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Development of immunotherapy for small cell lung cancer using novel modified antigens
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Development of immunotherapy for small cell lung cancer using novel modified antigens
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1
Development of Immunotherapy for Small Cell Lung
Cancer Using Novel Modified Antigens
By
Prerna Sehgal
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Medicine)
August 2017
2
Table of Contents
Dedication 5
Acknowledgements 6
Chapter 1: Introduction 7
1.1: Small Cell Lung Cancer 7
1.2: Origin of SCLC and SCLC cell lines 10
1.3: Mouse Models 11
1.3.1: Xenograft mouse models 11
1.3.2: Genetically engineered mouse models (GEMMs) 12
1.4: Paraneoplastic Neurologic Syndrome and ELAVL proteins 14
1.5: Isoaspartylation of ELAVL proteins 16
1.6: New studies in the field of SCLC 17
1.6.1: GEMM-derived embryonic stem cells (ESC-GEMMS) 17
1.6.2: Circulating tumor cells (CTCs) 17
Chapter 2: Preliminary Study conducted by Mario Pulido 19
Chapter 3: Development of syngeneic SCLC mouse models 22
3.1: Introduction 22
3.2: Materials and Methods 23
3.2.1: Generation of small cell lung cancer cell lines 23
3.2.2: Intratracheal intubations of 625 SCLC-cell line into healthy mice 24
3.2.3: Plasma Collection 25
3.2.4: Recombinant protein production and anti-ELAVL41-117 plasma reactivity
testing 26
3
3.2.5: 3D Imaging 28
3.2.6: Splenocyte and T-cell response 28
3.2.7: Statistical Analysis 31
3.3: Results 31
3.3.1: 3D Imaging 31
3.3.2: Anti-ELAVL41-117 antibody response 31
3.3.3: Splenocyte and T-cell response 32
3.4: Discussion 36
Chapter 4: Immunization studies with native and isoaspartylated ELAVL41-117 38
4.1: Introduction 38
4.2: Materials and Methods 40
4.2.1: Recombinant ELAVL41-117 production 40
4.2.2: Mouse Immunization 40
4.2.3: Plasma Collection 41
4.2.4: Anti-ELAVL41-117 plasma reactivity testing 41
4.2.5: Splenocyte and T-cell response 41
4.2.6: Statistical Analysis 42
4.3: Results 42
4.3.1: Immunogenicity of Isoaspartylated ELAVL41-117 42
4.3.2: Anti-ELAVL41-117 antibody response 42
4.3.3: Splenocyte and T-cell response 44
4.4: Discussion 48
Chapter 5: A cohort study for SCLC mouse model based on the previous study 50
4
5.1: Introduction 50
5.2: Materials and Methods 51
5.2.1: Generation of 477V SCLC cell line 51
5.2.2: Intratracheal intubations of 477V SCLC cell line in healthy mice 51
5.2.3: Plasma collection 51
5.2.4: Anti-ELAVL41-117 plasma reactivity testing 52
5.2.5: 3D Imaging 52
5.2.6: Weight and Paster score records 52
5.2.7: Statistical Analysis 52
5.3: Results 52
5.3.1: Culturing of SCLC cell lines and intratracheal intubations of mice 52
5.3.2: Survival Analysis 53
5.3.3: Paster scores and Weight curve 54
5.3.4: Lung Fixation 56
5.3.5: Anti-ELAVL41-117 Antibody response 56
5.4: Discussion 58
Chapter 6: Future Directions 61
References 65
Appendix 71
5
Dedication
To my parents, Rajeev and Sangeeta Sehgal for their constant and unconditional
support and to my siblings Pritika and Aamulya for their love, support and trust.
6
Acknowledgements
I would like to thank the members of my committee for their kind support. I would like to thank
Dr. Ite Laird-Offringa for her constant guidance and support throughout my master’s project. I
would also like to thank Dr. Zoltan Tokes for his guidance and support throughout the program
I would like to acknowledge Dagmar Bouwer’s equal contribution in this project. I would like to
express my appreciation to the current and previous members of the Laird-Offringa lab for their
helpfulness: Mario Pulido, Evelyn Tran, Chenchen Yang, Chunli Yan, Mihaela Campan, Theresa
Ryan Stueve, Romina Ortiz, Diane Lee, Daniel Mullen, Laura St Pierre, Anusha Muralidhar and
Madhura Lotlikar.
I would like to sincerely thank Dr. Crystal Marconett and her lab members, Vishaly Kumaran and
Jonathan Castillo for all their insights and encouragement.
I would also like to express thanks to Jiao Luo from Dr. Zea Borok’s lab for her help in sample
preparation for lung micro CT scan. I would also like to acknowledge Diane Da Silva and Joseph
Skeate from Dr. Martin Kast’s lab for assisting us with immunizations and T-cell assays. I would
like to also give my thanks to Daisy Flores from Amy Lee’s lab for teaching and helping me with
intratracheal intubations.
Lastly, I would like to thank Monica Pan for her advice and support throughout the program.
