 2 Hz, 1H).
44
4.3.2.2.3.2 6-alkyne-NAD
+
After the reaction mixture was checked by MS, the molecular ion peak showed: 701.2 (exact
mass = 701.12).

4.3.2.3 8-alkyne-NAD
+

4.3.2.3.1 8-alkyne-adenosine
The reaction was carried out with iodiadenosine (197 mg, 0.100 mmol) and following with the
above method to yield light yellow solid.
1
H NMR (400MHz, CD
3
OD) δ 8.35 (s, 1H), 5.93 (d, J =
5.6 Hz, 1H), 4.84-4.61 (m, 1H), 4.29-4.27 (m, 1H), 4.12 (s, 1H), 3.87 (d, J = 14.8Hz, 1H), 3.72 (d,
J = 14.8Hz, 1H), 0.234 (s, 1H).
After removal the TMS group, it yielded light yellow solid (66 mg, 45%).
1
H NMR (400MHz,
CD
3
OD) δ 8.35 (s, 1H), 5.93 (d, J = 6 Hz, 1H), 4.68-4.66 (m, 1H), 4.31-4.29 (m, 1H), 4.14-4.12
(m, 1H), 3.86 (dd, J = 12.4, 2.4 Hz, 1H), 3.72 (dd, J = 12.4, 2.4 Hz, 1H), 3.28-3.27 (m, 1H).
4.3.2.3.2 8-alkyne-AMP
No NMR or MS data were available.
4.3.2.3.3 8-alkyne-NAD
+

No NMR or MS data were available.
45

Figure 8:Nicotinamide riboside
46

Figure 9:β-Nicotinamide mononucleotide
47

Figure 10: 6-alkyne-adenosine
48

Figure 11: 6-alkyne-AMP morpholidate
49

Figure 12: 8-alkyne-adenosine with TMS
50

Figure 13: 8-alkyne-adenosine
51

Figure 14: MS result for 6-alkyne-NAD
+

52

Figure 15: HPLC for separation of 6-alykne-AMP
53
Chapter 5
Discussion & Conclusion & Future work
5.1 Aim 1: Generation of Antibody-Actin Fusion Antibody
Trastuzumab (Herceptin) is a recombinant humanized IgG1 targeting the extracellular domain
of the human epidermal growth factor receptor 2 (HER-2). Herceptin inhibits the proliferation of
tumor overexpressing HER-2 antigen. The interaction between the antibody and the HER-2
antigen is featured with high affinity and specificity (Magdelaine-Beuzelin et al., 2007).
Actin was designed for fusion into C-terminus of the Herceptin heavy chain with the (GGGGS)
2
flexible linker. The full-length heavy chain of Herceptin and human actin sequences were
amplified and spliced by PCR reaction. After transformation, positive clones were confirmed by
DNA sequencing.
The Herceptin-action fusion antibody was designed to be expressed by Expi293F™ expression
system. This fusion antibody was designed as a secretory protein that the plasmid included a
secretory signal sequence. However, the yield of the fusion protein was too low for detection by
SDS-PAGE. It did not show a correct size by using western blotting. The possible reasons for this
failure as follows:
First of all, the target fusion protein or at least most of target fusion protein could not be secreted
from the cells to the Expi293F™ medium due to its larger size compared with the full length
Herceptin antibody. The data indicated that some protein fragments were expressed with Herceptin
light chain in the cells. However, neither of them were the target fusion proteins.
54
Furthermore, the target fusion protein secreted from cells to the Expi293F™ medium. However,
most of them went to the degradation process during the purification which might explain why the
concentration was low but still remained a little.
As a result of above discussion, the future work should involve: 1) designing new DNA
constructs for Herceptin-actin fusion proteins with optimized secretory signal sequence. 2)
optimizing the methods of purification by avoiding the degradation. 3) designing new linker
sequences for conjugating Herceptin and actin protein sequences. 4) using other expression
systems to produce the fusion proteins.
5.2 Aim 2: Expression and Purification of Photox Toxin and ADP-
ribosyltransferase Activity
Although, the sample protein was identified as a 46 kDa toxin by SDS-PAGE and His-tagged
protein by western blotting. It could not efficiently ADP-ribosylates nonmuscle β- and γ-actin
mixture with normal NAD
+
. The possible reasons for this failure as follows:
First of all, the photox toxin used in this study was isolated from the supernatant of the cell lysis.
However, the photox toxin reported with highly ADP-ribosylation activity was purified from the
inclusion bodies. The enzyme from supernatant might have the same size and His-tag as the
reported enzyme with high efficient mono-ADP-ribosylation activity, but the enzyme from
supernatant might not have the enzymatic functions.
Second, actin used for the activity assay could not be recognized by the photox toxin. The
commercial nonmuscle β- and γ-actin mixture might include some impurity that might affect the
recognition by the photox enzymes.
55
Another possibility would be that HPLC system could not detect the generation of free
nicotinamide due to the low sensitivity of the HPLC detector.
Thus, the future work should include: 1) re-purification of the Photox toxin by using reported
method. 2) using other actin as the substrate for this assay. 3) optimizing the efficacy test of ADP-
ribosylation through the improving the sensitivity of detectable methods.
Furthermore, the future work may use fluorescein isothiocyanate (FITC)-NAD
+
as the cofactor.
In this way, proteins can be analyzed by SDS-PAGE and UV illumination. Another method may
be that ADP-ribosylation activity can be tested by using ‘clickable’ alkyne-tagged NAD
+

