University of Southern California Dissertations and Theses

Investigation of the synergistic effect of midostaurin, a tyrosine kinase inhibitor, with anti FLT3 antibodies-based therapies for acute myeloid leukemia

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Investigation of the synergistic effect of midostaurin, a tyrosine kinase inhibitor, with anti FLT3 antibodies-based therapies for acute myeloid leukemia

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Investigation of the synergistic effect of midostaurin, a tyrosine kinase inhibitor,
with anti FLT3 antibodies-based therapies
for acute myeloid leukemia

By

Mohammed Farayah Almatani

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

August 2021

Copyright 2021
Mohammed Farayah Almatani
ii

Dedication

With heartened gratitude, I dedicate my thesis
To my parents for inspiring me and believing in me
To my brother and sisters for all the courage and support you have provided me
To my in-laws and dear friends, for all their support and positivity
To my wife, Bashaer, for your love, for always being supportive and your unquestionable faith
in me

iii

Acknowledgments

We first thank the almighty God for giving us the strength and many blessings in our life to thrive,
learn, and help others for the sake of humanity and evolution on this earth and beyond.
With utmost appreciation and honor, I want to thank Dr. Houda Alachkar for providing me all the
knowledge and guidance for writing this thesis; Her kindness and warm welcome since the first
day in the lab. Dr. Alachkar is and consistently one of the influential people in my life.
I acknowledge the professors in my thesis committee, Dr. Okamoto and Dr. Zhang, for their
insightful contribution and guidance for my thesis.
I was fortunate to have my friends Pooja, Atham, Yang, Lena, Ophelia, and Aurora, who offered
their time and effort to help me with my research during my journey.
I want to acknowledge Dr. Zhang and his crew for their tremendous help in providing the FLT3-
scFv antibody to conduct my research.
Lastly, I am grateful to my wife, parents, brother, and sisters for believing in me to earn this
master's degree. Thank you all.
Statement
My first author review paper, Strategies targeting FLT3 beyond the kinase
inhibitors, is published in Pharmacology & Therapeutics journal. Therefore, the content of
this thesis will highlight some of the parts pertaining to the concept and strategy of
combining FLT3-based immunotherapy with currently FDA-approved tyrosine kinase
inhibitor, Midostaurin, to treat the FLT3-ITD + AML in an in-vitro model. The following is
the reference:
iv

Almatani, M. F., Ali, A., Onyemaechi, S., Zhao, Y., Gutierrez, L., Vaikari, V. P., &
Alachkar, H. (2021). Strategies targeting FLT3 beyond the kinase inhibitors.
Pharmacology & Therapeutics, 225, 107844. doi:
https://doi.org/10.1016/j.pharmthera.2021.107844

v

Table of Contents
Dedication ........................................................................................................................................ii
Acknowledgments........................................................................................................................... iii
List of Tables .................................................................................................................................. vii
List of Figures ................................................................................................................................ viii
Abbreviations .................................................................................................................................. ix
Abstract ............................................................................................................................................ x
Chapter 1: Introduction .................................................................................................................. 1
1.1. Acute Myeloid Leukemia ................................................................................................ 1
1.1.1. AML Diagnosis and symptoms .................................................................................... 1
1.1.2. Disease classification and genomic landscape ........................................................... 2
1.1.3. Risk stratification of AML ............................................................................................ 4
1.1.4. AML treatment ............................................................................................................ 6
1.2. FMS-Like Tyrosine-3 gene overview ............................................................................. 10
1.3. FLT3 mutations.............................................................................................................. 13
1.4. FLT3 inhibitors: treatment and challenges ................................................................... 15
1.4.1. FLT3 inhibitors ........................................................................................................... 15
1.4.2. Limitations of FLT3 inhibitors.................................................................................... 17
1.4.3. Resistance to FLT3 inhibitors .................................................................................... 21
1.5. FLT3-targeting immunotherapy .................................................................................... 24
1.6. The rationale for the dual therapy of FLT3 inhibitor and FLT3- based antibody:......... 29
Chapter 2: Materials and Method ................................................................................................ 31
2.1. Cell line and cell culture ................................................................................................ 31
2.2. Drug for treatment........................................................................................................ 31
2.3. FLT3-scFv ....................................................................................................................... 31
2.4. Viability assay ................................................................................................................ 33
2.5. Measurement of FLT3 surface expression level ........................................................... 33
2.6. Measurement of scFv binding in AML cell line ............................................................. 34
2.7. Apoptosis assay ............................................................................................................. 34
2.8. Statistical analysis ......................................................................................................... 35
Chapter 3: Results ......................................................................................................................... 36
3.1. Midostaurin promotes FLT3 surface expression .......................................................... 36
3.2. FLT3-scFv binds to the FLT3 positive cells..................................................................... 37
vi

3.3. The effect of Midostaurin treatment on FLT3-scFv binding to the FLT3 positive cells 38
3.4. The effect of midostaurin and FLT3-scFv combined treatment on the viability of the
FLT3-ITD+ cells .......................................................................................................................... 40
Chapter 4: Discussion .................................................................................................................... 43
4.1. Discussion........................................................................................................................... 43
4.2. Conclusion .......................................................................................................................... 46
References .................................................................................................................................... 47

vii

List of Tables

Table 1. 2016 revised WHO classification of AML .......................................................................... 3
Table 2. 2017 ELN classification of AML ......................................................................................... 5
Table 3. NCCN risk stratification by the genetics of AML ............................................................... 6

viii

List of Figures

Figure 1. Functional categories of commonly mutated genes in AML ........................................... 4
Figure 2. Illustration of FLT3 receptor. ......................................................................................... 12
Figure 3. Signaling pathways activated upon FLT3-WT activation ............................................... 13
Figure 4. FLT3 surface expression upon treating FLT3-ITD + Cell line (MV4-11) with different
concentrations of Midostaurin ..................................................................................................... 37
Figure 5. Binding signals of Anti-His tag secondary antibody to 0.5 and 1µM FLT3-scFv and their
mean fluorescence intensity ......................................................................................................... 38
Figure 6. Binding signals of Anti-His tag secondary antibody to 1 and 5µM FLT3-scFv and their
mean fluorescence intensity ......................................................................................................... 40
Figure 7. Relative viability of FLT3-ITD + cells in three-time points under different treatment
conditions of Midostaurin and/or 1, and 5µM FLT3-scFv. ........................................................... 42
Figure 8. Apoptosis of FLT3-ITD + cells (MV4-11) under different treatment conditions. ........... 42

ix

Abbreviations

AA, Amino Acid; ADCC, Antibody-Dependent Cellular Cytotoxicity; AML, Acute Myeloid Leukemia;
ATP, Adenosine Triphosphate; Bssc-antibody, bispecific single-chain antibody; DLT, Dose-Limiting
Toxicity; ELISA, Enzyme-Linked Immunosorbent Assay; ELN, European LeukemiaNet; FDA, U.S.
Food and Drug Administration; FL, FLT3 Ligand; FLT3, FMS like tyrosine kinase receptor 3; FLT3-
WT, wild type FLT3; FLT3-ITD, Internal Tandem Duplication FLT3 mutation; FLT3-TKD, Tyrosine
kinase Domain FLT3 mutation; FMS, macrophage-stimulating factor receptor; HSC,
Hematopoietic Stem Cells; ICCC, International Classification of Childhood Cancer; JMD,
Juxtamembrane domain; Ig, Immunoglobulin; MDS, Myelodysplastic syndromes; NCCN, National
Comprehensive Cancer Network; NK cells, Natural Killer Cells; NOS, Not Otherwise Specified;
ORF, Open Reading Frame; TKI, Tyrosine Kinase Inhibitor.

x

Abstract

Mutations in the FMS-related tyrosine kinase 3 (FLT3) receptor have been extensively
investigated over the past two decades due to their importance as prognostic markers and
therapeutic targets in AML. Two main types of recurrent mutations occur in the FLT3 gene:
Internal tandem duplication representing the majority of FLT3 mutations (FLT3-ITD about 25% of
AML cases), and tyrosine kinase domain mutation (FLT3-TKD, about 10% of AML cases). The FLT3-
ITD is associated with a high leukemic burden and an unfavorable prognosis. Therefore, in an
effort for targeted therapy, anti-FLT3 antibodies and small-molecule FLT3 tyrosine kinase
inhibitors were developed and reported promising preclinical and clinical efficacy. However, their
efficacy was suboptimal, and these treatments were associated with limited improvement in
overall survival. Accumulating evidence suggests that FLT3 inhibitors while downregulating the
phosphorylated mutant FLT3 they upregulate the surface expression of FLT3 receptor. This
increase in FLT3 expression on the surface may be leveraged to enhance the recognition for anti-
FLT3 antibody and potentially improve their antileukemia activity. in order to test this approach,
we investigated the dual-therapy effect of midostaurin with an in-house manufactured anti-FLT3
single-chain variable fragment (FLT3-scFv) to assess the in vitro ability of binding to FLT3 and
reducing the viability of FLT3-ITD cell line (MV4-11). Our findings indicate that the midostaurin
treatment increased the expression of FLT3 on the cell surface. In addition, the reduction of cell
viability was higher in dual therapy treated cells compared with either treatment alone. These
results provide the basis supporting the combination of TKI with FLT3-directed antibody
treatment and warrant further preclinical in vivo investigations to validate the in vitro studies.
1

Chapter 1: Introduction

1.1. Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy characterized by poorly
differentiated and highly proliferative hematopoietic cells accumulating in the bone marrow and
the blood (Döhner et al., 2015). In the United States, 3 to 5 cases of AML are reported per 100,000
population (De Kouchkovsky &Abdul-Hay, 2016). The incidence rate, reported from 2009 to 2013,
was 5.0 in males and 3.4 in females, with a mortality rate of 3.7 and 2.2, respectively. In 2020,
the estimated new AML cases of both genders in the US were 19,940 cases, where males’ cases
account for approximately 55% (n=11090) compared to 8850 cases in females. Also, the total
death of 11,180 patients in the United States is due to AML (Siegel et al., 2020).