7
Chapter 1: Introduction
1.1 Small Cell Lung Cancer
Lung carcinoma is the second most frequent cancer in both men and women, and is the
most prominent cause of cancer-related death in the USA (Figure 1A) (Seigel et al., 2017). Lung
cancer can be broadly classified into two major clinicopathological groups, non-small cell
carcinoma and small cell carcinoma (Figure 1B). The majority of new lung cancer cases are
represented by a heterogeneous group of non-small cell lung cancers (NSCLC) which accounts
85% of all diagnosed lung cancers, while SCLC accounts for about 10-15% (American Cancer
Society).
A
8
Small Cell Lung Cancer (SCLC) is an aggressive lung cancer subtype with a five-year
survival rate of 7% (Howlader et al., 2016). SCLC is thought to arise from pulmonary
neuroendocrine cells, which are rare sensory cells located in the branches of airways in the lung
(Sutherland et al., 2011, Calbo et al., 2011). Smoking is the major risk factor for SCLC;
approximately 95% of SCLC patients are current or previous heavy smokers. The duration and
intensity of smoking increases the risk of SCLC development in a dose-dependent manner (van
Meerbeeck et al., 2011). Due to the aggressive nature of the disease, patients are often diagnosed
after metastasis has already occurred. Although about two-thirds of the patients respond quite well
to initial chemotherapy of cisplatin/etoposide combined with prophylactic cranial irradiation,
B
Figure 1. Demographics of lung cancer. A. Lung cancer is the leading cause of cancer-related death and
second most common cancer among both males and females in the United States (Seigel et al., 2016). B.
Lung cancer can be broadly classified into non-small cell lung cancer (NSCLC) and small cell lung cancer
(SCLC) (Brody et al, 2014).
9
almost all patients experience a relapse as the cancer becomes resistant, and die within 1-2 years
of initial diagnosis.
Various studies suggest that SCLC exhibits genetic alterations, many of which arise by
smoking, such as somatic mutations, epigenetic abnormalities and genomic variability. 90% of
human SCLC cell lines are characterized by two key alterations, inactivation of Rb1 and loss of
heterozygosity in the tumor suppressor gene p53. In addition, Myc oncogenes are also frequently
overexpressed. These three alterations are the main genetic drivers of small cell carcinomas
(Sutherland et al., 2011, Wistuba et al., 2001). Infrequent mutations in other genes such as PTEN,
PIK3CA, EGFR and KRAS also occur in SCLC (Pleasance et. al., 2010) but these genetic
variations overlap with other lung carcinomas as well (Figure 2).
Recently, DNA-methylation profiling of
pathologically indistinguishable human small-
cell lung cancer was conducted and tumors were
classified into three subtypes based on their
DNA methylation pattern and gene expression
(Poirier et al., 2015). This approach can help
classification of SCLC tumor types. Further
epigenetic characterization of SCLC may help to
develop better diagnostic criteria and eventually,
develop personalized medicine approaches.
Research material from SCLC patients,
such as tumor tissue samples, is limited in its
availability compared to other solid tumors.
Figure 2. Genetic Alterations in different subtypes of lung
cancer (Pietanza et al., 2012).
10
Surgical intervention is usually not appropriate due to the high frequency of metastasis at
diagnosis, and diagnostic methods such as fine needle aspirates yield only small amounts of tissue
(Carter et al, 2014).
1.2 Origin of SCLC and SCLC cell lines
The localization of primary SCLC tumors in airway junctions and the expression of
characteristic markers in SCLC tumor cells suggests that SCLC originates from rare pulmonary
neuroendocrine cells (PNECs). These are specialized cells that appear within the epithelium and
are found in clusters at the junctions of the airways. They are implicated in sensory function,
secretion of serotonin, regulation of pulmonary blood flow, maintenance of the stem cell niche,
and expression of calcitonin gene-related peptide and the mitogen bombesin in response to hypoxia
and other triggers (Song et al., 2012, Park et al., 2011). Elegant studies using a genetically
engineered mouse model and specific promoters that cause inactivation of TP53 and RB1 in
specific cell types strongly support PNECs as the cells of origin of SCLC (Sutherland et al., 2011,
Calbo et al., 2011).
A number of cell lines have been established from human small cell lung cancers. Based
on appearance, morphology and expression of neuroendocrine biomarkers, these SCLC cell lines
can be sub-classified into 2 major classes: classical cell lines and variant cell lines. Classical cell
lines grow as tightly packed, floating ball-like aggregates and overexpress neuroendocrine markers
such as DDC, BLI, NSE, and CK-BB whereas variant cell lines form loose, adherent sheet-like
structures and show elevated expression of NSE and CK-BB but lack the other 2 markers (Carney
et al., 1985). Variant cell lines grow faster and are more invasive in nature than classical cells, as
shown by their colony forming efficiency in soft agarose and nude mice xenografts. It has been
11
speculated that variant SCLC cell lines are derived from later stages of small cell cancer.
Concomitant presence of classical and variant cell lines leads to loss of classical features which
suggests that tumor starts expressing variant phenotype as it progresses (Bepler et al., 1987).
