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Development of engineered antibodies as novel anti-cancer agents

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Development of engineered antibodies as novel anti-cancer agents

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Content Development of Engineered Antibodies as Novel Anti-Cancer Agents
By
Zekun Li
A Thesis Presented to the
FACULTY OF THE USC MANN SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2023
Copyright 2022 Zekun Li
ii

Acknowledgements
I would like to express my sincere appreciation to Dr. Yong (Tiger) Zhang for his exceptional
mentorship, insightful advice, and unwavering support throughout this journey. His guidance has
been invaluable to the success of this research.
I am also grateful to my committee members, Dr. Houda Alachkar and Dr. Roger Duncan, for
their dedicated effort and invaluable support in reviewing and providing valuable input on this
work.
Furthermore, I extend my heartfelt thanks to all the members of Dr. Zhang's laboratory,
including Jiawei Li, Dr. Xiao-Nan Zhang, Dr. Liang-Chieh Chen, Guoyun Kao, Elisa Stephens,
Hyo Sun (Sunny) Kim, Kimia Hariri, Po-Wen Yu, and Yuanteng Zhao. Their assistance and
support have been instrumental in the progress and completion of this study.
I am profoundly grateful to everyone mentioned above for their contributions, guidance, and
encouragement throughout this research endeavor.

Zekun Li
May 2022
University of Southern California

iii

Table of Contents
Acknowledgements………...………………………………………………...…….....….............ii
List of Figures ……………...………………………………………………...……..…..………..v
Abbreviations………………………………………………………………….….….…..…........vi
Abstract………………………………………………………………………....….…..….……viii
Chapter 1: Introduction………………………….…………..……...………………...….....……..1
1. Target-specific therapy and prostate cancer………………………………….....………….1
2. Prostate-Specific Membrane Antigen (PSMA)………………………………….…………2
3. J591 Monoclonal Antibody………………………………………………………....……...3
4. ADP-ribosyl cyclase–enabled Antibody-Drug Conjugate …………………………..….…5
5. Acute myeloid leukemia (AML)………………………………………………….………..8
6. Potential targets for AML treatment…………………………………………………….….9
Purpose of this study……………………………………………………………….…….....…10
Chapter 2: Material and Methods………………………...…………..………….….…...….……12
1. Rationale of ARC-ADC…………………………………………………………………..12
2. The design and preparation of J591 plasmid construct……………………………………12
3. The design of J591-CD38 plasmid construct……………………………………………...12
4. Target construct amplification…………………………………………………………….13
iv

5. Expression and purification of J591 fusion protein………………………………………..14
6. ELISA binding assay for J591 fusion protein……………………………………………..15
7. Expression and purification of scFvs……………………………………………..………16
Chapter 3: Results……………………………………..…………………………………………17
1. J591-CD38 Construct……………………………………………………………………..17
2. Purification of CD99, FLT3 and CD99/FLT3 ScFvs……………………………………..25
Chapter 4: Discussion………………………………………………………..…………….……..28
1. Preparation of J591 fusion protein………………………………………….……………..28
2. Purification of CD99-FLT3 fusion protein………………………………………………..29
Chapter 5: Conclusion and Future Direction…………………………….……….……...…….....32
References………………………………………………………………………………………..34
v
List of Figures
Figure 1. The 3-part structure schematic of PSMA homodimer………………...…...…….....…....5
Figure 2. Structure of CD38 covalent inhibitor 2′-F-araNMN and schematic
representation of 2′-F-araNMN/CD38 complex……………………………..…….…………..….7
Figure 3. The catalytic reaction of 2′-F-araNAD+/CD 38 stable complexes…...…….………........8
Table 1. List of designed primers……………………………………………..…….………........14
Figure 4. Amplification of J591 heavy, light chains and Herceptin heavy, light chains……..........20
Figure 5. Overlap extension PCR of J591 construct……………………………………..….........21
Figure 6. SDS-PAGE for J591 fusion protein………………….………………...………..…..….21
Figure 7. The graph of J591 fusion protein showed dose-dependent manner
against recombinant PSMA by ELISA………………………………………….………….….…22
Figure 8. Non-linear regression fitting curve for J591 fusion protein…………..………………...23
Figure 9. Amplification of heavy chain and light chain CD38………………….…………….….24
Figure 10. Overlap extension PCR of J591-CD38 Fc and Fab regions………..………………...24
Figure 11 Overlap extension PCR of J591-CD38 heavy chain light chain with fewer
non-specific DNA fragments.…………………………………...……………..…………………25
Figure 12 SDS-PAGE of two ScFvs: anti-CD99 and anti-CD99/FLT3.....................................…27
Figure 13. SDS-PAGE of three ScFvs…………………………………………………...……….27
vi

Abbreviation
PSMA: Prostate Membrane Specific Antigen
MAbs: Monoclonal antibodies
PSA: Prostate-specific antigen
GCPII: Glutamate carboxypeptidase II
NAAG: N-acetylaspartylglutamate
SPECT: Single-Photon Emission Computerized Tomography
CT: Computerized Tomography
MRI: Magnetic resonance imaging
ADP:Adenosine diphosphate
NAD
+
: Nicotinamide adenine dinucleotide
ADC: Antibody-drug conjugate
ARC-ADCs: ADP-ribosyl cyclase–enabled antibody-drug conjugate
HER2: Anti human epidermal growth factor receptor 2
ELISA: Enzyme-linked immunosorbent assay
PBST: Phosphate buffered saline with Tween 20
E.Coli: Escherichia coli
AML: Acute Myeloid Leukemia
vii