analogues as the cofactor. Thus, the ADP-ribose unit labeled proteins can be analyzed by western
blotting based on click chemistry with high sensitivity.
5.3 Aim 3: Chemically Synthesis of ‘Clickable’ Alkyne-tagged NADs
Analogues
6-alkyne-NAD
+
: the final product was under purification.
8-alkyne-NAD
+
: The molecular ion peak of 8-alkyne-AMP was detected by MS. However, 8-
alkyne-AMP was not able to be purified by HPLC system. There still remained impurity within
the product. Without further purification, the crude 8-alkyne-AMP was used to synthesis 8-alkyne-
NAD
+
by using three different methods. Unfortunately, MS could not detect the molecular ion
peak and other fragment peak of 8-alkyne-NAD
+
. The reasons as follows:
First of all, the impurity in 8-alkyne-AMP could affect the synthesis of 8-alkyne-NAD
+
.
56
Another possible reason is that 8-alkyne-NAD
+
was synthesized, however, it went to
degradation during the purification and MS experiment due to possible unsuitable separation
methods.
Above all, the future work should include: 1) purifying and lyophilizing crude 8-alkyne-AMP
to obtain the pure product by using ion exchange chromatography. 2) optimizing HPLC and
LC/MS separation methods in order to avoid possible degradation.

57
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Abstract (if available)

Abstract Monoclonal antibodies possess exquisite specificity to their cognate antigens. By covalently linking cytotoxic drugs to monoclonal antibodies, the resulting antibody-drug conjugates (ADCs) represent a promising approach for targeted drug delivery. Numerous methods have been developed for generating ADCs through chemically conjugating small-molecule drugs to surface amino acid residues of monoclonal antibodies, which often leads to heterogenous products due to non-specific chemical reactions. To make homogenous ADCs with site-specifically labelled drug molecules, enzyme-catalyzed reactions could possibly offer a new solution. Using nicotinamide adenine dinucleotide (NAD⁺) as a co-substrate, photox, a mono-ADP-ribosyltransferase from Photorhabdus luminescens, catalyzes ADP-ribosylation of actin. It was envisioned that photox could enzymatically add ADP-ribose groups with alkyne tags to antibodies fused with actin polypeptides. The resulting antibody-actin fusions modified with alkyne-labelled ADP-ribose could thus enable site-specific conjugation of cytotoxic drugs through well-established click chemistry. Herein the photox enzyme was recombinantly expressed and purified from bacteria. By genetically fusing human actin to an anti-HER2 monoclonal antibody, an antibody-actin fusion protein was expressed from mammalian cells. Catalytic activities of purified photox were examined with actin and the antibody-actin fusion. In addition, NAD⁺ analogues with alkyne tags were attempted for chemical synthesis. Through identification of active NAD⁺ analogues for the photox enzyme, antibody-actin fusions labeled with alkyne tags will likely be created for site-specific conjugation of monoclonal antibodies.

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Asset Metadata

Creator Lu, Yanran (author)

Core Title Development of new approaches for antibody modification

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/17/2017

Defense Date 04/17/2017

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

Tag ADP-ribosylation,antibody-drug conjugates,NAD analogues,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zhang, Yong (committee chair), Okamoto, Curtis T. (committee member), Shen, Wei-Chiang (committee member)

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