1.3 Mouse Models
Animal models are useful for studying the interaction between a tumor and its micro-
environment, and for understanding the underlying molecular mechanisms involved in tumor
growth and development, resistance to therapy and, invasive and metastatic properties (Hanahan
et al., 2000, Hanahan et al., 2011, Sakamoto et al., 2015). Especially given the scarcity of patient
samples, it was of crucial importance to find a suitable mouse model for SCLC. A variety of mouse
models are currently being used in SCLC research. They can be grouped into two categories:
xenograft mouse models and genetically engineered mouse models.
1.3.1 Xenograft Mouse Models
Xenograft mouse models involve transplantation of human cells ectopically or
orthotopically in an immunocompromised murine environment to prevent rejection of these cells
(Jung et al., 2014, Seol et al., 2013). The advantage of such models is that they utilize human tumor
tissues and better mimic the tumor-host interactions as observed in human disease. Earlier
subcutaneous xenograft mouse models were widely used but these models prevented tumor
metastases due to tissue barrier. Thus, many orthotopic xenograft SCLC mouse models were
developed to study SCLC’s metastatic potential. In one such patient-derived xenograft (PDX)
model, the DMS273 cell line, a variant cell line developed from the pleural effusion of a SCLC
patient, was transplanted in nude mice. As variant cell lines are more aggressive in nature, it
12
allowed distant macro- and micro metastases formation (Sakamoto et al., 2015). Advanced in vivo
imaging techniques, such as small animal PET/CT scans and bioluminescence imaging (BLI),
allow the metastatic potential of SCLC to be very well studied in these models (Iochmann et al.,
2012). The drawback of this mouse model is that it lacks an immune system and limits our
understanding of immune system-tumor interaction. To overcome this disadvantage, genetically
engineered mouse models were developed for SCLC (Taromi et al., 2016).
1.3.2 Genetically engineered mouse models (GEMM)
Numerous transgenic mouse models have been generated to replicate human cancers in
mice. To develop an ideal tumor model, it is imperative to understand clinicopathological
characteristics of the disease, its cell of origin, and major genetic alterations involved (Taromi et
al., 2016). One of the most powerful GEMM for SCLC, was the one created in the Berns lab. In
this mouse, loxP sites were inserted into the Trp53 and Rb1 genes. Adenoviral vectors carrying a
cytomegalo virus (CMV) promotor-driven Cre gene (Adeno-Cre) were delivered intratracheally
in the mouse lungs, resulting in homozygous deletion of exons in these genes to create a p53/Rb1
double knock out (KO). Tumors became detectable after 6 months and showed characteristics very
similar to human SCLC in terms of histology, pathology, and metastatic behavior. Experiments
with these mice also indicated that loss of both genes plays a synergistic role and is required for
the development of small cell carcinoma. Rb1 is important for tumor formation and development
and p53 is important for tumor progression. The Berns lab also confirmed PNECs as the cells of
origin for SCLC (Meuwissen et al., 2003, Sutherland et al., 2011, Calbo et al., 2011) (Figure 3).
13
To address some of the drawbacks of this
model, such as the long latency times, newer
versions have been developed. Loss of a single
Pten gene along with homozygous Rb/p53 KO
led to development of small cell lung cancer but
loss of both Pten genes shifted the histological
spectrum to lung adenocarcinomas (Cui et al.,
2014). Song et al. also generated a triple
homozygous KO mice in which Rb1, Tp53 and
Pten were simultaneously deleted. Cre was
fused with estrogen receptor allowing the
tamoxifen induced Cre expression. This fusion
construct was driven by PNE-specific calcitonin
gene-related peptide (CGRP) promoter.
Although the tumor latency period was shorter in this model, Cre expression was not exclusive to
PNECs causing the animals to develop thyroid carcinomas. A triple homozygous KO of
p130/Rb/p53 resulted in faster manifestation of SCLC (Schaffer et al., 2010). Myc and NFIB
overexpression works collectively with Rb/p53 deletions in mice for promoting SCLC tumor
progression (Kim et al., 2016, Wu et al., 2016). Despite these advances, there remains a
considerable latency period in tumor development (Taromi et al., 2016). To overcome this barrier,
we have come up with an adaptation of the Berns mouse model which will be discussed in Chapter
2.
Figure 3. SCLC cell of origin. Trp53 and Rb1 were
deleted in different lung cell types. Loss of Trp53 and
Rb1 in CGRP positive neuroendocrine cells were found
to be the cells which gave rise to SCLC. PNECs are the
cell of origin for SCLC. (Sutherland et al., 2011).