1.1.1. AML Diagnosis and symptoms

In general, AML results from abnormal myeloblasts accumulation that primarily resides in
the bone marrow, which ultimately leads to bone marrow failure and death (Estey, 2018). AML
patients typically have anemia-like symptoms (Estey, 2012). In addition, fatigue, hemorrhage,
infections, and fever due to decreases in red cells, platelets, or white cells, respectively, are
typical in AML (Löwenberg et al, 1999). Besides, various symptoms such as bone pain,
hepatomegaly, splenomegaly, leukemia cutis in the skin, lymphadenopathy, and other tissues
might be affected due to leukemic infiltration (Löwenberg et al., 1999).
According to WHO criteria, AML is diagnosed based on the multiparameter flow
cytometry revealing about 20% or more of myeloblasts in marrow or blood with myeloid lineage,
except for cytogenetic AML with t(15;17), t(8;21), inv(16) or t(16;16) where the diagnosis of AML
2

is irrelevant to the percentage of blasts (Estey, 2018; Döhner et al., 2010; Arber et al., 2016).
Moreover, some cases referred to “mixed phenotype acute leukemia” in which different myeloid
or lymphoid populations, T- or B-cell markers exist with myeloid blasts. AML cases with more
than 20% blasts and no T- or B-cell markers are considered acute undifferentiated leukemia yet
managed as AML (Estey, 2018).

1.1.2. Disease classification and genomic landscape

Cytogenetic profiling is an integral part of classifying AML for better comprehension of
risk and treatment modalities (Döhner et al., 2015; Daver et al., 2019). According to WHO
classification, AML was categorized into four main groups: AML with recurrent genetic
abnormalities, AML with myelodysplasia-related changes, Therapy-related myeloid neoplasms,
and AML not otherwise specified (AML, NOS) (Table. 1) (Arber et al., 2016). The new genomic
analysis techniques such as next-generation sequencing facilitate identifying emerging genes
mutated in AML (fig. 1). For example, the Cancer Genome Atlas Research Network have
conducted a genomic analysis of 200 AML patients via whole-genome sequencing, whole-exome
sequencing, RNA and microRNA sequencing, and DNA-methylation analysis; The identified genes
that are significantly mutated grouped into different functional categories (fig. 1). In addition,
whole-genome sequencing revealed that clonal heterogenicity and the presence of founding
clone during diagnosis and subclones in relapse might contribute to therapeutic resistance (Ding
et al., 2012).
The mutation acquisition and presence of preleukemic stem cells is also an important
factor in clonal evolution and resistance to chemotherapy (Corces-Zimmerman et al., 2014).
Studies identified recurrent mutated DNMT3a-bearing HSC at high allelic frequency concomitant
3

with NPM1 mutation in AML patient samples as these mutations presented multilineage
repopulation when compared to non-mutated HSC in xenografts (Shlush et al., 2014). In addition,
mutant DNMT3a-HSC were found in remission samples suggesting the escape from
chemotherapy. Thus, the early rise of such mutations mostly affecting epigenetic regulators
contributes to the so-called pool of preleukemic-HSC that leads to clonal evolution and in turn
impacting resistance and relapse (Corces-Zimmerman et al., 2014).

Table 1. 2016 revised WHO classification of AML (Arber et al., 2016)

4

Figure 1. Functional categories of commonly mutated genes in AML (Döhner, Weisdorf, & Bloomfield,
2015).
[A] ITD and TKD mutation in signaling genes such as the class III tyrosine kinase receptor gene FLT3
promote proliferation. [B] RUNX1 and transcription factor fusions by chromosomal rearrangements
mutations that lead to transcriptional deregulation and impaired hematopoietic differentiation. [C] NPM1
mutations result in the aberrant cytoplasmic localization of NPM1 and NPM1-interacting proteins. [D]
SRSF2, SF3B1, U2AF1, and ZRSR2 mutilations are involved in deregulated RNA processing. [E] STAG2 and
RAD21 mutations might impair accurate chromosome segregation and transcriptional regulation. [F]
Several genes such as ASXL1, EZH2, and KMT2A–MLLT3 in which their mutations are involved in the
epigenetic homeostasis of cells. [G] Several mutations to genes such as DNMT3A, TET2, IDH1, and IDH2
can lead to the deregulation of DNA methylation. [H] Mutation of TP53 can lead to transcriptional
deregulation and impaired degradation through the mouse double minute 2 homologue (MDM2) and the
phosphatase and tensin homologue (PTEN). Data are from (Ley et al., 2013).

1.1.3. Risk stratification of AML

5

Classifications of the National Comprehensive Cancer Network (NCCN) and European
LeukemiaNet (ELN) stratify patients according to the risk associated with the cytogenetic and
molecular abnormalities they carry into favorable, intermediate, and adverse risk group (Table 2
and 3) (Daver et al., 2019). This thesis focuses on AML caused by recurrent mutation of the gene
FLT3 that resides on chromosome 13q12. FLT3 gene encodes the FMS like tyrosine kinase
receptor 3 (FLT3). Out of all the cases in AML mutations, the FLT3 gene mutations account for
approximately 30%. Of FLT3 mutation, the most common type is internal tandem duplication
(FLT3-ITD) which accounts for approximately 25% of all AML patients, whereas point mutation in
the tyrosine kinase domain (FLT3-TKD) at codon D835 and I836 occurs in 7-10% of patients (Daver
et al., 2019). Both, NCCN and ELN consider high FLT3 allelic ratio confers poor prognosis and
carries a leukemic burden in AML patients (Daver et al., 2019).
Table 2. 2017 ELN classification of AML (Döhner et al., 2017)

6

Table 3. NCCN risk stratification by the genetics of AML (Adapted from NCCN ver.3 2021)

1.1.4. AML treatment
The therapeutic strategy to treat AML has not been dramatically changed in the past
three decades. Patients require an initial assessment, including demographic, laboratory, and
cytogenetic profiling work up to check the compatibility of chemotherapy or HSC
transplantation (Döhner et al., 2010). In addition, a post-remission regimen is implemented
once remission is achieved.

Induction therapy
Cytarabine continuous infusion for seven days with anthracycline is the mainstay of
induction regimen (NCCN ver.3 2021) (Döhner et al., 2015). In the clinical trial
(#ISRCTN55675535), adding daunorubicin (Anthracycline) of either 60 or 90 mg per square meter
adds no statistically significant difference in terms of rate of complete response or the overall
7

survival (OS) (Burnett et al., 2015). For FLT3 mutated intermediate-risk patients, 50mg
midostaurin is administered orally every 12 hours from day 8 to day 21 (NCCN ver.3 2021).
Approximately, 60 to 85% of complete response in patient age 60 year or younger is achieved,
yet patients older than 60 years usually have inferior clinical outcome due to poor prognostic
factors, genetic abnormalities, or clinically significant co-exciting conditions; These factors
negatively contribute to the dismal benefit from induction therapy, yet they may be candidates
for investigational therapy (Döhner et al., 2015).

Consolidation therapy
Because most patients with AML will relapse within few months of achieving remission
after induction therapy, consolidation therapy is critical for achieving long-term survival.
Conventional chemotherapy, as high- or intermediate-dose cytarabine and hematopoietic cell
transplantation (HCT) are considered the standard post-remission strategies. These strategies are
recommended based on the leukemia genetic-risk profile, the scores of scales that predict the
risk of treatment-related death, and specific transplantation-associated factors in the patient. In
patients 60 years or younger, a range of 2 to 4 cycles of high- or intermediate-dose cytarabine is
preferred. Nevertheless, the dose appropriateness and the number of cycles is inconclusive
(Löwenberg, 2013). Monitoring patients with/or without leukemic cells below the threshold of
detection (i.e., minimal residual disease) by conventional morphologic methods via RT-PCR assay
can guide the use of allogeneic-HCT (Döhner et al., 2015). Moreover, patients, irrespective of age
as a prognostic factor, who are unlikely to have extended complete remission with conventional
approaches may undergo the allogeneic-HCT (Sorror et al., 2014).

8

Relapsed/ Refractory therapy
Disease occurrence is likely in AML patients within three years post-diagnosis. However,
many factors influence the outcomes after relapse, like short remission duration, poor general
health, prior allogeneic transplantation, and adverse genetic factors. Therefore, clinical trials are
preferred as they are scarce in involving patients with relapsed/ refractory AML (R/R AML)
(Döhner et al., 2015). It is worth noting that patients with R/R AML harboring FLT3-ITD mutation
have benefited from tyrosine kinase inhibitor (gilteritinib), which resulted in more prolonged
survival and high remission rate than salvage therapy (Perl et al., 2019). In addition, targeted
therapy or chemotherapy followed by allogeneic-HCT are also options for treatment of R/R AML.
Also, the repeat of the initial successful induction regimen is recommended if more than 12
months past the induction therapy (NCCN ver.3 2021). Low-intensity therapy or best supportive
care is reserved for physically unable patients to receive intensive salvage therapy (Thol et al.,
2015).