LSCs: Leukemic stem cells
CD99: Cluster of differentiation 99
FLT3: FMS-like tyrosine kinase 3
RTK: Receptor tyrosine kinase
FL: FLT3 ligand
ITDs: Internal-tandem duplications
TKD: Tyrosine kinase domain
ScFvs: Single-chain variable fragments

viii

Abstract
Prostate-specific membrane antigen (PSMA) has appeared as a promising target for both therapy
and diagnosis of prostate cancer. As a transmembrane protein predominantly expressed in prostate
epithelial cells, particularly in prostate carcinomas. Elevated levels of PSMA have been linked to
the aggressiveness of prostate cancer, making it an attractive target for anti-PSMA targeted therapy
and radioactive-labeled anti-PSMA imaging agents. Typically, anti-PSMA drug candidates in
research are designed to bind to PSMA extracellularly. This extracellular binding has been shown
to enhance the rates of internalization, facilitating greater drug accumulation when using antibody-
drug conjugates. In this study, we propose an innovative approach by developing an ADP-ribosyl
cyclase–enabled antibody-drug conjugate based on the fusion of J591 and CD38. This construct is
expected to exhibit potent and selective activity against the PSMA extracellular domain.
Furthermore, we aim to achieve site-specific conjugation to improve the stability and
pharmacological activity of resulting antibody-drug conjugates, utilizing the enzymatic activity of
CD38.

CD99 and FLT3 have emerged as promising targets for therapy in acute myeloid leukemia (AML).
CD99 is a transmembrane protein expressed on normal hematopoietic cells, and its overexpression
has been correlated with the reactivity of self-renewing leukemic stem cells (LSCs). These LSCs
are resistant to conventional chemotherapy and may contribute to AML recurrence. FLT3, an
FMS-like tyrosine kinase 3, possesses mutations that continuously activate downstream pathways,
leading to myeloproliferation and potentially resulting in mortality. In this context, we hypothesize
that bispecific single-chain variable fragment (scFv) antibodies targeting both CD99 and FLT3
could exhibit superior inhibitory activity against AML compared to conventional targeted drug
ix

molecules. Thus, our study focuses on the expression and production of an in-house anti-
CD99/anti-FLT3 bispecific ScFv for subsequent cellular and animal studies.
1
Chapter 1: Introduction
1. Target-specific therapy and prostate cancer
In the past, pharmaceutical drugs or medicines were viewed as chemical substances that caused
biological changes in individual organs or the body when administered. These substances were
derived from plant extractions or synthesized compounds created in laboratories. They played
crucial roles in human survival, with notable examples such as penicillin, discovered by Alexander
Fleming in 1928, which saved millions of lives, particularly during wartime (Laxminarayan et al.,
2013). However, as time passed and life expectancy increased, the discovery of new diseases and
the presence of more complex illnesses highlighted the limitations of traditional medicines. This
realization led to the establishment of the concept of the "magic bullet" (Zauberkugel) in the early
1900s by immunologist Paul Ehrlich. He envisioned drugs that specifically targeted pathogens
without harming surrounding tissues, providing a promising avenue for future therapeutic
developments (Valent et al., 2016). Ehrlich's pioneering work laid the foundation for advances in
targeted therapies, which continue to benefit research today.
It was not until 1973 that Jerrold Schwaber and his colleagues reported the production of
monoclonal antibodies (mAbs) as we know them today (Schwaber and Cohen, 1973). Over the
past 20 years, monoclonal antibodies have proven to be versatile tools for research, cancer
diagnosis, therapeutic development, and other applications.
Prostate cancer is the most common cancer diagnosed in US men with estimated 165,000 cases
and 17.5% of mortality in 2018 (Siegel et al., 2018). And it was also reported that certain race and
family with prostate cancer history have greater risk of prostate cancers (National Cancer
institution, 2017). Conventionally, potential prostate cancer incidence and recurrence can be
detected through prostate-specific antigen (PSA) screening. PSA is a serine protease produced in
2

both normal and prostate cancer cell. For malignant cells, serum PSA can be elevated. As a result,
PSA screening may indicate prostate cancer or condition related to prostate which provide
invaluable time to treatment and prevention. However, studies showed factors such as age, race,
family history, certain medications and other causes may interfere the test result and may raise
false alarm (Center for Diseases Control and Prevention, 2021). After further investigation,
prostate-specific membrane antigen (PSMA) was discovered to express in all forms of prostate
cells, and it was suggested that PSMA can be utilized in developing diagnostic tools and
therapeutics (Chang, 2004).

2. Prostate-Specific Membrane Antigen (PSMA)
PSMA, also known as glutamate carboxypeptidase II (GCPII), was initially characterized by the
monoclonal antibody 7E11-C5.3 and classified as a type II membrane protein. Encoded by the
FLOH1 gene located on human chromosome 11p11-p12, PSMA exhibits an 18-amino acid
intracellular region and a large 700-amino acid extracellular domain involved in antibody
recognition and binding (Figure 1). Originally identified as a novel biomarker on the surface of
prostate cells, including malignant ones, PSMA garnered attention as a promising target for
therapeutic and diagnostic applications (Chang, 2004). However, subsequent research revealed
that PSMA is not exclusive to prostate cells, contrary to its name. It is also present in the kidney,
brain, small intestine, nervous system, and neovasculature of various tumors, expanding its
research significance (Evans et al., 2016).

3

In the brain, PSMA indirectly influences neurotransmitter release by catalyzing the hydrolysis of
N-acetylaspartylglutamate (NAAG), leading to increased N-acetyl-aspartate and glutamate levels.
Inhibiting PSMA could potentially provide neuroprotection by reducing glutamate concentrations
(Mesters et al., 2006). Nonetheless, a substantial amount of PSMA remains predominantly
expressed on prostate epithelial cells, particularly in prostate cancer. Although the exact role of
PSMA in prostate cancer progression is yet to be fully elucidated, it has been observed that
overexpression of PSMA is associated with the advancement of the disease. Membrane-bound
proteins such as PSMA, unlike secretory proteins like PSA, offer improved diagnostic accuracy
and serve as better targets for monoclonal antibody therapies (Akhtar et al., 2012).