14
1.4 Paraneoplastic Neurologic Syndromes and ELAVL proteins
One of the hallmarks of SCLC is the presence of paraneoplastic neurologic syndromes
(PNS), which are cancer-associated neurological diseases. PNS are neurological manifestations
resulting from indirect effects of the tumor on the immune system (Pignolet et al., 2013, Gozzard
et al., 2015). They are characterized by an auto-immune response against neuronal proteins
aberrantly expressed in the tumor. The neuronal proteins ectopically expressed in SCLC tumors
are referred to as onconeuronal antigens. SCLC-associated autoantigens include neuronal ELAVL
RNA-binding proteins (previously known as Hu proteins), SOX transcription factors, voltage-
gated calcium channels and recoverin, among others (Kazarian et al., 2011). Paraneoplastic
encephalomyelitis/sensory neuronopathy (PEM/SN) is one of the autoimmune diseases associated
with SCLC and involves neuronal loss and perivascular infiltration of lymphocytes in various parts
of the brain and spinal cord (Schiller et al., 1993). 85% of PEM/SN cases are associated with small
cell lung cancer (Pignolet et al., 2013). PEM/SN is rare, occurring in ~1% of SCLC patients and
is characterized by antibodies against neuronal ELAVL proteins (Dalmau et al., 1995, Kazarian et
al., 2009) (Figure 4).
ELAVL proteins are family of four RNA-binding proteins. ELAVL2 (HuB/Hel-N1),
ELAVL3 (HuC), and ELAVL4 (HuD) are SCLC-associated antigens and are normally exclusively
expressed in the nervous system and gonads, while the fourth, ELAVL1 (HuR), is ubiquitously
expressed. Neuronal ELAVL proteins are involved in neuron-specific RNA processing and neural
development (Szabo et al., 1991, Good et al., 1995, Kazarian et al., 2011, Pulido et al., 2016).
ELAVL proteins contain an unstructured N-terminus followed by three RNA recognition domains,
RRM1, RRM2 and RRM3. Studies has shown that immunogenicity resides in the N-terminal
region containing RRM1 (1-117aa) (Manley et al., 1995, Sillevis Smitt et al., 1996).
15
Due to abnormal expression of neuronal
ELAVL proteins in SCLC, the immune system
somehow identifies them as foreign, leading to
generation of anti-ELAVL (anti-Hu)
autoantibodies. These autoantibodies have been
shown to be mainly directed against
ELAVL4/HuD as it is the most frequently
expressed neuronal ELAVL protein in SCLC
tumors (Manley et al., 1995, Kazarian et al., 2011,
Pignolet et al., 2013, Pulido et al., 2016). Patients
suffering from PEM/SN produce high titers of
antibodies against ELAVL proteins which results
in an immune system-mediated response against
SCLC tumors. SCLC patients with PEM/SN can
show spontaneous regression of their tumors,
suggesting that some aspect of the immune
response is protective in nature (Dalmau et al., 1992, Pignolet et al., 2013). Interestingly, 15% of
SCLC patients without PEM/SN have low titers of anti-ELAVL4 antibodies. Such patients have
been shown to exhibit an improved survival, suggesting that even low level immune response
might inhibit tumor growth (Dalmau et al., 1990, Kazarian et al., 2011, Gozzard et al., 2015).
Figure 4. Expression of different neo-antigens in
SCLC and their associated paraneoplastic diseases,
(A) anti-Hu, (B) anti-voltage-gated calcium
channels, (C) anti-SOX1, and (D) anti-recoverin
autoantibodies (Kazarian et al., 2011)
16
1.5 Isoaspartylation of ELAVL proteins
One of the questions that arises, is why the anti-ELAVL immune response is present in
only a fraction of SCLC patients, when all SCLC tumors express ELAVL onconeuronal proteins
(Voltz et al., 1997, Pittock et al., 2004). One hypothesis is based on the fact that abnormal
expression of self-antigen alone is not enough to generate an immune response. The antigen may
have to undergo some kind of mutation or modification to activate the immune system and
ultimately, break tolerance. Our laboratory has recently shown that ELAVL proteins undergo
spontaneous isoaspartylation under physiological conditions and that this modification makes the
protein highly immunogenic (Pulido et al., 2016). Isoaspartylation results in a kink in the
polypeptide chain at asparagines (N) or aspartates (D). Accumulation of isoaspartyl modification
is a sign of protein damage and has been reported to elicit immune responses (Mamula et al., 1999,
Doyle et al., 2006). This naturally occurring modification is repaired by an enzyme called PIMT1
(Protein-L-isoaspartate O-methyltransferase) (Yang et al., 2006). All living organisms, from
bacteria to mammals, carry such a repair enzyme, indicating that repair of this type of damage is
essential. Expression of PIMT is particularly high in the brain (Kim et al., 1997) and PIMT
knockout mice die at 4 weeks of age of seizures and apparent brain inflammation (Doyle et al.,
2006). This suggests that neuronal proteins or the environment in the nervous system is particularly
prone to isoaspartylation. Conditions in the tumor, such as insufficient PIMT, may prevent proper
repair of isoaspartylated ELAVL proteins.
17
1.6 New approaches in SCLC studies
Novel ways of studying SCLC have come up in recent years which show promise for
pushing the research in this field forward. Some of the revolutionizing studies will be mentioned
in this section.