Targeted Therapy

Recent research advances aimed at understanding the molecular pathogenesis of AML
have led to the development of molecularly targeted approaches (Krause, & Van Etten, 2005).
Considering the heterogeneity of AML and that several genetic/epigenetic aberrations contribute
to the disease etiology, targeting one single mutated protein is unlikely to abolish the leukemic
clone (Döhner et al., 2010; Darnell, 2002). While some targeted molecular therapies presented
antileukemic effects, often these targeted therapies have shown better clinical benefit when
9

combined with standard chemotherapy. Here are some modalities that have approached phase
3 clinical trial development for treatment of AML.
Cytosine analogs like azacitidine and decitabine are demethylating agents approved by
the FDA to treat Myelodysplastic syndromes (MDS). A phase 3 randomized trial has reported that
azacitidine had prolonged OS compared with conventional care regimens in patients with
intermediate-2 or high-risk MDS. It is worth noting that one-third of these patients (n=113) in the
trial were classified as having AML under current WHO criteria (blast counts 20%-30%) (Fenaux
et al., 2009). Of note, the 2-year overall survival (OS) was 50% and 16% with azacitidine in these
patients compared with conventional treatment regimens, respectively. Azacitidine has been
approved for older AML with 20% to 30% blasts based on these results (Fenaux, 2010) (Döhner
et al., 2010).
Ivosidenib and Enasidenib are isocitrate dehydrogenase (IDH-i) inhibitors targeted IDH1
and IDH2 that are commonly mutated in AML, respectively. Their plausible safety and efficacy
profile in R/R AML led to their approval (NCT02074839, DiNardo et al., 2018) (NCT01915498,
Stein et al., 2017). One caveat in terms of side effects is that IDH inhibitors can promote serious
cytokine effect referred to ‘cytokine storm’, yet it can be avoided by discontinuing the treatment.
FLT3 inhibitors such as midostaurin, gilteritinib, and lestaurtinib are examples of targeted
therapies. FLT3 inhibitors have shown an antileukemic effect. Gilteritinib is proven to have
antileukemic activity as a single agent in relapsed/refractory FLT3 mutated AML (Perl et al., 2019).
Midostaurin was approved in combination with standard chemotherapy in FLT3 mutated AML
(Stone et al., 2017).
10

Venetoclax is B-cell lymphoma-2 (BCL-2) inhibitor approved to be used in combination
with azacitidine in AML patients who are unable to receive standard chemotherapy. The longer
overall survival with azacitidine-venetoclax group compared to control group led to its FDA-
approval (NCT02993523, DiNardo et al., 2020).
Gemtuzumab ozogamaicin (GO) is a humanized anti-CD33 monoclonal antibody that
inhibits DNA synthesis and induces apoptosis (Döhner et al., 2010). GO is approved in the United
States and Japan but not in Europe for relapsed AML in older patients who are not considered
candidates for other cytotoxic therapies (Sievers et al., 2001). The addition of GO to younger and
older patients with favorable- and intermediate-risk reduced the risk of relapse and improved
survival. Nevertheless, GO is not standard in AML therapeutic regimen, yet its regulatory status
needs to be revised (Hills et al., 2014).

1.2. FMS-Like Tyrosine-3 gene overview

FLT3 receptor is a ligand-activated transmembrane kinase that has an essential role in the
early stages of development of myeloid and lymphoid lineage (Daver et al., 2019). The receptor
is encoded by the FLT3 gene that has 24 exons covering about 96kb. The transcript length is 3700
base pairs, which contains pseudogene in the ORF of 2979 bp. The FLT3 protein contains 933
amino acids with a molecular weight of 155 to 160 kDa. This protein then undergoes passage
from the endoplasmic reticulum and Golgi compartments for glycosylation via the addition of N-
acetylglucosamine, galactose, fucose, and mannose at the N-terminus that subsequently
promotes the plasma membrane localization (Grafone et al., 2012).
11

Typically, in the bone marrow, FLT3 is expressed in the CD34+ hematopoietic stem cells
and immature hematopoietic progenitors, including the B-lymphoid progenitors, the myeloid
precursors, and monocytes. Besides, FLT3 is absent in the erythroid progenitors. FLT3 plays an
essential role in the regulating processes of early hematopoiesis (Rosnet et al., 1993). Other
organs such as spleen, liver, and brain have FLT3 expression, yet the expression in these organs
is not actual and may be due to contamination from hematopoietic cells (Stirewalt & Radich,
2003).
The FLT3 receptor comprises four parts, including the extracellular N-terminal domain, a
transmembrane domain, a juxtamembrane domain (JMD), and a C-terminal intracellular domain
(Fig. 2) (Grafone et al., 2012). The N-terminal domain has 541 amino acids and contains 5 Ig-like
domains; three domains situated outward from the plasma membrane that are highly
glycosylated and deemed for FLT3 ligand (FL) binding (Griffith et al., 2004). The remaining
proximal domains are involved in receptor dimerization. The C-terminal has 431aa with a kinase
domain that has two substructures known as tyrosine kinase 1 and 2 (TK1, TK2) (Agnès et al.,
1994; Kiyoi et al., 2002). FLT3-WT remains in the inactive monomeric form due to the steric
inhibition mediated by the juxtamembrane domain. Upon binding of its ligand (FL), it induces
receptor dimerization that promotes the phosphorylation of the tyrosine kinase domain leading
to the activation of the receptor and consequently the downstream effectors. Following the
activation of the dimerized receptor, it gets rapidly internalized and degraded. In the tyrosine
kinase domain, FL binding and subsequent dimerization expose the phosphorylated acceptor site.
The two substructures, the N lobe (TK1) and C lobe (TK2), are connected by a flexible peptide
12

that promotes kinase domain rotation; The rotation of the N lobe towards or away from the C
lobe determines the activation or inactivation of the receptor, respectively (Grafone et al., 2012).

Figure 2. Illustration of FLT3 receptor, [a] inactive conformation of the receptor where [b] represents the
active conformation upon ligand binding.

FLT3 plays an essential role in regulating hematopoietic cell proliferation and
differentiation (Markovic, MacKenzie, & Lock, 2005; Stirewalt, & Radich, 2003). Once it is
activated, FLT3 subsequently activates a network of signal transduction pathways via PI3K and
RAS cascade leading to the activation of Akt, STAT, and ERK (Figure 3). The three distal domains
in the extracellular region process the ligand binding, which is essential in regulating FLT3
receptor activity, causing conformational changes that help kinase domains for tyrosine residue
autophosphorylations. The latter activation aid in the binding and phosphorylation of several
13

signaling pathways such as STAT5, RAS, and PI3K leading to cell proliferation and survival (Kiyoi
et al., 2002; Daver et al., 2019; Grafone et al., 2012).

Figure 3. Signaling pathways activated upon FLT3-WT activation (Adapted from Grafone et al., 2012).

1.3. FLT3 mutations

The most omnipresent somatic mutation in AML is FLT3 mutation. There are two main
types of mutation: Internal tandem duplication (ITD), which account for the majority of FLT3
mutated AML cases, and the point mutation of aspartic acid 835 reside in the kinase domain
(Small, 2006); Other residue substitutions might take place as a secondary mutation that confers
resistance after therapy. The ITDs are variable numbers of duplications of base pairs. These
duplications are a multiple of three and reserving the reading frame coding to exon 14 and 15,
14

where mostly occur at the 5’ of the JM domain in exon 14 (Nakao et al., 1996; Breitenbuecher et
al., 2009). The duplications are varied in length as they span from 3 to more than 400 base pairs
that result from the insertion of repeated amino acid sequences at different sites of the JM
domain (Small, 2006; Stirewalt, & Radich, 2003). When ITD mutations occur, they result in ligand-
independent dimerization and phosphorylation of the WT receptor leading to continuous
activation, even in the absence of FLT3 ligand (Griffith et al., 2004; Kottaridis et al., 2003). Unlike
the mutated receptor, the FLT3-WT has a helical conformation at the JM domain that blocks the
activation and halts the self-dimerization (Kottaridis et al., 2003). The ITD mutation leads to
different patterns of signaling transduction resulting in cytokine-independent proliferation and
block of the myeloid differentiation of hematopoietic progenitors (Chung et al., 2005). Although
the mutated receptor activates the RAS and PI3K pathways similarly to FLT3-WT, the STAT5
pathway is only phosphorylated by FLT3-ITD (Hayakawa et al., 2000). Nevertheless, activation of
STAT5 is crucial for cell growth in association with the activation of MAPK, and its anti-apoptotic
function is mediated by transcriptional regulation of cycline D1, BCL-XL, 59 PIM serine-threonine
kinases, p21WAF1/CIP1, and c-MYC (Takahashi et al., 2004; Kim et al., 2005; Nosaka et al., 1999).
The high allelic ratio of FLT3-ITD is associated with poor prognosis and leukemic burden that
manifested negatively in AML management. Also, FLT3-ITD levels were significantly higher at first
relapse than de novo AML patients at diagnosis (Shih et al., 2002).
The TKD mutations found in the FLT3 receptor result from a substitution of an amino acid
residue in the tyrosine kinase domain caused by a missense mutation in exon 20 that mainly
involves aspartic acid 835 (D835) and isoleucine 836 (I836). The D835 codon can have a
15

replacement to tyrosine, histidine, valine, glutamate, and asparagine. On the other hand, the
I835 codon is replaced by methionine and asparagine (Yamamoto et al., 2001).

1.4. FLT3 inhibitors: treatment and challenges

Considering the high recurrent FLT3 mutations in de novo AML and its negative impact,
FLT3 is considered a promising target for leukemia-directed therapies (Ley et al., 2013). Due to
their encouraging preclinical and clinical antileukemia activity, FLT3 inhibitors have been
extensively studied and utilized to combat the oncogenic signaling produced by FLT3 mutations
(Daver et al., 2019).