Furthermore, PSMA undergoes internalization and recycling through clathrin-dependent
trafficking. Binding of PSMA-specific monoclonal antibodies to its extracellular domain enhances
the rate of internalization. This phenomenon can be exploited for therapeutic and imaging
applications, enabling greater accumulation of drugs, toxins, or radioactive imaging agents within
PSMA-positive cells via endocytosis (Gong et al., 1999). Recent advancements include the
approval of two PSMA-targeted imaging agents for prostate cancer diagnosis, albeit with limited
indications, highlighting the potential of PSMA-targeted agents (Evans et al., 2016).

3. J591 Monoclonal Antibody
The advent of 7E11-C35 monoclonal antibodies (mAbs) marked the beginning of PSMA-specific
antibody research. Capromab pendetide (ProstaScint; Aytu BioScience, Inc) became the first
application of anti-PSMA mAbs for diagnosing soft tissue metastases. This imaging agent utilizes
4

111In-labeled 7E11-C35 for Single-Photon Emission Computerized Tomography (SPECT) scans,
exhibiting higher sensitivity and specificity compared to traditional computerized tomography (CT)
and magnetic resonance imaging (MRI) scans (Lamb and Faulds, 1998). However, its utility is
limited due to the constraints of SPECT imaging, the inability to detect PSA, and the detection of
prostate cancer lesions.

Moreover, the epitope of 7E11-C35 resides in the cytoplasmic domain of PSMA, impeding its
therapeutic development as it lacks specificity towards viable prostate cells (Donin and Reiter,
2018). To overcome these limitations, several mAbs have been developed, including J415, J533,
and J591. These mAbs exhibit high specificity for the extracellular domain of PSMA, offering
potential for therapeutic applications targeting viable tumors. Among them, J415 and J591 have
shown superior binding affinity and improved localization compared to J533, making them the
preferred mAbs for prostate cancer research (Smith-Jones et al., 2003).

5

4. ADP-ribosyl cyclase–enabled Antibody-Drug Conjugate
CD38 is a type II transmembrane glycoprotein that was initially discovered on thymus cell
lymphocytes in 1980. It belongs to the adenosine diphosphate (ADP)-ribosyl cyclase family and
Figure 1. The 3-part structure schematic of PSMA homodimer. Each
monomer (100kDa) includes 44-750 amino acid-extracellular region
consisting of the epitope for J591 mAb, while the1-18 amino acid--
intracellular domain epitope can be specifically bound with 7E11-C5.3
mAb and a 19-43 amino acid transmembrane domain (Evans., et al,
2016).
6

plays diverse roles in biological processes. One of its crucial functions is its role as a receptor that
specifically binds to CD31, a highly glycosylated protein found on endothelial cells, platelets, and
macrophages. Upon activation, CD38 initiates intracellular signaling pathways, leading to the
production of various cytokines, such as interleukin 6, interferon-γ, interleukin 10, and
granulocyte-macrophage colony-stimulating factor. Through these immune signaling cascades,
CD38 modulates immune responses (Nooka et al., 2019).

CD38 also exhibits enzymatic activities involved in the regulation of nicotinamide adenine
dinucleotide (NAD+) levels. It catalyzes the conversion of NAD+ into ADP ribose and cyclic ADP
ribose. Notably, specific inhibitors of CD38, namely 2′-F-arabinose nicotinamide mononucleotide
(2′-F-araNMN) and 2′-F-arabinose NAD+ (2′-F-araNAD+), form highly stable complexes with
CD38 (Figure.2). This interaction occurs through the formation of an acylal ester bond between
the glutamate 226 residue (E226) and either 2′-F-araNMN or 2′-F-araNAD+ (Liu et al., 2008; Jiang
et al., 2009). Exploiting this unique characteristic, researchers have developed site-specific
antibody-drug conjugates (ADCs) known as ADP-ribosyl cyclase–enabled ADCs (ARC-ADCs).
These ARC-ADCs have found applications in various fields, including immunofluorescence
imaging (Figure.3) and drug delivery, among others.

In a recent study by Dai et al. (2020), an innovative approach was described for the design of an
ARC-ADC targeting human epidermal growth factor receptor 2 (HER2). The CD38 sequence was
strategically incorporated into an anti-HER2 antibody, either at the N or C terminus, resulting in a
bifunctional antibody-CD38 fusion protein. This fusion protein was covalently bound with a CD38
7

covalent inhibitor. The resulting anti-HER2 ARC-ADC exhibited remarkable cytotoxicity,
surpassing that of recombinant Herceptin, and demonstrated exceptional specificity against HER2-
positive breast cancer cells in both in vitro and in vivo experiments (Dai et al., 2020). These
findings highlight the potential of ARC-ADCs as promising therapeutic agents in the field of
targeted cancer treatment.

Figure 2. (A) Structure of CD38 covalent inhibitor 2′-F-araNMN. (B)
Schematic representation of 2′-F-araNMN/CD38 complex. Catalytic
residue E226 on CD38 is linked with 2′-F-araNMN by a stable ester
bond that is represented by sticks between the two different colored
CD38 domains. (Liu et al., 2008)
8

5. Acute myeloid leukemia (AML)
Acute myeloid leukemia (AML) is a hematopoietic disorder characterized by bone marrow failure
and the abnormal proliferation of immature myeloid cells. These disease-causing cells, known as
leukemia blasts, are present in the same locations where normal hematopoietic cells reside. As the
disease progresses, these leukemia blasts can expand within the bone marrow and peripheral blood
system (Saultz and Garzon, 2016).
Figure 3. The catalytic reaction of 2′-F-araNAD+/CD 38 stable
complexes. Purple sphere indicates the presence of fluorophore for
imaging purpose. CD 38 is conjugated to 2′-F-araNAD by covalent
binding of CD38 at Glu 226 residue. Schematic below represent the
structure of 2′-F-araNAD+ conjugated with rhodamine through click
chemistry. (Jiang et al., 2009).
9

Leukemic stem cells (LSCs) play a critical role in AML as they possess the ability to self-renew
and generate non-self-renewing cells that give rise to the majority of leukemic blasts. Conventional
chemotherapy approaches have limited effectiveness against LSCs, and it is believed that the
persistence of LSCs contributes to disease relapse. Therefore, the development of therapeutics with
the capability to selectively eliminate disease-causing stem cells, including LSCs, is crucial for
achieving effective treatment strategies in AML.