1.6.1 GEMM derived embryonic stem cells (ESC-GEMMs)
Traditionally, GEMMs were created by using wild-type mouse strain embryos and adding
genetically modified embryonic stem cells (ESCs), which, when they gave rise to the gonads,
would generate the desired GEMM. By using stem cells derived from existing GEMMs, additional
genes can be rapidly added (Huijbers et al., 2014). This approach was successfully used to add an
oncogenic MycL1 gene to the Trp53
fl/fl
/Rb1
fl/fl
GEMM SCLC model and demonstrated that MycL1
is an oncogenic driver in SCLC (Huijbers et al., 2014). Along with new gene editing technologies,
such as CRISPR-Cas9, this ESC-GEMMs approach can reduce the time spent in developing novel
mouse models. It is anticipated that this procedure will accelerate the elucidation of the molecular
pathways involved in SCLC pathogenesis and metastasis.
1.6.2 Circulating Tumor Cells (CTCs)
Circulating tumor cells are cancer cells shed by the tumor into the blood stream and can be
harvested with minimal invasiveness for diagnostic and research purposes. In SCLC, CTCs can
serve as a “liquid biopsy” for determining specific biomarkers by molecular profiling to develop
personalized treatments for SCLC patients (Normanno et al., 2016, Hamilton et al., 2016, Hamilton
et al., 2016). CTCs can also help in determining whether the cancer has become chemo resistant
or not. Higher number of CTCs after treatment indicates that the cancer has become unresponsive.
18
A novel approach of using CTC-derived xenografts can be another stepping stone in developing
treatments tailored to an individual (Hodgkinson et al., 2014). Recently, a rare CTC sub-population
expressing endothelial markers was found in SCLC patients. These tumor cells acquired
endothelial cell behavior which enabled them to generate tumor vasculature. This ability is known
as vasculogenic mimicry. It gives a certain advantage to the tumor cell, such as the ability to
generate its own vasculature, facilitation of epithelial-to-mesenchymal transition (EMT),
decreased tumor latency and resistance to cisplatin treatment (Williamson et al., 2016). Many
studies are exploring CTCs, and promise to provide further insight into SCLC and move us one
step closer to curing it.
19
Chapter 2: Preliminary study conducted by Mario Pulido
A major limitation of the Berns lab mouse model is that it takes many months to develop
full-blown small cell lung cancer. Though many different mouse models have been developed,
none of them have resulted in a major decrease in the tumor latency period. The goal of our lab
was to develop a faster and more reproducible mouse model as a research tool. A novel adoptive
transfer model was designed in our lab by previous PhD student Mario Pulido. Adeno-Cre virus
was delivered by intratracheal instillation in the lungs of the p53/Rb1 floxed mice to develop
SCLC. SCLC cell lines were derived from the lung tumors. Two subtypes of these cell lines were
observed, which mirrored the phenotypes of human classical and variant SCLC cell lines. The
classical cell lines are floating with suspended ball-like aggregates. On the other hand, variant cells
grow as attached sheet-like layers and propagate much faster than the classical cell lines. Four cell
lines were generated by Mario. Two of them were classical cell lines, 625 and 486, and 2 showed
a variant morphology, 477 and 494 (Figure 5). These cell lines can be transplanted back into the
lungs of syngeneic mice intratracheally.
Classical cell line Variant cell line
Figure 5. Classical and Variant phenotype of SCLC cell lines. Bright field
images of classical and variant SCLC-cells in culture. Classical SCLC-cells
form floating, round clusters. Variant SCLC-cells are adherent in nature and
grow at least three times faster than classical SCLC-cell lines (Pulido thesis,
2015).
20
A preliminary study was carried out by Mario which was aimed at developing a syngeneic
mouse model for SCLC by instilling cell lines into the lungs of the mice. In his experiment, mice
were instilled with 4 cell lines: 2 classical cell lines (625 and 486) and 2 variant cell lines (477 and
494), both by intratracheal intubation and by tail vein injections. 24 mice were used in each group.
12 mice for each cell line treatment were exposed to 0.1 million cells and the remaining 12 to 1
million cells. The tail-vein injection study was not promising as the variant cells got attached to
the site of injection and caused necrosis of the tail of the mice, requiring euthanasia. However, the
intratracheal instillation study was successful. There was not a big survival difference between
mice instilled with the different cell numbers and different phenotypes, though the variant cell
lines showed slightly faster propagation. Mice intubated with the 477 variant cell line died as early
Figure 6. Kaplan Meier survival analysis of intratracheally intubated mice with SCLC
classical and variant cell lines. The median survival with both the cells types was comparable
though we do observe earlier tumor development in case of variant cell lines (Pulido thesis, 2015).
21
as 25 days. The median survival of mice with classical and variant cells observed was 67 and 60
days, respectively (Figure 6).
Variant cell lines also appeared to show a more frequent antibody response against
ELAVL4. Plasma collected from all 24 mice showed that 5 out of 12 mice injected with variant
cell line exhibited an ELAVL4 antibody response and only 1 out of 12 mice for classical cell line.
Thus, analysis of antibody response suggested a more common immune response against ELAVL4
in variant cell lines than in classical cell lines.
The antibody response was observed against both monomeric and multimeric form of the
ELAVL41-117 polypeptide. ELAVL proteins tend to form aggregates or multimers which are only
weakly detected by coomassie stain. These aggregates are seen in both native and isoaspartylated
peptide but increase upon incubation under isoaspartylation conditions. The anti-ELAVL
autoantibodies react with both monomeric and multimeric form of the protein (Pulido et al., 2016).