1.4.1. FLT3 inhibitors

FLT3 inhibitors are tyrosine kinase inhibitors (TKI) that have an inhibitory effect against
FLT3 receptors and are classified into two generations, first- and second-generation inhibitors,
based on where the inhibitory effect occurs on the receptor.
First-generation TKIs including lestaurtinib, sunitinib, sorafenib, and midostaurin are
multitargeted kinase inhibitors that affect FLT3-ITD mutated AML (Stone et al., 2005; Smith et al.,
2004; Daver et al., 2019). Hence, they are nonspecific for FLT3 receptors and present with an off-
target effects. Conversely, the second-generation FLT3 inhibitors such as quizartinib, crenolanib,
and gilteritinib are more selective and superior for inhibiting FLT3 over first-generation FLT3
inhibitors (Wander et al., 2014).
As per the mode of action, FLT3 inhibitors are classified into type I and type II inhibitors
based on how they interact with the FLT3 receptor (Daver et al., 2019). Type I inhibitors like
16

lestaurtinib, midostaurin, gilteritinib, and crenolanib can bind to FLT3 either within proximity to
the ATP-binding pocket in inactive or active conformation (Ley et al., 2013). Due to their mode of
action, type I FLT3 inhibitors are effective against both FLT3-ITD and other TKD (Ke et al., 2015).
On the other hand, type II inhibitors such as sorafenib and quizartinib bind only when the
receptor is inactive, and this binding occurs within proximity to ATP binding domain in the
hydrophobic region. Therefore, type II inhibitors have a superior effect over FLT3-ITD mutations
than FLT3-TKD mutations, which might resemble the resistance (Ambinder & Levis, 2021).
Currently, midostaurin and gilteritinib are the only FDA-approved in the US for treating
AML. Other inhibitors are currently under development to treat AML at the time of this writing
(Daver et al., 2019). Midostaurin is the first FDA-approved FLT3 inhibitor to treat newly diagnosed
FLT3-mutated AML patients in the USA. The FDA approval was based on the results of the clinical
trial reporting on the improved OS rate in patients with AML treated with midostaurin together
with traditional therapy (Stone et al., 2017). Gilteritinib was approved by the FDA in 2018 and
has a single agent activity used to treat adult patients who have relapsed/refractory (R/R) FLT3-
mutated AML (Perl et al., 2019). Quizartinib is another orally active type II FLT3 inhibitor. Due to
some safety issues and suboptimal survival benefits, quizartinib has not been FDA-approved in
the US and European Union (Garcia-Horton, & Yee, 2020). Nevertheless, quizartinib is currently
approved for use in Japan due to its effectiveness in improving the OS in patients with R/R FLT3-
ITD mutations (Cortes et al., 2019). Moreover, crenolanib, tandutinib, and cabozantinib are either
undergoing clinical trials or still in the development process as FLT3 inhibitors for AML patients
(Hou, & Tien, 2020).

17

1.4.2. Limitations of FLT3 inhibitors

Even though some FLT3 inhibitors present some therapeutic activity, the majority poses
limited antileukemic activity when used as a monotherapy. Thus, better efficacy was observed
upon combining with standard chemotherapy. Midostaurin approved in 2017 to be used only in
combination with standard chemotherapy as first-line therapy for newly diagnosed FLT3-ITD AML
patients. Thus, the phase 3 clinical trial that included 717 patients (NCT00651261) concluded that
OS was significantly higher in the midostaurin group with a median of 74.7 months compared to
25.6 months in the placebo group. Moreover, the disease-free survival was significantly higher in
the midostaurin group than placebo, with a median of 26.7 months versus 15.5 months,
respectively. Upon the sensitivity analysis of overall survival in patients undergoing allogeneic
therapy, the midostaurin group had a death risk of 24.3% lower than the placebo group, and no
significant change was observed per the 4-year overall survival. Indeed, the midostaurin
benefited patients who had transplantations only after first remission (Stone et al., 2017). Other
preclinical studies concluded that midostaurin has multi-targeted inhibition of different kinases
along with FLT3. When administered as monotherapy, midostaurin showed poor clinical
outcomes in managing AML (Levis, 2017; Davis et al., 2011).
Gilteritinib is another FDA-approved FLT3 inhibitor as a single agent in R/R FLT3 mutant
AML. However, results from a phase 3 clinical trial showed that only 37% of gilteritinib-treated
group survived more than one year (NCT02421939) (Perl et al., 2019). Another clinical trial phase
1/2 first-in-human to assess the safety, PK, and tolerability of dose escalation concluded that
gilteritinib was generally well tolerated and improved response rate in all dose groups. In
addition, the better response was essentially observed in doses of 80mg or higher. As per the
18

antileukemic effect, the reported overall response rate was higher in patients harboring mutated
FLT3 compared to WT of 49% versus 12%, respectively. Furthermore, higher bone marrow
reduction is superior in FLT3 mutants than in FLT3-WT. Unfortunately, several patients had
disease progression, and some has developed sepsis which led to the discontinuation of the study
(NCT02014558 Perl et al., 2017). Thus, gilteritinib was proven to have potency as a single agent,
yet the limited capacity even in newly diagnosed patients urges the need to investigate its
efficacy in combination with other chemotherapeutic regimens. Several clinical trials are
currently active; one trial in phase 1 studying the dose-limiting step of gilteritinib and ascertaining
the maximum tolerated dose in combination with induction-consolidation therapy in newly
diagnosed FLT3 mutated AML patients (NCT02236013). Another study in phase 3 is comparing
gilteritinib alone versus combination with azacitidine to assess the longevity of newly diagnosed
FLT3 mutated AML patients (NCT02752035). Sorafenib was tested in another randomized
controlled trial (NCT00893373), including 267 patients aged 18-60 years with newly diagnosed,
previously untreated AML and reported that the event-free survival median was 9 months versus
21 months in the placebo compared to the sorafenib arms, respectively; The sorafenib group had
an improved 3-year event-free survival of 40% compared with the 22% for the placebo group.
Nevertheless, the toxicity profile manifested as grade 3 adverse events like fever, diarrhea,
bleeding, cardiac event, and rash occurred more frequently in the sorafenib cohort than in the
placebo group. Therefore, the investigators concluded that despite sorafenib antileukemic
activity, the increased toxicity profile warranted investigating long-term overall survival further
to reduce the toxic events and better clinical evaluation of the role of sorafenib (Röllig et al.,
2015).
19

Sunitinib, sorafenib, and ponatinib are currently tested in an active clinical trial
(NCT02551718), including 34 patients with relapsed or refractory AML to evaluate the drug of
choice based on high throughput ex vivo drug sensitivity assay in combination with mutation
analysis. In addition, current active trials, including the pilot clinical trial (NCT01578109) assessing
the side effect of sorafenib maintenance therapy before- and post-donor bone marrow
transplantation. Another phase 1 trial (NCT01861314), including 15 participants assessing the
side effects and determining the biologically effective and tolerable dose (BETD) dose of
bortezomib/sorafenib tosylate combination when given together with decitabine, cytidine
analog chemotherapy that inhibits nucleic acid synthesis, in treating patients with AML.
Lestaurtinib, previously known as CEP-701, was approved as an orphan drug (Smith et al.,
2004). A prospective randomized assessment study has reported that no significant difference in
5-year relapse-free survival RFS (40% vs. control, 36%) or OS (46 vs. control, 45%) upon the
addition of lestaurtinib to standard chemotherapy in newly diagnosed younger AML patients
(Knapper et al., 2017). Reports from another clinical trial have shown that the lestaurtinib
following salvage chemotherapy in the first relapse did not result in statistically significant
differences in complete remission (CR), complete remission with incomplete platelets recovery
(CRp), and the CR/CRp ratio compared to patients treated with chemotherapy only
(NCT00079482) (Levis et al., 2011). Although there was no significant difference in the adverse
events between the two groups, 24% of lestaurtinib and 7% of control have discontinued from
planned therapy due to developed adverse events. In addition, serious adverse events were
associated with the lestaurtinib group more than in the control group (55% and 45%,
respectively) (Levis et al., 2011). Also, a higher incidence of infectious death was observed in the
20

lestaurtinib group (32%) versus the Control group (21%). Also, 15 out of 79 patients whose
lestaurtinib level measured for the aplasia assessment had plasma concentration of more than
20µM, which is higher than the anticipated levels. Even though some patients in this trial have
benefited from lestaurtinib and some of them equal in number were harmed, investigators in
this trial have concluded that the pharmacokinetic complexity and off-target effects associated
with lestaurtinib limits its utility in relapsed FLT3 mutated AML (Levis et al., 2011).
Quizartinib, a next-generation TKI, was tested in a phase 3 multicenter, randomized
controlled open-label trial (QuANTUM-R NCT02039726) as a monotherapy compared to salvage
therapy to assess if it can prolong the overall survival of R/R FLT3-ITD AML patients within six
months period. They have concluded that quizartinib is a potent inhibitor and comprises a safety
profile. Quizartinib group had a prolonged OS compared to that of a chemotherapy group with a
median OS of 6.2 and 4.7 months. However, some patients underwent HCT transplantation
during the study, some relapsed, some had no response, or the disease progressed; all led to
discontinuing the initial quizartinib treatment. As patients progressed to transplantation, 32% of
patients in the quizartinib group underwent HSC transplantation versus 11% of patients from the
chemotherapy group. This high proportion of patients who underwent HSC transplantation in the
quizartinib group was attributed to the high response rate, the duration of composite complete
remission, and safety profile. However, the decision for patients to undergo HSC transplantation
and resume therapy after transplantation was under the investigators' discretion; Thus,
investigators in the QuANTUM-R trial could not conclude in this regard the benefit of HSC
transplantation or differences between the two treatment groups (Cortes et al., 2019).
Quizartinib is still an experimental drug and has not been approved yet for treating FLT3 mutated
21

AML in the US and European Union. Currently, several clinical trials are testing or assessing
quizartinib as monotherapy or in combination with omacetaxine mepesuccinate, azacitidine, or
cytarabine regarding ascertain certain results for remission or event-free survival (NCT03135054)
(NCT02668653) (NCT01892371).
The majority of TKIs are effective when combined with other chemotherapies. In this
regard, several clinical trials are pointed to investigate the efficacy of FLT3 inhibitors in a
combinatorial manner with induction-consolidation therapy or with azacitidine in newly
diagnosed patients with FLT3 mutated AML (NCT02236013) (NCT02752035) (NCT03135054)
(NCT01892371).