6. Potential targets for AML treatment
CD99 is a transmembrane protein expressed on leukocytes, including the abnormal cells found in
acute myeloid leukemia (AML). It participates in various cellular processes, such as T-cell
differentiation, transendothelial exchange, and cell adhesion (Vaikari, 2019). In AML, CD99 is
overexpressed in leukemic stem cells (LSCs) compared to normal hematopoietic cells. Its presence
can be utilized for identifying AML recurrence by detecting leukemic cells in bone marrow
specimens of AML patients. Additionally, CD99 has been reported to selectively enhance LSC
activity, and monoclonal antibodies (mAbs) targeting CD99, such as clone H036-1.1, have
demonstrated direct anti-leukemic cytotoxicity. Therefore, CD99 represents a specific and
promising target for imaging and therapeutic research as a disease stem cell marker (Chung et al.,
2017).

FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase (RTK), is a frequently
mutated gene found in AML, with approximately 35% of AML patients exhibiting FLT3 mutations.
10

Normally, FLT3 expression is confined to immature hematopoietic progenitors. It plays a crucial
role in downstream signaling upon binding with FLT3 ligand (FL) and participates in monocyte
differentiation, thereby exerting significant influence on human hematopoiesis. Like CD99, FLT3
is overexpressed in AML. Two major FLT3 mutations have been identified in AML: internal
tandem duplications (ITDs) and tyrosine kinase domain (TKD) mutations. AML patients positive
for FLT3-ITD exhibit constant activation of FLT3, leading to myeloproliferation through sustained
activation of downstream pathways. FLT3-TKD mutations have similar effects. Mutations in
FLT3 result in aberrant FLT3 expression, which has stimulated research interest, and the
development of FLT3-targeted monoclonal antibodies (mAbs) is in high demand. Several
inhibitors have been developed to interfere with the activity of mutated FLT3. Combination
therapy with midostaurin and decitabine or azacitidine has shown significant benefits, particularly
in older patients (Kindler, 2010).

Purpose of this study
1. Over the past two decades, the discovery of prostate-specific membrane antigen (PSMA) has
garnered significant research interest. In 2020, the first PSMA-specific positron emission
tomography (PET) imaging agent, Gallium 68 PSMA-11, received FDA approval for the
detection of potential metastatic prostate cancer. Unlike conventional imaging modalities such
as computed tomography (CT) scans, magnetic resonance imaging (MRI), and bone scans,
Gallium 68 PSMA-11 administration enables the visualization of prostate cancer lesions
through PET scanning (Hennrich & Eder, 2021). Subsequently, the second PSMA-targeting
imaging agent, piflufolastat F 18 (brand name: Pylarify), gained FDA approval six months
later. Pylarify is indicated for patients with suspected prostate cancer metastasis and potential
11

recurrence (Keam, 2021). Although significant advancements have been made in PSMA-
targeted diagnostics, the therapeutic application in this field is still awaiting a breakthrough.

Therefore, our objective is to develop a highly specific and potent humanized fusion protein
that targets PSMA-positive prostate cancer. Subsequently, we aim to utilize this fusion protein
to construct an effective antibody-drug conjugate (ADC) known as an ADP-ribosyl cyclase–
enabled ADC (ARC-ADC) by incorporating CD38 enzymatic activity. It is anticipated that the
ARC-ADC, with its drug payload internalized through the aid of the anti-PSMA antibody, will
induce cell death specifically in PSMA-positive prostate cancer cells.

2. An anti-CD99/anti-FLT3 bispecific antibody was developed with the aim of achieving
enhanced elimination or suppression of disease-causing stem cells. The rationale behind this
design stems from the observed correlation between FLT3-ITD mutation and the
overexpression of CD99. The bispecific single-chain variable fragment (scFv) antibody was
constructed by genetically fusing the scFv targeting CD99 with the scFv targeting FLT3 into
a single-chain format. Experimental findings demonstrated that the anti-CD99/anti-FLT3
bispecific scFv exhibited significant inhibitory effects on leukemic stem cells (LSCs). It was
hypothesized that the bispecific scFv would outperform single-target therapies in terms of
efficacy. To assess this hypothesis, large-scale expression, and purification of both bispecific
and monospecific scFv antibodies, targeting CD99 and/or FLT3, were conducted for
subsequent in vitro and in vivo studies.

12

Chapter 2: Materials and Methods
1. Rationale of ARC-ADC
J591 is an antibody that specifically targets the extracellular domain of PSMA. While it shares
reactivity characteristics with 7E11-C35, J591 offers additional advantages such as improved rates
of internalization in vivo and enhanced accessibility to PSMA on viable cancer cells and tumors.
To construct the full-length monoclonal antibody, the scaffold of an anti-HER2 antibody,
trastuzumab (Herceptin), was utilized. For site-specific drug conjugation, the CD38 sequence can
be genetically incorporated into the terminal region of the resulting antibodies. Our hypothesis
revolves around the creation of a potent and highly specific ARC-ADC, capable of delivering drug
payloads into PSMA-positive prostate cancer cells through endocytosis upon extracellular binding
to PSMA.

2. The design and preparation of J591 plasmid construct
To generate the desired construct, the variable fragments of J591 were amplified and genetically
linked to the N-terminus of the constant regions of both the heavy chain and light chain of
trastuzumab (Herceptin). After transfection, this fusion resulted in a novel fusion protein that
combines the antigen specificity of J591 with the Fc regions of trastuzumab.