The study also showed a fluctuation in the antibody response, i.e., the response would
decrease or die out and then bounce back very strongly. Fluctuation was also seen in the health
and the weight of the mice. The mice would appear sick but would become healthy again. This
oscillation was concurrent with variations in antibody reactivity.
22
Chapter 3: Development of Syngeneic SCLC mouse model
3.1 Introduction
To study SCLC, several different mouse model systems are available which include
patient-derived xenografts and genetically engineered mice. Each type of model has its own
drawbacks. Xenograft mouse models lack a competent immune system (Taromi et al., 2016). Since
SCLC is associated with autoimmune disorders, such a model would be unable to recapitulate the
interaction between the tumor and the host immune response (Isobe et al., 2013). So, a better
approach to study SCLC is using GEMMs. The Berns group in the Netherlands have developed
the most powerful transgenic SCLC mouse model to date (Meuwissen et al., 2003). It is based on
the fact that 90% of human SCLCs lack the RB1 and TP53 genes. In this genetically engineered
mouse model, Rb1 and Tp53 are engineered to contain loxP recombination sites in both alleles.
This allows the intervening genetic sequence to be homozgously deleted by intratracheally
administering adenovirus carrying Cre recombinase in the lungs. Several adenoviral vectors were
generated by this research group wherein Cre expression was under the control of different lung
cell type-specific promotors (Sutherland et al., 2011). These experiments confirmed that
pulmonary neuroendocrine cells (PNECs) are the progenitors to SCLC. The only limitation this
model suffers from is that it takes almost 6-7 months until tumors become detectable (Meuwissen
et al., 2003).
Novel diverse models were built on the Berns model to overcome this hurdle. Another lab
generated a triple KO mice in which Rb1, Tp53 and Pten were simultaneously deleted (Song et al.,
2012). In the mice, the investigators engineered the PNE-specific calcitonin gene-related peptide
(CGRP) promoter to drive tamoxifen-responsive fusion of Cre with the estrogen receptor (CreER)
allowing induced targeted expression of Cre. Although this model resulted in faster emergence of
23
tumors (within 2-4 weeks), Cre expression was not exclusive to PNE cells and the animals
developed thyroid carcinomas, complicating interpretation (Song et al., 2012). A recent study
showed hemizygous inactivation of Pten, along with loss of Rb1 and Tp53, resulted in much faster
SCLC development but homozygous deletion resulted in adenocarcinomas (Cui et al., 2014). This
model requires further investigation of the role of Pten in SCLC.
To overcome the limitation of long tumor latency associated with Berns model, a novel
syngeneic mouse model was developed by Mario in our lab. In this model, SCLC cell lines derived
from the tumors of the mice were transplanted back into the lungs of syngeneic mice
intratracheally. His preliminary data showed that mice implanted with variant cell lines and
classical cell lines developed SCLC as soon as 25 days and 42 days respectively. None of the mice
survived more than 130 days. This model not only accelerates small-cell lung cancer modeling in
vivo, but also enables us to understand the immune response directed towards the tumor.
To build on Mario’s preliminary study, we designed a 4-week pilot study. The study was
designed to serve two purposes: to measure the immune responses developed by mice with
intratracheally instilled SCLC cells and profile the timeline growth of SCLC tumors in the lungs.
3.2 Materials and Methods
3.2.1 Generation of small cell lung cancer cell lines.
Four cell lines were generated previously by Mario in our lab. Two of them were classical
cell lines, 625 and 486, and 2 showed a variant morphology, 477 and 494 (Figure 5). These frozen
cell lines were thawed and cultured in modified HITES media (DMEM/F12 (1:1), hydrocortisone
(4 µg/ml) (sigma), murine EGF (5 ng/ml) (gibco), 1X of insulin-transferrin-selenium mix (Gibco),
fetal bovine serum (10%), and penicillin-streptomycin added for infection control) (Linnoila et al.,
1993, Seol et al., 2013, Pulido et al., 2016). After eight to ten passages, all of the cell lines showed
24
mixed population of classical and variant cells. These cells were split according to the subtypes to
generate eight new cell lines; 625C, 625V, 486C, 486V, 477C, 477V, 494C and 494V. These eight
cell lines were frozen down after six to eight passages and stored in liquid nitrogen. ELAVL
expression was determined by western blot for cell line lysates. It was found that 625C showed
the highest ELAVL4 expression. To classify any difference between the immune responses
generated and tumor growth between different SCLC cell line subtypes, we decided to use 625C
and 625V cell lines for this study. The mice were intubated with 625C, 625V and PBS as control
for two different time points: 2 weeks and 4 weeks (Figure 7).
3.2.2 Intratracheal Intubations of 625 SCLC-cell line into healthy mice
Eighteen 12-14-week-old FVB/N Tp53fl/fl; Rbfl/fl transgenic mice were used in this study.