1.4.3. Resistance to FLT3 inhibitors

Several resistance mechanisms against FLT3 inhibitors are likely to contribute toward the
limited long-term effectiveness of FLT3 inhibitors in a clinical setting (Ding et al., 2012).
Furthermore, in addition to primary resistance seen in patients lacking response to FLT3
inhibitors, secondary resistance may emerge following FLT3 inhibitor administration (Alotaibi et
al., 2021).
Resistant clones sometimes presented to an extent that could not be detected or found
at low levels at diagnosis which then expanded following FLT3 inhibitors treatment. A recent
study used next-generation sequencing had identified several emergent mutations, involving
FLT3, in relapsed bone marrow samples following treatment with FLT3 inhibitors (Alotaibi et al.,
2021).
22

Prolonged exposure to FLT3 inhibitor could develop AML-resistant cell lines in which
selective phosphorylation in sunitinib-treated resistant cell lines for S6K and AKT, mTOR
substrates was observed. These findings suggest that survival protein expression via the
PI3K/mTOR pathway activation promotes further resistance to FLT3 inhibitors (Lindblad et al.,
2016).
FLT3 inhibitors work better for peripheral blasts with limited effectiveness on bone
marrow blasts (Yang et al., 2014). However, the leukemia stem cells responsible for disease
relapse in the bone marrow niche are out of reach from the FLT3 inhibitors (Smith et al., 2004).
In addition, the mesenchymal stromal cells provide a protective environment for AML blasts in
bone marrow, becoming resistant to FLT3 inhibitor monotherapy (Yang et al., 2014).
Furthermore, as FLT3 inhibitors showed a considerable reduction in circulating blasts, it does not
reflect the actual residual leukemic blasts protected by stromal cells in bone marrow, adding
another layer of resistance against FLT3 inhibitors and subsequently promoting relapse. In this
context, the presence of bone marrow mesenchymal stroma attenuates FLT3 inhibitors and
prevents apoptosis of AML blasts (Chen et al., 2016; Ghiaur & Levis, 2017). Also, drug-
metabolizing enzymes play a significant role in bone marrow microenvironment protection in
which bone marrow expresses CYP enzymes, CYP3A4 notably, in a similar amount to hepatocytes
that participate in chemoprotection to FLT3-ITD and FLT3-WT blasts. (Alonso et al., 2015).
Secondary TKD mutation emerges following FLT3 inhibitors can aid in resistance (Heidel
et al., 2006; Alvarado et al., 2014; Smith et al., 2012). AML Patients harboring secondary
mutations failed to respond to the second line of FLT3 inhibitors (Alvarado et al., 2014). In
addition, several mutations identified at the kinase domains confer resistance to several FLT3
23

inhibitors, affecting their stability and binding to the receptor due to conformational alteration
to the protein (Smith et al., 2012; Heidel et al., 2006; Daver et al., 2015; Zhang et al., 2014). Some
point mutations induce the phosphorylation of ERK, AKT, STAT5, and S6K, even when FLT3
receptor is inhibited, resulting in dismal efficacy of FLT3 inhibitors (Ghiaur & Levis, 2017; Smith
et al., 2012; Zhang et al., 2014).
Several kinases like Janus kinase (JAK) and receptor tyrosine kinase (AXL) can promote
signaling pathway activation of mTOR, AKT, and MAPK pathway in which they contribute as
mechanisms of resistance (Lindblad et al., 2016; Rummelt et al., 2020; Park et al., 2015; Piloto et
al., 2007).
Upregulation of specific genes such as tescalcin (TESC) (Man et al., 2014) and CCL5
(Waldeck et al., 2020) are contributing to FLT3 inhibitors resistance. The binding protein TESC,
encoded by the TESC gene, is expressed in primary and immortalized human hematopoietic cells
and activates Na1 /H1 exchanger type-1 (NHE1) that subsequently leading to cell proliferation,
reduce apoptosis, and facilitate drug resistance (Man et al., 2014). Another study found that the
cytokine CCL5, a protein encoded by the CCL5 gene, expression was upregulated in induced
midostaurin-resistant cell lines compared to TKI-sensitive cells. Resistant AML cell lines have high
CCL5 secretion that can upregulate AKT1, AKT3, STAT1, and STAT2 pathways that are crucial in
survival and proliferation. In addition, the study also noted that CCL5 could serve as a biomarker
to track TKI drug resistance (Waldeck et al., 2020).

24

1.5. FLT3-targeting immunotherapy

Although, midostaurin and gilteritinib were recently approved to treat patients with
the FLT3-ITD mutation (Stone et al., 2018; Dhillon, 2019), their clinical benefit is still limited since
half of the patients die within five years (Fischer et al., 2010). Therefore, the TKI’s suboptimal
efficacy and several resistant pathways; urge alternative therapeutic strategies.
A number of FLT3 antibodies have been developed and tested, and have been shown to
have promising anti-leukemic effects (Li et al., 2004; Piloto et al., 2005). IMC-EB10 (IgG1, κ) is a
fully human anti-FLT3 neutralizing antibody that was isolated and constructed from a human Fab
phage display library against soluble FLT3-Fc protein. Upon ELISA binding and blocking assays,
IMC-EB10 was able to block and compete for the binding of the FLT3 ligand (FL) on the cell-surface
FLT3 receptor. The binding affinity of IMC-EB10 was 158 pM compared to FL which was 200-500
pM. Besides, no binding was observed of IMC-EB10 to fibroblast growth factor FGF-Fc fusion
protein or other kinases such as VEGF-(R1 and R2), PDGF-(Ra and Rb), which manifests its
selectivity to FLT3 receptor. Given that IMC-EB10 can recognize cell-surface FLT3, flow cytometry
analysis showed that IMC-EB10 is bound to BaF3-ITD and EOL-1, cells that express FLT3-ITD and
FLT3-WT, respectively. Furthermore, a dose-dependent manner ability of IMC-EB10 to inhibit the
FL-induced phosphorylation and subsequently downstream pathway of MAPK, PI2K/AKT, and
STAT5 (Li et al., 2004). However, in phase 1 clinical trial treating with IMC-EB10 antibody weekly
for participants with AML has been terminated early since maximum tolerated dose (MTD) was
inferior to the required level of efficacy. Those patients were enrolled based on the failure to
achieve complete remission after standard induction regimen, relapsed after response to
25

previous antileukemia therapy, or are not eligible for potentially selected or approved salvage
options (NCT00887926).
Compared to IMC-EB10, another monoclonal antibody has been constructed and named
IMC-NC7 that selectively binds and inhibits FLT3 expressing cells. IMC-NC7 binds both FLT3-WT
and FLT3-ITD on EOL-1 and BaF3-ITD cell lines, similar to IMC-EB10. Also, IMC-NC7 competes for
FL binding and subsequently reduced phosphorylation of AKT and MAPK downstream pathways.
However, in vitro assessment by increasing the amount of FL showed that EM3 cells treated with
IMC-EB10 regain the FLT3 activation compared to cells treated with IMC-NC7 suggesting that
IMC-NC7 is more effective in blocking FL-mediated FLT3 activation. Additionally, IMC-EB10 and
IMC-NC7 had potent inhibition of FLT3 phosphorylation of primary AML blasts expressing either
FLT3-WT or FLT3-ITD. Nevertheless, both monoclonal antibodies could not affect proliferation or
induce apoptosis, yet IMC-EB10 incubation with NK cells was more effective than with IMC-NC7
to induce antibody-dependent cell-mediated cytotoxicity. The latter observation made the
investigator only assessing IMC-EB10 in vivo. Therefore, the NOD/SCID mouse model showed
that IMC-EB10 significantly prolongs survival compared to PBS-treated mice, yet the median
survival over 100 days has not been reached (Piloto et al., 2005).
Genetic engineering for optimizing the Fc-region to produce antitumor antibodies has
been developed and profusely improved ADCC in lymphoma. Besides, the feasible approach of
drug accessibility and monitoring advantages found similar in both leukemia and lymphoma.
Therefore, another approach optimizing human IgG1 of two FLT3 antibodies, BV10 and 4G8, by
adding amino-acid exchanges S239D and I332E (SDIEM modification) (4G8-SDIEM and BV10-
SDIEM) to facilitate both in vitro and in vivo detection as well as improving ADCC. The parental
26

antibodies 4G8 and BV10 are bound to domains 4 and 2 of the FLT3 receptor, respectively. At
lower concentrations, 4G8-SDIEM had significant ADCC than BV10-SDIEM due to either different
FLT3 epitope recognition or low binding avidity of BV10-SDIEM. Thus, 4G8-SDIEM was the main
focus for upcoming characterization. Further ADCC evaluation of 4G8-SDIEM revealed no
cytolytic activity more than what was observed with NK cells on three PBMCs of healthy donors.
In addition, ADCC-mediated activity was less pronounced on three AML-blast samples. Currently,
4G8-SDIEM (FLYSYN) is investigated in phase 1/2 trial as monotherapy on 31 participants who
had minimal residual disease (MRD) AML in terms of safety, tolerability, efficacy,
pharmacokinetics, and pharmacodynamics (NCT02789254) (Hofmann et al., 2012).
Another approach to overcome the shortcomings of the Fc-optimized antibody is
developing a bispecific antibody termed fabsc, named Anti-FLT3 X CD3, in which 4G8 antibody is
the FLT3 antibody and UCHT1 serves as the anti-CD3 antibody. Despite the identical specification
of fc-optimized antibody (Fabsc) and bispecific single chain (Bssc) format, the binding affinity of
Fabsc antibody is higher to CD3-expressing Jurkat cells which are attributed to the fact that the
N-terminal FLT3 antibody is expressed in the more physiologic fab compared to a bispecific single
chain. Further characterization when comparing 4G8 X UCHT1 to Fc-optimized 4G8-SDIEM via
incubating PBMC of 10 AML patients with these antibodies and measuring the blasts percentage
revealed that T-cells were activated by CD69 or CD25 and proliferated in all samples treated with
4G8 X UCHT1 after three days of incubation. Moreover, a substantial decrease in FLT3-expressed
leukemic cells as well. Of note, both Fabsc- and bssc-antibody were comparable in terms of T-cell
activation and blasts reduction. In contrast, 4G8-SDIEM was only effective in one AML sample,
and that was attributed to diminished activity of natural killer (NK) cells in the case of PBMC
27