3. The design of J591-CD38 plasmid construct
The generated J591 fusion protein demonstrated robust binding affinity towards recombinant
human PSMA, indicating its potential as a selective targeting agent. To facilitate site-specific
13

conjugation of a drug-linker to the antibody, novel constructs were designed by fusing CD38 to
the C-terminus of both the heavy and light chains of the J591 construct. This strategic fusion was
predicted to leverage the enzymatic activity of CD38, enabling precise and controlled conjugation
of the drug-linker to the antibody molecule.

4. Target construct amplification
Primers for individual amplicon were designed using SnapGene Software (2020). The constant
regions of Herceptin, encompassing the fragments for both the heavy chain and light chain, were
amplified using designed primers (Thermo Fisher, Table 1) and AccuPrimeTM Pfx polymerase
(Invitrogen). Overlap extension polymerase chain reactions (PCR) were performed to fuse the
amplified J591 variable fragments or CD38 to the N-terminus or C-terminus, respectively.
Individual fragment amplifications for J591 variable region and Herceptin Fc region were
performed with similar protocols with slight modification on melting temperature and extension
time based on the primer sequences and fragment sizes, respectively. All amplifications, including
the following overlap extension steps were performed using PTC-200 Thermal Cycler (MJ
Research). The resulting plasmid constructs, namely J591 or J591-CD38 fusion protein fragments,
were subjected to digestion using the restriction enzymes EcoRI and NheI (New England Biolab)
at 37 ℃ overnight. The digested products were isolated using DNA gel extraction kits (Zymogen),
and the DNA fragments were ligated with pFuse backbones using T4 DNA ligase (New England
Biolab). Subsequently, the complete fusion protein DNA vectors were transformed into DH10B
electro-competent cells following electroporation. Colonies containing the vectors were cultured,
and minipreps were performed to extract the plasmids. The extracted plasmids were subjected to
14

sequencing to confirm their integrity and fidelity. The expression vectors were then extracted from
bacterial cultures using the ZymoPure plasmid Miniprep kit (Zymo Research).

Table1. List of designed primer designs for fragment amplification and overlap extension PCR.
J591 variable region
Heavy
chain
Forward 5'-CACGAATTCGGAAGTTCAACTGCAACAATCTGGCC-3'
Reverse 5'-GGAGGCGGAACTCACGGTCAGTGTCGTG-3'
Light
chain
Forward 5'-CACGAATTCGGACATCGTTATGACTCAATCCCACAAGTTC-3'
Reverse 5'-GGTGCAGCCACAGTTCGTTTCAAATCCAACATTGTACCAGCACCG-3'
Herceptin constant region
Heavy
chain
Forward 5'-CCGTGAGTTCCGCCTCCACCAAGGGCCC-3'
Reverse 5'-CCAGCTAGCACTTATCATTTACCCGG-3'
Light
chain
Forward 5'-GGTACAATGTTGGATTTGAAACGAACTGTGGCTGCACCATC-3'
Reverse 5'-GCCAGCTAGCACTTATCAACACTCTC-3'
J591CD38 overlap region
Heavy
chain
Forward 5'-CTCCGGGTAAAGGGGGTGGCGGAAGCAGATGG-3'
Reverse 5'-CTTCCGCCACCCCCTTTACCCGGAGACAGGGAGAG-3'
Light
chain
Forward 5'-GGGGAGAGTGTGGGGGTGGCGGAAGCAGATGG-3'
Reverse 5'-CTTCCGCCACCCCCACACTCTCCCCTGTTGAAGCTC-3'
CD38 reverse primer
Heavy
chain
5'-CCAGCTAGCACTTATCAGATCTCACTAGTACATGAACTATCCTCTGG-3'
Light
chain

5. Expression and purification of J591 fusion protein
The expression vector encoding J591 monoclonal antibody was introduced into DH10B cells
through transformation. The transformed DH10B cells were cultured in 250 mL of low-salt LB
broth at 37 ℃ and 220 RPM overnight. The J591 expression vector was then extracted using
ZymoPure plasmid Maxiprep kits (Zymo Research).
15

Subsequently, the extracted J591 expression vector was transiently transfected into Expi293 cells.
The resulting supernatants were subjected to dialysis three times in 4L of equilibrium buffer (250
mM NaCl, 25 mM Hepes, pH=7) either overnight or for a duration of 6 hours. The collected
supernatants containing J591 fusion protein were purified using protein G affinity chromatography,
resulting in a yield of 5.0 mg of purified antibody. The purified J591 fusion protein was stored at
-80℃ for further use.

6. ELISA binding assay for J591 fusion protein
A sandwich enzyme-linked immunosorbent assay (ELISA) was performed using a total volume of
200 µL. A 96-well high binding plate was utilized for the assay. The plate was initially incubated
overnight with 2.0 µg/mL recombinant PSMA in PBST (phosphate-buffered saline with Tween
20) after blocking with a 3% BSA blocking buffer.

A primary antibody, J591 fusion protein, was added to the plate in a concentration gradient with
3-fold serial dilution in 9 wells, ranging from 100 µg/mL to 0.015 µg/mL. A positive control,
consisting of anti-PSMA-biotin, and a negative control, consisting of PBST, were included.

Subsequently, a secondary antibody, goat anti-human Kappa light chain, was added to wells
containing the primary antibody and negative control. For wells containing the positive and
negative controls with the primary antibody, rabbit anti-goat horseradish peroxidase (HRP) was
16

added, while for the anti-PSMA positive and negative controls, PBS and biotin-streptavidin-HRP
were added to each well, respectively.
To detect the enzymatic activity, QuantaBlu substrate was added to each well, and the resulting
fluorescence intensity was monitored for a period of 10 minutes. Experiment was done in duplicate
and the for the second experiment, biotin-streptavidin-HRP was added to PSMA antibody positive
well. Fluorescence data for the second experiment was normalized for binding activity analysis.