SCLC was induced by intratracheal intubation of 2 autologous mouse cell lines. 6 of the mice were
instilled with the 625 classical cell line, 6 with the 625 variant cell line and, the remaining 6 with
PBS as control. Each of the 18 mice were exposed to 1 million cells. 12 of these mice were allotted
Figure 7. Schematic describing the strategy implemented to assess the response of different SCLC cell
lines at different time points on development of tumors and corresponding immune response in mice.
25
for ELAVL4 antibody studies and 3D lung imaging and the remaining six mice were used to study
the T-cell response (Figure 8). The cell lines were mycoplasma tested before administration.
For intratracheal instillation, mice were given intraperitoneal injections of an anesthetic
concoction containing Ketamine (Ketaject) and Xylazine (AnaSed) before administering the cells
in the lungs. The cells were counted and resuspended in PBS before instillation.
3.2.3 Plasma Collection
Mouse plasma samples were obtained from twelve of the mice at two different time points.
These 12 mice were divided into three groups: 4 with 625C, 4 with 625V and 4 with PBS. The
three groups were further divided into two subgroups based on euthanasia time point. 2 mice from
each group were euthanized at the two-week time point and the rest were euthanized at the four-
week time point (Figure 9). Plasma was also collected from mice at euthanasia by cardiac puncture
for blood collection using a 25G needle attached to a 1 mL plastic syringe (~500µl). Blood was
Figure 8. Schematic to show the different groups of mice used in the intubation study. 12 mice were used
to study antibody response and 6 mice were used to study T-cell response.
26
collected in K-EDTA-coated capillary blood collection tubes (BrainTree Scientific). Plasma was
separated by centrifugation at 13,000 rpm for 10 minutes at 4°C, aliquoted and stored at -80°C.
3.2.4 Recombinant protein production and anti-ELAVL41-117 plasma reactivity testing
A pET bacterial expression vector system was used to produce the N-terminally
hexahistidine-tagged ELAVL4-fragment containing the unstructured N-terminal region and the
first globular RNA-recognition motif (RRM1) (amino acids 1-117). This vector was transformed
in competent BL21 (DE3) cells carrying a plasmid with two rare tRNAs (J57, chloramphenicol
resistance) for protein production. Transformed cells were selected by ampicillin and
chloramphenicol resistance on LB agar plates. For setting a 1 liter culture for protein production,
a 100 ml of LB containing ampicillin and chloramphenicol was inoculated with one of the
transformed cell colonies and cultured overnight at 37° C. This culture was added to 900 ml of LB
with appropriate amounts of ampicillin and chloramphenicol antibiotics and cultured until the
Figure 9. Division of mice based on different experimental conditions and end-time points. Out of 12 mice, 4
mice were instilled with 625C, 4 with 625V and remaining 4 with PBS. 2 mice from each of these three groups were
euthanized at 2-week time point and the remaining 2 mice at 4-week time point.
27
OD600 was between 0.6-0.8. Protein production was induced by the addition of 1mM IPTG and
cells were then cultured for 4 hours. The culture was pelleted by centrifuging it at 6000 rpm for 10
minutes at 4°C. Supernatant was discarded and the pellet was resuspended in 10 ml of sonication
buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100) containing 1 protease
inhibitor tablet. The cells were lysed by sonication and then sheared with 18G needle as an extra
measure. The sample was spun at 13,000 rpm for 10 minutes at 4° C and the supernatant was
saved. 400 µl of Ni-NTA Agarose beads (Qiagen) were added to the supernatant and the protein
was allowed to bind to them by rotating the sample overnight at 4° C. The beads were washed in
wash buffer (Sonication buffer + 10% glycerol) and eluted in 50mM and 500mM concentrations
of Imidazole. The protein concentration was determined by Bradford assay.
To induce isoaspartylation, ELAVL41-117 was “aged”, i.e. incubated at 37° C in 50 mM K-
HEPES (pH 7.4), 1.0 mM EGTA, 0.02% (w/v) sodium azide, and 5% (w/v) glycerol for 7 days
(Johnson et al., 1989, Pulido et al., 2016). Blots were prepared from 14% SDS-gels run at 100
volts for 2 hours, with transferred unaged (day 0) and aged, isoaspartylated (day 7) recombinant
ELAVL4 protein 1-117 (~1µg). Transfer was carried out at 100 volts for 1 hour at 4° C in Towbin
buffer to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked
in 5% milk. The collected plasma samples were tested at a 1:200 dilution in a solution of 5% milk
in TBST (20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) overnight. Each blot was
washed 3 times with 3 ml of TBST for 15 minutes. For signal acquisition, a secondary goat anti-
mouse IgG 1:5000 (Santa Cruz, sc-2005) conjugated to horseradish peroxidase (HRP) in a solution
of 3% milk in TBST solution was used. The blots were washed 3 times with 3 ml of TBST for 5
minutes and probed with Super Signal West Femto Maximum Sensitivity Substrate (Fisher
28
Scientific). Each blot was exposed for a maximum of 2 minutes and visualized using a Biorad
ChemiDoc XRS+.