preparations of AML patients (Durben et al., 2015). Nevertheless, a short half-life and elevated
risk of immunogenicity diminished the FLT3 X CD3 bispecific antibody promising activity (Durben
et al., 2015; Yeung et al., 2020). Therefore, another group of researchers optimized a full-length
anti-FLT3-CD3 antibody regarding domain four (D4) as the optimal targeting region due to the
observed efficacy upon screening. Thus, they generated a bispecific antibody, named 7370 Anti-
FLT3 CD3 Bispecific IgG. This bispecific antibody had 49 pM and 27 nM dissociation constant of
FLT3 and CD3 proteins, respectively. In addition, it demonstrates T-cell-specific binding
irrespective of the magnitude of FLT3 expression. Epitope mapping studies showed that arginine
substitution at D4 residue serine 370 abrogated 7370 Anti-FLT3 CD3 bindings to D4, confirming
the specificity to FLT3 D4 (Yeung et al., 2020). Moreover, the short-term cytotoxic assay showed
that 7370 could induce lysis in 14% and 33% of MV4-11 and EOL-1 cell lines, respectively, at
effector to target (E: T) ratio of 1:20. Also, the average half-maximal effective concentration
(EC50) to redirect healthy donor T-cells towards AML was 14.6 ± 5.6 pM and 1.4 ± 0.7 pM for
MV4-11 and EOL-1, respectively. Also, interferon-  (IFN- ), tumor necrosis factor-alpha (TNF-α),
and interleukin-2 (IL-2) cytokines from healthy donor T cells in the presence of EOL-1 and MV4-
11 cell lines were potently induced by 7370. Of note, 7370 does not induce cell killing in cells
lacking FLT3 expression. Altogether, 7370 bispecific antibodies have in-vitro ability to redirect T-
cells and activating them along with secreting cytokines against tumor cells. Furthermore, 7370
bispecific antibodies have an expression-dependent manner when it comes to in vivo efficacy;
assessing NSG mice xenograft model of three cell lines showed that a single dose of 7370 exerted
complete elimination of EOL-1 and MOLM-13 cell lines and all mice remained tumor-free for at
least 40 days after tumor implantation. Regarding expression-dependent FLT3 reduction, a low
28

dose of 7370 (0.1mg/kg) had complete efficacy in EOL-1 models compared to MOLM-13 models
(0.03 mg/kg). In addition, models with the MV4-11 cell line, which has the least FLT3 expression,
manifested a reduction of leukemic burden after a single dose of 0.03 mg/kg of 7370, though it
did not fully eradicate AML cells. In addition, 0.5 nM or higher of 7370 was able to induce dose-
dependent proliferation of T-cells (Yeung et al., 2020).
Another antibody that targets FLT3 named α-FLT3-A192 fusion protein. This protein is
composed of a single-chain variable fragment conjugated with elastin-like polypeptide (ELP). This
fusion protein was assessed for its specificity by incubating rhodamine-labeled α -FLT3-A192 at
two different concentrations at 10 and 25µM, with FLT3-ITD cell lines, MOLM13 and MV4-11, as
well as an FLT3 negative cell line (U937). A dose-dependent increase was observed in the
fluorescence signals in both MOLM13 and MV4-11, but no binding to U937 was observed. The
viability assays show a significant decrease of viable cells by 17% and 67% at 1µM for MOLM13
and MV4-11, respectively. Also, the higher the concentration of α-FLT3-A192, the higher the
decrease in viable cell count. In addition, western blot analyses show that α-FLT3-A192 treatment
resulted in significant reduction in the phospho-STAT5 and phospho-ERK in MOLM13 and MV4-
11 compared to control. Both STAT5 and ERK are downstream targets of FLT3-ITD (Park et al.,
2020).
In vivo, NSG mice were engrafted with MOLM13 cells and then grouped based on
treatment regimen, α-FLT3-A192, A192, or midostaurin. After euthanasia, α-FLT3-A192-treated
mice had a significantly smaller spleens and less leukemia cell engraftment than those treated
with A192. Mice treated with midostaurin had smaller spleen and reduced leukemia engraftment
compared with A192 as well, but there were no differences between midostaurin and α-FLT3-
29

A192 in terms of reducing the leukemic burden. Thus, although α-FLT3-A192 had excellent
stability, pharmacokinetic profile, and therapeutic potential, it requires further preclinical
development for clinical use (Park et al., 2020).

1.6. The rationale for the dual therapy of FLT3 inhibitor and FLT3- based antibody:

FLT3-ITD or TKD (D835Y) mutant receptors are sequestered intracellularly in the
perinuclear endoplasmic reticulum, whereby TKIs such as quizartinib, midostaurin, and sorafenib
promote their surface expression similar to that of FLT3-WT (Reiter et al., 2018). Moreover,
surface expression of FLT3 was elevated in FLT3 mutants-cell lines than in cells harboring FLT3-
WT such as THP-1, and OCI-AML3, which was barely changed. In vivo, a similar trend of surface
expression of FLT3 was seen in two AML patients’ samples with mutated FLT3 after sorafenib
treatment in which the FLT3 expression was higher than at the time of diagnosis. Also, T-cell
mediated cytotoxicity (TCMC) assay using the combination of bispecific FLT3xCD3 antibody with
quizartinib revealed that mutated-FLT3 had a significant change in its cytotoxicity pattern. In
contrast, the FLT3-WT cell line (HL60) had no significant change in FLT3 surface expression upon
dual therapy. Aside from the observed additive effect, it is worth mentioning that FLT3xCD3
antibody alone could eradicate CD33+ upon treating the heterozygous FLT3-ITD cell line (MOLM-
13). However, the additive effect from a combined treatment was inconclusive. Altogether,
investigators had hypothesized a promising benefit of the dual therapy of TKI with FLT3-directed
immunotherapy in which TKI will upregulate FLT3 antigen from one side, and the FLT3
immunotherapy will exclusively affect the antigen (Reiter et al., 2018).
30

As novel α-FLT3-A192 was proven to have an antileukemic effect in both in vitro and -vivo,
we hypothesized that in-vitro treatment of FLT3-ITD + cell line (MV4-11) with the FDA-approved
tyrosine kinase inhibitor (midostaurin) would upregulate the FLT3 surface marker that leads to a
superior effect by FLT3-based immunotherapy (FLT3-scFv) to halt mutated-FLT3 activation.
Therefore, the aim of this thesis is based on midostaurin upregulating the surface marker
FLT3, which then the FLT3-scFv induce a synergistic or added effect in the preclinical cellular
model.
31

Chapter 2: Materials and Method
2.1. Cell line and cell culture

MV4–11 cells were purchased from the American Type Culture Collection (ATCC).
Authentication of cell lines was done at the University of Arizona Cell Authentication Core. All cell
lines were cultured in Roswell Park Memorial Institute1640 (RPMI 1640) medium (Thermo Fisher,
MA, USA) in addition to 10% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic (Thermo
Fisher, MA, USA).

2.2. Drug for treatment

Midostaurin was used to treat the MV4-11 cell line. Midostaurin was stored at -20
o
C at a
stock concentration of 10mM. The 10nM dose was diluted from the starting stock and used as
the main treating dose and other doses of 5, 50, and 100nM for investigational purposes. The
treatment period for midostaurin-treated samples was six hours and they were incubated inside
a 5% CO 2, 37
o
C incubator during the mentioned period.

2.3. FLT3-scFv

The FLT3-scFv was designed and produced in collaboration with Dr. Zhang’s lab at the
University of Southern California. The antibody had an approximate molecular weight of 27.8 Kd.
The DNA encoding α-FLT3 scFv was cloned into the pFUSE backbone (InvivoGen, CA) using
primers:
32

ATL225-TGTCACGAATTCGTATCCATACGACGTACCGGATTATGCAGAAGTCCAACTGGTCCAGTCAGGC
and ATL256-TGGCCAGCTAGCTTATTAATGGTGGTGGTGATGGTGCTTGATTTCCAAGCGAGTCCCCTG
to introduce an N-terminal HA-tag and a C-terminal His6 tag. Then the purified PCR products and
pFUSE vectors were subjected to double digestion using restriction enzymes EcoRI and NheI
before ligation into a with T4 ligase (New England Biolabs, MA, USA). Plasmids were transformed
into TOP10F’ E. coli (Thermo Fisher, MA, USA) and plated onto LB agar plates with 25 µg mL-1
Zeocin (InvivoGen, CA, USA) (Park et al., 2020).
The encoded DNA sequence of α-FLT3 scFv is as follows (Park et al., 2020):
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGTATCCATACGA
CGTACCGGATTATGCAGAAGTCCAACTGGTCCAGTCAGGCGCTGAGGTAAAGAAGCCGGGCGCAAGCG
TGAAAGTATCTTGTAAGGCTTCCGGGTACACTTTTACTTCCTACTATATGCACTGGGTGCGTCAAGCCCCC
GGACAGGGCCTGGAGTGGATGGGGATTATTAACCCCTCGGGCGGTAGTACATCGTACGCCCAAAAGTT
TCAAGGCCGCGTAACTATGACGCGCGATACCTCCACAAGTACAGTATATATGGAACTTAGCAGTTTGCG
TTCCGAGGATACCGCCGTTTATTACTGCGCTCGCGGCGTAGGTGCACACGATGCTTTCGACATCTGGGG
CCAAGGGACAACAGTTACAGTCAGTAGTGGTGGAGGTGGCTCGGGTGGCGGCGGCAGCGGAGGCGGT
GGAAGCGACGTCGTAATGACGCAATCTCCCCTTTCTTTGCCTGTTACGCCCGGCGAGCCTGCTTCCATTT
CCTGTCGCTCCAGTCAATCTTTGTTACATTCAAACGGGAACAATTATCTGGATTGGTACTTGCAAAAACC
AGGCCAAAGTCCTCAGCTGTTGATTTACTTGGGGAGCAATCGTGCATCGGGGGTGCCTGACCGCTTTTC
AGGTAGCGGGTCAGATACCGATTTCACGCTGCAAATCTCCCGTGTTGAGGCCGAAGACGTTGGCGTGTA
TTACTGCATGCAGGGAACTCATCCAGCAATCTCATTTGGTCAGGGGACTCGCTTGGAAATCAAGCACCAT
CACCACCACCAT
33

The encoded amino acid sequence of α-FLT3 scFv is as follows (Park et al., 2020):
MYRMQLLSCIALSLALVTNSYPYDVPDYAEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPG
QGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGAHDAFDIWGQG
TTVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGNNYLDWYLQKPGQSP
QLLIYLGSNRASGVPDRFSGSGSDTDFTLQISRVEAEDVGVYYCMQGTHPAISFGQGTRLEIKHHHHHH

2.4. Viability assay

Cell viability was analyzed by treating 5 × 10
5
MV4-11 cells first with 10nM midostaurin
and then incubated inside a 5% CO 2, 37
o
C incubator for six hours. After incubation, samples were
spun down via centrifuged for 3 minutes at 1300 RPM and the sample’s pellets were prepared
and labeled either alone (i.e. midostaurin-only group) by resuspending it with RPMI1640 media
or in combination with either FLT3-scFv at 1 or 5μM for 30 min at room temperature and then
topped off to the corresponding volume with RPMI1640 media. After that, samples were seeded
at a concentration of 5 × 10
5
/mL in a 24 well plate.
The number of live cells then were counted at 24-, 48-, and 72-hours using trypan blue
(Cat # 15250–061, Life Technologies, Carlsbad, USA), and cell viability were determined by the
ratio of the number of live cells in treated samples to that in untreated cells.