7. Expression and purification of scFvs
The expression vector encoding the anti-CD99/anti-FLT3 bispecific single-chain variable
fragment (scFv) was transformed into DH10B cells, along with the expression vectors for the anti-
CD99 and anti-FLT3 scFvs. The transformed DH10B cells were cultured in 250 mL of low-salt
LB broth at 37 ℃ and 220 RPM overnight. The resulting plasmids were concentrated using the
Zymopure plasmid Maxiprep kit (Zymo Research).

Afterwards, the three scFvs were transiently transfected into Expi293 cells, and the supernatants
containing the scFvs were collected. The collected supernatants were dialyzed three times in a 4L
equilibrium buffer (250 mM NaCl, 25 mM Hepes, pH=7) overnight or for 6 hours. The scFv-
containing supernatants were then purified using Ni-NTA affinity chromatography.

The yields obtained from the purification were 9.2 mg for anti-CD99, 27.3 mg for anti-FLT3, and
8.3 mg for the bispecific scFv. The purified scFvs were stored at -80℃ for further use.
17

Chapter 3: Results
1. J591-CD38 Construct
The fragments corresponding to the constant regions of Herceptin and the variable regions of J591
were amplified and purified using gel electrophoresis (Figure.4). Overlap extension PCR was
performed to obtain the J591 fusion protein fragment, which was then purified through
electroporation (Figure 5).

The purified J591 fusion protein was subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) analysis. The protein displayed a high overall purity, with two
distinct bands observed for the J591 fusion protein collected on day 3 and day 6, respectively
(Figure 6). The upper bands corresponded to the J591 heavy chain (~60 kDa), while the lower
bands represented the light chain (~30 kDa). It is important to note that the generation of the
J591CD38 construct was still in progress and not yet completed.

The binding activity of the J591 construct to PSMA was assessed using sandwich enzyme-linked
immunosorbent assay (ELISA). The intensity of the signal was measured in a dose-dependent
manner, and the data were fitted to a non-linear regression model (Figure 7). The results
demonstrated clear and increasing signals in response to recombinant PSMA, indicating the
successful binding of the J591 fusion to its target.

18

Another ELISA was conducted to compare the relative binding affinity of the J591 fusion protein
with a humanized PSMA monoclonal antibody. In this experiment, a dye was added to the PSMA
antibody positive control instead of PBS. Figure 8 presents the results, showing a slight
discrepancy in the relative binding activity of the J591 fusion protein at lower concentrations (≤100
nM). However, beyond 100 nM, the relative binding activity of the J591 fusion protein increased
significantly, consistent with the model's predictions. This trend supports the significant
fluorescence signal increase observed in Figure 7, suggesting a much higher saturation
concentration for recombinant PSMA

Additionally, the fluorescence of the PSMA monoclonal antibody positive sample served as a
reference for the analysis of relative binding affinity. Figure 8 demonstrates that the strength of
the J591 fusion protein at 600 nM rivals the binding activity of 1 μM of the PSMA monoclonal
antibody. This observation further highlights the excellent potency of the J591 fusion protein

It is important to note that the production of the J591-CD38 construct for evaluation was not
completed at the time of analysis. Amplification of individual fragments were successful as Figure
9 depicted, even though impurities were shown as, target bands still had acceptable intensity and
yields. Potential reasons for the failure include issues with the quality of the overlap products or
the digestion products necessary for generating the expression vector. The most likely issue may
be suboptimal primer design, as the substantial difference in melting temperature (6 °C) between
the two primers suggests. Insufficient digestion time or other factors could contribute to the
smearing tail observed in the gel electrophoresis results for the J591-CD38 construct (Figure 10).
19

Further investigation and optimization are required to address these challenges and successfully
complete the production of the J591-CD38 construct for future evaluation

20

Figure 4. Overlap extension of J591 Fab region and Herceptin Fc
region. (A) The bands of J591 Herceptin heavy chain (top left) with
estimated size of 345 bps. (B) J591 Herceptin light chain band (top
right) with a estimated size of 321 bps. (C) The bottom panel include
bands for Herceptin heavy chain (left) and light chain (right) with
estimiated size of 993 bps and 321 bps, respectively.

21

J591
day 3
J591
day 6
Figure 5. Overlap extension PCR for J591 fusion protein heavy chain
fragment (Left) and light chain fragment (Right). The bands of two
amplicons were clearly defined with great intensity and correct sizes.
These are correlated to the absence of impurity and high yield after
the fragments were extracted.

Figure 6. SDS-PAGE for J591 fusion protein collected in day 3
(Left) and day 6 (Right) which had relatively high purity with correct
sizes for heavy chain (~60kDa) and light chain (30kDa). Bands were
clearly defined and no apparent impurity bands were observed while
the band intensity is similar between day 3 and day 6 post-
transfection.
22

Figure 7. The graph of J591 fusion protein demonstrated a dose-
dependent manner against recombinant PSMA by ELISA. Control 1
represents antibody negative control while Control 2 represents anti-
PSMA negative control. Both showed no fluorescence signals for
recombinant PSMA. Concentrations of J591 fusion protein was
increased proportionally while the fluorescent increased
significantly when concentration was increased from 33.3 μg/ml to
100 μg/ml.
23

R² = 0.8247
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 100 200 300 400 500 600
Normalized Relative activity
Concentration (nM)
Non-linear regression of J591 Fusion Protein Inhibition
Figure 8. Non-linear regression fitting curve for PSMA binding by
J591 fusion protein based on ELISA data. Normalized relative
activity is performed using fluorescence intensity of each sample
against humanized recombinant PSMA monoclonal antibody.
Similar activities were observed when concentration of the fusion
protein is ≤100 nM. Trend shows the dose dependent activity of
J591 fusion protein with a incerasing slope. This indicates the
saturation concentration could be much greater. 600 nM of J591
fusion protein reaches the activity that ressembling to 1 μM of anti-
PSMA antibody.
24