2.5. Measurement of FLT3 surface expression level

The expression of surface FLT3 was analyzed by incubating 1×10
6
FLT3-ITD+ (MV4–11)
cells with midostaurin at 5, 10, 50 and 100nM for 6 hours. Cells were then washed to remove the
34

media by PBS two times and then incubated with APC-conjugated Anti-Hu CD135 (FLT3)
(Invitrogen Cat # 17-1357-42) for 20 min at room temperature. Cells were then washed with PBS
two times and then were analyzed by assessing the shift representing the mean fluorescence
intensity (MFI) compared to respective control. Mean fluorescence intensity normalized to
unstained cells and data were analyzed using the LSRII BD Fortessa X20 flow cytometer and
analyzed using Flow Jo software (BD, Franklin Lakes, NJ, USA).

2.6. Measurement of scFv binding in AML cell line

The binding of FLT3-scFv to cell-surface FLT3 was assessed by incubating 1×10
6
FLT3-ITD+
(MV4–11) cells with either FLT3-scFv 1 and 5μM for 30 min at room temperature. The scFv-
treated cells were either alone (i.e., untreated) or pre-treated with 10nM midostaurin. Cells were
then washed to remove the media by PBS and incubated with PE-conjugated Anti-His tag mouse
IgG 1 (R&D Systems Cat # IC050P) for 30 min on ice. Cells were then washed with PBS to remove
unbound antibody. Bound Anti-His tag labeled FLT3-scFv were measured by assessing the shift
representing the mean fluorescence intensity (MFI). Mean fluorescence intensity normalized to
unstained cells and data were analyzed using the LSRII BD Fortessa X20 flow cytometer and
analyzed using Flow Jo software (BD, Franklin Lakes, NJ, USA).
2.7. Apoptosis assay

In vitro apoptosis assay was performed using the Annexin V and PI APC kit according to
the manufactures protocol (Invitrogen, Cat # 17–8007-74). The percentage of Annexin V APC +
35

cells were compared among groups. Data were analyzed using the LSRII BD Fortessa X20 flow
cytometer and processed using Flow Jo software (BD, Franklin Lakes, NJ, USA).

2.8. Statistical analysis

All the data are presented as mean ± standard deviation (SD). Student t-test was used to
determine if the difference in means between two groups was statistically significant. P-value
less than 0.05 was considered significant. All graphs and p-values in the results section was
processed by Microsoft Excel. FLT3 Receptor expression assay and proliferation experiments
were performed in triplicate. The binding assay was performed two times each have different
conditions, yet the 1µM FLT3-scFv with or without 10nM midostaurin is the common condition
in these two experiments. The apoptosis assay was done once.

36

Chapter 3: Results
3.1. Midostaurin promotes FLT3 surface expression

Previous reports showed that midostaurin has FLT3-inhibitory effect on cells-expressing
FLT3-ITD (Weisberg et al., 2002). In addition, Midostaurin promotes membrane localization of
FLT3-ITD similarly to FLT3-WT due to a change in glycosylation pattern on the receptor and in
turn elevates FLT3 surface expression (Reiter et al., 2018). In order to confirm the increased
expression of FLT3 surface marker, I treated FLT3-ITD cells (MV4-11) with midostaurin at 5, 10,
50, and 100nM concentrations. The selection of MV4-11 as appropriate cell model to study this
concept is due to the fact that this cell line carries homozygous FLT3-ITD mutation (Spiekermann
et al., 2003; Reiter et al., 2018).
Flow cytometry analysis showed that midostaurin successfully upregulates FLT3 surface
markers in treated cells compared with respective naïve control (Fig. 4A). Moreover, there was a
statistically significant increase in FLT3 expression in cells treated with 50nM midostaurin when
compared to control group (P<0.01), and 10nM midostaurin group (P= 0.02) (Fig. 4B), yet there
was no significant change measured at 100nM. Large variability existed in this sample, possibly
due to some cell death at this high concentration. Of note, a dose-dependent pattern of increased
FLT3 expression was observed, yet no dependence was observed beyond 50nM group; whether
this is due to the saturation of the FLT3 receptor expressed on the surface or variability due to
cell apoptosis is not clear (Fig. 4).

37

Figure 4. FLT3 surface expression upon treating FLT3-ITD + cell line (MV4-11) with different concentrations
of Midostaurin; [A] The FLT3 expression shift under different midostaurin concentrations; [B] Mean
fluorescence intensity of FLT3 surface expression when cells were treated with different midostaurin
concentrations. The 5nM midostaurin was not indicated for comparison as they were tested once, while
other concentrations were tested in triplicate; The error bar indicate standard deviation (SD)

3.2. FLT3-scFv binds to the FLT3 positive cells

To confirm that FLT3-scFv binds specifically to surface FLT3, we tested the binding using
several concentrations of FLT3-scFv at 0.5, 1, and 5µM. A dose-dependent increase in binding
was observed when comparing the 0.5 and 1µM treated groups, yet the significance could not
be calculated for the 0.5µM group, since it was tested only once (fig. 5 A & B).
In addition, no significant difference in binding was observed when comparing the 1µM
group to the 5µM group, and 1µM group compared with control group in terms of a dose-
38

dependence pattern (P=0.22) (Fig 5 D & E). The latter could result from the fact that the
concentration of anti-His tag antibody while conducting the assay was fixed in all treatment
conditions and did not adjust for the increase level of the scFvs added.

Figure 5. [A] Binding signals of Anti-His tag secondary antibody to 0.5 and 1µM FLT3-scFv and [B] their
corresponding mean fluorescence intensity. [C] Binding signals of Anti-His tag secondary antibody to 1
and 5µM FLT3-scFv and [D] their corresponding mean fluorescence intensity; [E] The binding of 1µM FLT3-
scFv to surface FLT3 compared to control.

3.3. The effect of Midostaurin treatment on FLT3-scFv binding to the FLT3 positive cells

39

Since midostaurin upregulated the FLT3 surface marker, next, we tested if adding
midostaurin in combination with FLT3-scFv, at concentrations of 0.5, 1, and 5µM, would increase
the binding of scFv to surface FLT3.
Our findings showed that 10nM midostaurin combined with 1µM FLT3-scFv have no
statistically significant increase in binding to FLT3 when compared with cells treated with 1µM
FLT3-scFv alone (P=0.2) (Fig 6C). The latter statistically insignificant difference might be attributed
to the low concentration of anti-His tag antibody used during the binding assay. It is also possible
that during the 30 min. incubation period with FLT3-scFv, the FLT3 receptor might be internalized,
yet the mechanism of the FLT3 internalization remains to be investigated (Kellner et al., 2020).
Besides, the 0.5µM and 5µM concentrations were tested once, therefore no statistical testing
could be performed.

40

Figure 6. [A] Binding signals of Anti-His tag secondary antibody to FLT3-scFv using 0.5 and 1µM FLT3-scFv
with or without 10nM midostaurin with their corresponding mean fluorescence intensity. [B] Binding
signals of Anti-His tag secondary antibody to FLT3-scFv using 1 and 5µM FLT3-scFv with or without 10nM
midostaurin with their corresponding mean fluorescence intensity. [C] Comparison of mean fluorescence
intensity of binding to FLT3-scFv upon dual therapy of 1µM FLT3-scFv and 10nM midostaurin compared
to respective treatment conditions.

3.4. The effect of midostaurin and FLT3-scFv combined treatment on the viability of the
FLT3-ITD+ cells

Next and in order to examine the antileukemic effect of the combined therapy of
midostaurin with anti-FLT3 scFv; we treated cells for 72 hours with different conditions at 1µM
FLT3-scFv, 5µM FLT3-scFv, 10nM midostaurin, or in combination. We used a low dose of
midostaurin (10nM) in order to induce only limited viability reduction in cells as the direct effect
of midostaurin only.
Our compiled viability experiments showed a statistical significance reduction after 72
hours of treatment in viability between dual therapy of 10nM midostaurin combined with 1µM
41

FLT3-scFv when compared with cells in the midostaurin alone group (P=0.03) or 1µM FLT3-scFv
alone group (P=0.01) (fig. 7C). Moreover, a statistical significance reduction was observed in cells
treated with dual therapy of 10nM midostaurin with 5µM FLT3-scFv after 24 and 48 h of
treatment when compared to 10nM midostaurin alone group (P<0.01), and (P=0.02), respectively
(fig. 7 D & F). No other significant reduction was observed among other treatment groups in
which some groups under the same conditions were tested once.
Apoptosis analysis using the Annexin V and PI staining assay measured by flow cytometry
revealed a slight increase in apoptosis at 72 hours in MV4-11 cells treated with midostaurin
combined with FLT3-scFv, yet no statistically significant apoptotic induction was observed since
the assay was performed only once (Fig. 8).

42

Figure 7. Relative viability of FLT3-ITD + cells in three-time points under different treatment conditions of
midostaurin and/or 1 (A: 24 hours, B: 48 hours, and C: 72 hours) and 5µM FLT3-scFv (D: 24 hours, E: 48
hours, and F: 72 hours). The data were presented as a mean of three different repeated experiments, and
the error bars represent standard deviation (SD)

Figure 8. Apoptosis of FLT3-ITD + cells (MV4-11) was measured by flow cytometry assessment of annexin
V and PI staining of cells treated with midostaurin or anti-FLT3 scFv or the combination for 72 hours. under
different treatment conditions

43

Chapter 4: Discussion
4.1. Discussion

AML is a blood cancer characterized by poor differentiation of hematopoietic stem cells
and a negative clinical prognosis. The standard therapeutic regimen for AML has not been
changed dramatically in the last three decades as a high relapse rate and death from the disease
has persisted. FLT3 is expressed on the surface of immature HSC and hematopoietic progenitors.
Due to its importance in hematopoiesis regulation, FLT3 is considered a promising target to treat
AML (Rosnet et al., 1996). Recurrent FLT3 mutations mainly the ITD and TKD pose a leukemic
burden and comprise a challenge in AML management due to their association with a high risk
of relapse and treatment resistance. Therefore, new therapies targeting FLT3 beyond standard
therapy are highly needed (Small, 2006; Shih et al., 2002; Yamamoto et al., 2001).
Many treatment modalities have been introduced, including small molecule FLT3 kinase
inhibitors, that showed a plausible effect, yet they are still suboptimal. TKIs had presented a
plausible therapeutic strategy, yet the outcomes remain unforthcoming due to clinical challenges
including relapse, lack of specificity, limited monotherapy effect, and developed resistances.
Therapeutic regimens capable of solving these shortcomings are needed to improve the outcome
of AML patients (Yeung et al., 2020). As of now, two FLT3 inhibitors received FDA approval for
treating FLT3-ITD AML patients. Midostaurin is approved for the treatment of pre-transplant
patients in combination with standard therapy. Still, half of these patients die of their disease
within four years. Gilteritinib is approved for R/R AML in patients with FLT3-ITD AML.
Nevertheless, according to the phase III ADMIRAL trial, less than 40% of treated patients survived
longer than one year (Daver et al., 2019; Stone et al., 2017). Altogether, these limitations support
the need to investigate different modalities in a combinatorial manner with a more specific
44

approach to achieve higher efficacy and low off-target effect. Previously, IMC-EB10 and IMC-NC7
FLT3-mAb had a promising antileukemic effect both in vitro and in FLT3-ITD murine models
(Piloto et al., 2005). Nevertheless, the clinical trial (NCT00887926) in phase 1 to determine the
safety of IMC-EB10 in patients with relapsed or refractory AML was terminated since the
maximum tolerated dose was beneath the desired level of efficacy. Although it remains unclear
why IMC-EB10 failed clinical testing, it was speculated that pharmacological properties and
dismal therapeutic concentration may play a significant role in FLT3-mAb shortcomings. The
novel fusion protein (α-FLT3-A192) is designed to target FLT3. Thus, several stability and
pharmacokinetic shortcomings that observed with scFv alone had been addressed when ELP
fused with scFv antibody. Moreover, α-FLT3-A192 have exerted in-vitro antileukemic activity and
inhibition of FLT3 mediated downstream signaling kinases (Park et al., 2020). In vivo, leukemic
burden reduction and improved survival in NSG mice was also reported upon treating with α-
FLT3-A192. In addition, when α-FLT3-A192 retained in the bone marrow, it enables it to target
the peripheral blood and bone marrow cells, which are the omnipresent sites of leukemogenesis
in AML. Altogether, α-FLT3-A192 has a superior pharmacokinetic profile and efficacy in preclinical
models (Park et al., 2020; Almatani et al., 2021).
Compiled studies showed that FLT3 immunotherapies could promote antileukemic
activity irrespective of FLT3 mutational status as they can abrogate resistance mechanisms
associated with the use of TKIs. Other studies have reported that FLT3 inhibitors promote the
surface expression of mutated FLT3 that is often sequestered intracellularly in the perinuclear
endoplasmic reticulum (ER) (Reiter et al., 2018). Also, samples from patients with FLT3 mutated
AML have shown higher surface expression of FLT3 after TKIs treatment than at the time of
45

diagnosis (Reiter et al., 2018). These observations suggest a potential benefit of the combined
use of FLT3 inhibitor with FLT3-directed immunotherapy in which FLT3 inhibitor helps in
upregulating FLT3 surface marker and facilitate the recognition by FLT3-targeting
immunotherapy (Reiter et al., 2018). In our study, we hypothesized the promising effect of dual
therapy in an in-vitro cellular model by using an FLT3 single-chain variable fragment (FLT3-scFv)
in combination with the FLT3 inhibitor (midostaurin) to be tested on FLT3-ITD positive cell line
(MV4-11) to address the synergistic or additive effect. The results showed that the efficacy of the
combined therapies have a promising enhanced antileukemia activity in FLT3-mutated AML.
Considering the encouraging data generated with our initial studies with the anti-FLT3 scFv,
expanding these studies to α-FLT3-A192 combination with midostaurin or other TKIs present a
promising interest in AML management.
Several limitations of this study have been noted, including the use of a single dose for
treatment with midostaurin which was 10nM. The rationale lies in the fact that we want to
observe the effect of FLT3-scFv in order to induce only limited viability reduction in cells as the
direct effect of midostaurin only. However, considering that higher doses of midostaurin resulted
in better upregulation of FLT3 surface level, so investigating different doses would be important
to further validate the initial observations. in addition, multiple doses should be tested to assess
the apoptotic effect of the combined drugs, which was limited using the currently used doses.
Similarly, testing different doses of both drugs should be considered in order to determine
whether the combined effect of the two treatments is synergistic or additive. Since different FLT3
inhibitors were able to upregulate surface FLT3 marker other than midostaurin, it is worth
considering testing other TKIs with FLT3-scFv. Another caveat in this thesis was using one cell line
46

to conduct the assays which were insufficient and comprises a challenge to address and compare
the specificity of FLT3-scFv for treating other FLT3-WT or FLT3 negative cell lines. Additionally,
treating cell lines only is not sufficient as these cells are authenticated and processed with a
certain biological capacity. Thus, testing primary AML blasts should be implemented as well as
validation of the viability results should be transferred to the preclinical in vivo model via treating
immunocompromised mice model and assessing their survival, organ size of the spleen, and liver
to determine the leukemic burden. Altogether, these limitations are encouraging the upscale of
this research.

4.2. Conclusion

FLT3 is an essential target in treating AML due to its role in the AML prognosis. Although
FLT3 inhibitors have been presenting a feasible therapeutic effect, yet factors such as relapse,
lack of specificity, dismal monotherapy efficacy, and the emergence of resistance constitute
clinical challenges. Even with the latest FDA approval to some FLT3 inhibitors, they are still
therapeutically limited due to half of these patients dying of their disease within five years.
Therefore, combining more than one drug has the benefit of filling the gaps in treatment
regimens, yet the toxic adverse effects present an additional challenge. Thus, the need for
alternative therapeutic strategies surpassing the current modalities is high (Almatani et al., 2021).
This study presented some evidence of the improved effect of midostaurin and anti-FLT3
antibody-based therapy combination, yet further investigation is warranted on both levels of in-
vitro and in-vivo.
47

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2052

Abstract (if available)

Abstract Mutations in the FMS-related tyrosine kinase 3 (FLT3) receptor have been extensively investigated over the past two decades due to their importance as prognostic markers and therapeutic targets in AML. Two main types of recurrent mutations occur in the FLT3 gene: Internal tandem duplication representing the majority of FLT3 mutations (FLT3-ITD about 25% of AML cases), and tyrosine kinase domain mutation (FLT3-TKD, about 10% of AML cases). The FLT3-ITD is associated with a high leukemic burden and an unfavorable prognosis. Therefore, in an effort for targeted therapy, anti-FLT3 antibodies and small-molecule FLT3 tyrosine kinase inhibitors were developed and reported promising preclinical and clinical e?cacy. However, their efficacy was suboptimal, and these treatments were associated with limited improvement in overall survival. Accumulating evidence suggests that FLT3 inhibitors while downregulating the phosphorylated mutant FLT3 they upregulate the surface expression of FLT3 receptor. This increase in FLT3 expression on the surface may be leveraged to enhance the recognition for anti-FLT3 antibody and potentially improve their antileukemia activity. in order to test this approach, we investigated the dual-therapy effect of midostaurin with an in-house manufactured anti-FLT3 single-chain variable fragment (FLT3-scFv) to assess the in vitro ability of binding to FLT3 and reducing the viability of FLT3-ITD cell line (MV4-11). Our ?ndings indicate that the midostaurin treatment increased the expression of FLT3 on the cell surface. In addition, the reduction of cell viability was higher in dual therapy treated cells compared with either treatment alone. These results provide the basis supporting the combination of TKI with FLT3-directed antibody treatment and warrant further preclinical in vivo investigations to validate the in vitro studies.

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Asset Metadata

Creator Almatani, Mohammed Farayah (author)

Core Title Investigation of the synergistic effect of midostaurin, a tyrosine kinase inhibitor, with anti FLT3 antibodies-based therapies for acute myeloid leukemia

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Degree Conferral Date 2021-08

Publication Date 07/23/2021

Defense Date 06/18/2021

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

Tag AML,FLT3,FLT3-directed antibody,midostaurin,OAI-PMH Harvest,TKI

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Alachkar, Houda (committee chair), Okamoto, Curtis (committee member), Zhang, Yong "Tiger" (committee member)

Creator Email almatani@usc.edu,malmelfi@kku.edu.sa

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC15619460

Unique identifier UC15619460

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Rights Almatani, Mohammed Farayah

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

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