Figure 9. Amplification of heavy chain (Left two bands) and light
chain (Right two bands) CD38. Both fragments have similar sizes.
Impurity bands were presented below the targeted bands as marked
by black arrow indicating problems during annealing steps.
Figure 10. Overlap extension PCR of J591-CD38 heavy chain
(Estimated size: 2118 bps) and light chain (Estimated size: 1425
bps). Smear tails were present for both fragments while the light
amplicon has more non-specific products during the overlap and
extension process.
25

2. Purification of CD99, FLT3 and CD99/FLT3 ScFvs
After cell transfections of CD99, FLT3 and CD99/FLT3 plasmids, supernatants were purified
extensively with dialysis and column filtration. The purified single-chain variable fragments
(scFvs) were subjected to both quantitative and qualitative analysis using sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The findings substantiated that the obtained
FLT3 scFv displayed favorable overall quality, characterized by distinct and well-defined bands
corresponding to the two scFvs under investigation (Figure 12). Notably, the protein yields for
both the anti-CD99 and bispecific scFvs exhibited significant lower yields compare to FLT3, as
illustrated in Figure 12. After optimizations with dialysis and column filtration, the yield for anti-
CD99 scFv escalated from 2.1 mg to 9.2 mg per liter of cell suspension, while the bispecific scFv
Figure 11. Overlap extension PCR of J591-CD38 heavy chain
(marked by blue arrows, estimated size: 2118 bps) and light chain
(marked by red arrows, estimated size: 1425 bps) with fewer non-
specific DNA fragments due to lowered melting temperature.
26

yield rose from 3.0 mg to 8.3 mg per liter of cell suspension. Specifically, the previous samples
were transfected using 500 mL culture flasks containing 240 mL of media, whereas the latter
samples were transfected using 250 mL culture flasks with 120 mL of media. Moreover, the
optimization steps involving an additional dialysis cycle prior to purification, aimed at
eliminating excessive EDTA trace presented in cell culture medium, resulted in enhanced
recovery of the scFvs. Furthermore, an additional filtration through nickel beads columns for
these two samples based on the established protocol with scFvs eluted each time after a filtration
step. This optimized protocol can be readily adapted for other protein purification experiments
encountering low protein yields, providing a robust approach to enhance overall protein recovery
in a reliable manner.

27

Figure 12.SDS-PAGE of three scFvs: anti-CD99, anti-FLT3 and
anti-CD99/FLT3. The anti-FLT 3 has the highest concentration and
bispecific scFv has the least yield. Protocol was optimized to
increase scFv yield.
Figure 13. SDS-PAGE of two purified scFvs: anti-CD99 scFv and
anti-CD99/FLT3 bispecific scFv. Samples for SDS-PAGE were
diluted. Optimized protocol increased scFvs yield by 2 folds.
28

Chapter 4: Discussion
1. Preparation of J591 fusion protein
PSMA, an integral transmembrane glycoprotein, is primarily found in prostate epithelial cells.
However, it is also present in various other tissues such as the brain, small intestine, and kidney,
indicating potential applications beyond prostate cancer. Currently, only two imaging drugs have
been approved for prostate cancer diagnosis, but their use is limited. To address the need for
PSMA-targeted therapy, we proposed developing a highly specific antibody-drug conjugate
(ARC-ADC) that combines the extracellular targeting capability of PSMA with the enzymatic
activity of CD38. This approach aims to create a site-specific ADC with cytotoxic drug payloads.

The design of the J591 and J591-CD38 constructs involved utilizing Herceptin as a scaffold and
genetically grafting the J591 variable region and CD38 enzymatic activity onto the N- or C-
terminus of the Herceptin heavy and light chains. While the generation of the J591-CD38 ARC-
ADC was unsuccessful, the produced J591 fusion protein exhibited dose-dependent binding
activity against PSMA. It was anticipated that the J591-CD38 ARC-ADC would demonstrate
improved inhibitory activity in vivo due to its enhanced internalization rate.

The failure to prepare the J591-CD38 fusion vectors could be attributed to several factors. One
significant factor could be the suboptimal quality of the overlap extension products. Analysis of
the DNA agarose gel (Figure 9, 10) revealed non-specific DNA products and smeared bands,
possibly resulting from low PCR stringency or excessive repetitive sequences in the amplicons.
Notably, the 5' end of the CD38 fragment contains a 25-base pair GC-rich sequence (72% GC
29

content), which may contribute to these issues. Additionally, the significant difference in melting
points between the forward and reverse primers, approximately 6 ℃, could hinder primer binding
to the target sequence and affect subsequent overlap extension. To address these challenges, it is
advisable to use redesigned primers if the problems persist.

Unsuccessful overlap extension PCR, leading to incomplete amplification, can also result in low
yields during the overlap extension PCR and digestion steps, thereby yielding poor ligation
products and a lack of amplified bands in the following colonial PCR analysis. To overcome these
obstacles, the primer melting point was decreased from 57℃ to 55℃. Consequently, the
subsequent gel analysis of the overlap extension PCR step exhibited fewer non-specific fragments
compared to previous results, particularly for the light chain overlap extension PCR product
(Figure 11). However, with the optimization and improved results, bacterial colony was still absent
in E.Coli expression system. Potential reason caused the issue might be the failure in plasmid
ligation with fragments or inefficient digestion. So that the vector transformed to E.Coli system
showed no transformant. To troubleshoot, gel electrophoresis after digestion and ligation step
might help us to pinpoint the problem.

2. Purification of CD99-FLT3 fusion protein
The bacterial transformation step in the E. coli expression system resulted in a high number of
colonies. The determined plasmid concentrations for CD99, FLT3, and CD99/FLT3 were similar.
However, it was observed that CD99 and CD99/FLT3 scFvs exhibited suboptimal protein yields,
while FLT3 showed significantly higher yields, reaching double or triple the concentration in the
30

same 240 mL cell suspension. One possible explanation for this observation could be related to
the sequence design, which might have affected the proper formation of complexes between the
plasmid and the transfection reagent. It is possible that the FLT3 plasmid is more likely to be
condensed by the transfection reagent due to sequence features, resulting in increased transfection
efficiency and elevated protein yields compared to CD99 and CD99/FLT3.

To address the issue of low protein yield for CD99 and CD99/FLT3, the incubation time during
transfection was extended by an additional day to enhance protein yields. However, upon
performing SDS-PAGE analysis, it was observed that impurity bands were present alongside the
improved intensity of CD99 and CD99/FLT3 scFvs. This suggested that a significant amount of
protein was lost during the following dialysis and filtration steps.

Based on the observations and assumptions made, several optimizations were undertaken to
enhance the protein yields of CD99 and CD99/FLT3 scFvs. One potential reason for the low
protein yields could be the inefficiency of the nickel beads column in capturing the proteins present
in the cell supernatant. It is possible that the affinity of CD99 and CD99/FLT3 scFvs to the column
beads is weaker compared to FLT3 due to differences in protein folding.

To verify this hypothesis, an extended duration of filtration for the supernatant containing CD99
and CD99/FLT3 was implemented. After each filtration step, the proteins were carefully eluted to
prevent the saturation of the nickel beads. As a result of these modifications, the protein yields of
CD99 and CD99/FLT3 increased by 20% compared to the established protocol. It should be noted
31

that the presence of trace amounts of EDTA in the suspension cell medium might have contributed
to the lower protein capture. To mitigate this issue, the collected cell supernatants underwent an
additional 6-hour dialysis using Tris-HCl buffer. Subsequently, the supernatants were triple
filtered through a nickel column.

By implementing these optimizations, the final protein yields improved and slightly exceeded
previous protocol, with clearer bands observed on SDS-PAGE analysis (Figure 13). Notably, this
was achieved even though the amount of transfected plasmid used in the experiments was only
half of the previous amount.

These findings suggest that the modifications in the filtration process, including longer filtration
duration and dialysis, along with multiple filtrations through the nickel column, effectively
improved the protein yields of CD99 and CD99/FLT3 scFvs. These optimized steps help ensure
the capture of a higher proportion of the target proteins and minimize protein loss, leading to
enhanced overall protein recovery.

32

Chapter 5: Conclusion and Future Direction
The J591 fusion protein demonstrated strong and dose-dependent binding to human PSMA,
highlighting its potential for the development of novel therapeutics and imaging agents. However,
further optimization of the J591-CD38 fusion protein during the molecular cloning stage is
necessary to investigate its binding to PSMA more effectively. Redesigning the primers and
optimizing the current overlap extension PCR protocol are crucial steps to improve the generation
of the J591-CD38 fusion protein, ensuring higher specificity and yields of the desired product.
Once the J591-CD38 fusion protein is prepared, J591-CD38 ARC-ADC can be generated by
conjugation of linker to J591-CD38 fusion protein via CD38 enzymatic reactive domain, and
future research should focus on evaluating its in vitro reactivity and specificity in PSMA-positive
cell lines and even incorporate the system into nanomedicine formulation, such as exosome, to
improve stability. Additionally, increasing number the genetically grafted CD38 can be explored
to enhance the drug loading capacity. Furthermore, conducting in vivo studies to assess the potency
and toxicity of the J591-CD38 ARC-ADC in PSMA positive animal models might also need
modifications for the current fusion protein.

On another note, the production and purification of both mono- and bispecific scFvs in large scales
have been successful. Substantial improvements in the yields of these scFvs were achieved through
protocol optimization. Future research should primarily concentrate on assessing the biological
activity of the bispecific scFv towards AML cell lines and evaluating its in vivo cytotoxicity
against AML cells in animal models. Additionally, it would be beneficial to compare the
advantages of the newly developed fusion scFvs with established anti-CD99/anti-FLT3 bispecific
33

scFvs. These investigations will provide valuable insights into the potential applications and
advantages of the novel fusion scFvs in the context of AML therapy.

34

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Abstract (if available)

Abstract Prostate-specific membrane antigen (PSMA) has appeared as a promising target for both therapy and diagnosis of prostate cancer. Elevated levels of PSMA have been linked to the aggressiveness of prostate cancer, making it an attractive target for anti-PSMA targeted therapy and radioactive-labeled anti-PSMA imaging agents. In this study, we propose an innovative approach by developing an ADP-ribosyl--cyclase–enabled antibody-drug conjugate based on the fusion of J591 and CD38. This construct is expected to exhibit potent and selective activity against the PSMA extracellular domain. Furthermore, we aim to achieve site-specific conjugation to improve the stability and the pharmacological activity of resulting antibody-drug conjugates, utilizing the enzymatic activity of CD38. CD99 and FLT3 have emerged as promising targets for therapy in acute myeloid leukemia (AML). CD99 is a transmembrane protein expressed in normal hematopoietic cells, and its overexpression has been correlated with the reactivity of self-renewing leukemic stem cells (LSCs). These LSCs are resistant to conventional chemotherapy and may contribute to AML recurrence. FLT3, an FMS like tyrosine kinase 3, possesses mutations that continuously activate downstream pathways, leading to myeloproliferative and potentially resulting in mortality. In this context, we hypothesize that bispecific single-chain variable fragment (scFv) antibodies targeting both CD99 and FLT3 could exhibit superior inhibitory activity against AML compared to conventional targeted drug molecules. Thus, our study focuses on the expression and production of an in-house antiCD99/anti-FLT3 bispecific ScFv for subsequent cellular and animal studies.

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Asset Metadata

Creator Li, Zekun (author)

Core Title Development of engineered antibodies as novel anti-cancer agents

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2023-08

Publication Date 08/02/2023

Defense Date 08/01/2023

Publisher University of Southern California. Libraries (digital)

Tag antibody-drug conjugate,fusion protein,Molecular Biology,OAI-PMH Harvest,protein purification,scFv

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