3.2.5 3D Imaging
Lungs from SCLC-induced and control mice were collected. Mice were given
intraperitoneal injections of 200-250 µl euthanasia-III solution (Med-pharmacy, Inc.). This causes
the mice to die by decreasing the heart rate of the mice. Mice are dissected to expose the lung
cavity. The lungs of the mice were washed by 15-20 ml PBS by injecting the still slow beating
heart with a 25G syringe until they become clean and white. An incision is made on the trachea to
inject the lungs of the mice with 4% paraformaldehyde to fix it from inside. These insufflated
lungs are fixed overnight in 4% Paraformaldehyde. After overnight fixation, lungs were cleaned
and rinsed with PBS in a petri-dish to remove any material sticking to the lungs. The lungs are
incubated through an ethanol gradient (70%, 80%, 90% and 100%) for 2 hours each at 4°C.
Following this, lung samples were incubated with 100% hexamethyldisilazane overnight before
air drying. The 3D imaging was done at Molecular Imaging center, USC.
3.2.6 Splenocyte and T-cell response
Spleens were harvested from six of the mice at two different time points. These six mice
were divided into three groups: 2 intubated with 625C, 2 with 625V and 2 with PBS. The three
groups were further divided into two subgroups based on euthanasia time point. 1 mouse from
each group was euthanized at the two-week time point and the rest were euthanized at the four-
week time point (Figure 10). T-cell responses were assessed against both the antigens and SCLC
cells and quantified by 2 assays, a proliferation assay and an ELISPOT assay.
29
In the proliferation assay, 100 µl of each antigen at different concentrations and 100 µl of
SCLC cells were first added to wells of a 96 well plate. The aged and unaged ELAVL41-117 protein
antigens were plated at three different concentrations: 0.03 µg/ml, 0.3 µg/ml and 3 µg/ml. 100,000
cells from the two cell lines, 625C and 625V, were plated. These cells were irradiated before
plating. 100 µl of splenocytes (500,000 cells) were added in six replicates to the wells. 100 µl
Concanavalin A (Con A) (1µg/ml) was used as a positive control. All the materials were plated in
T-cell medium (RPMI-1640, 10% heat-inactivated FBS, 2 mM Glutamine, 20 µg/mL Pen/Strep
solution, 50 M 2-ME, 1 mM Sodium-Pyruvate, 0.1 mM non-essential amino acids). The plate
was centrifuged at 1000 rpm for 5 min at room temperature and was incubated for 4 days at 37°C,
5% CO2. 50 µl of supernatant was removed after 4 days and 50 µl of fresh medium with 1 µCi 3H-
thymidine was added per well and incubated overnight. The next morning, cells were harvested
using Cell Harvester to collect intact DNA onto 96 well filter plates. The plate was dried overnight
Figure 10. Division of mice based on different experimental conditions and end-time points. Out of 6 mice,
2 mice were instilled with 625C, 2 with 625V and remaining 2 with PBS. 1 mouse from each of these three groups
was euthanized at 2-week time point and the remaining 1 mouse at 4-week time point.
30
and the bottom was sealed. 25 µl of scintillation fluid was added and the plate was read in counts
per min (CPM) using Packard Top Count NXT Scintillation and Luminescence Counter to record
3H-thymidine (beta emission).
For the ELISPOT assay, 100 l of IFN capture Ab (BD Biosciences rat anti-mouse IFN ,
Clone AN18 (Cat #551309)) was added in each well of a Millipore Multi-screen HTS plate. The
plate was incubated overnight at 4 C. The capture antibody was discarded and the plate rinsed
with PBS three times. T-cell media was added as blocking solution. 100 µl of antigen and SCLC
cells were added to wells of a 96 well plate. The aged and unaged ELAVL41-117 protein antigens
were plated at three different concentrations: 0.03 µg/ml, 0.3 µg/ml and 3 µg/ml. 100,000 cells
from the two lines, 625C and 625V, were also plated. These cells were irradiated before plating.
100 µl of splenocytes (500,000 cells) were then added in triplicates to the well. 100 µl of
Concanavalin A (Con A) (1µg/ml) was used as a positive control. The plate was incubated for 20
hours at 37° C, 5% CO2. The plate was then rinsed with 0.05% PBST (PBS + 0.05% Tween-20)
five times. 1 g/ml of biotin IFN antibody was prepared and 100 µl of the solution was applied
in each well. The plate was incubated for 2 hours at room temperature away from light. The
antibody was discarded and the plates were rinsed with 0.05% PBST five times. 100 µl of
Streptavidin-HRP conjugate diluted at 1:4000 in PBS/0.5% BSA solution was then applied to each
well. The plate was incubated for 1 hour at room temperature away from light, then rinsed with
0.05% PBST six times and with PBS three times. 100 l of AEC (3-amino-9-ethyl-carbazole)
substrate was applied to each well and the plate was incubated for 5 minutes. The reaction was
stopped by washing extensively with water. The plate was air dried overnight in a dark drawer.
The next day, spots were counted in wells using Zeiss KS ELISPOT reader.
31
3.2.7 Statistical Analysis
Statistical Software Prism 5 was used to generate the graphs for histograms for T-cell
assays.
3.3 Results
3.3.1 3D Imaging
SCLC cell lines were intratracheally deposited in 12 of the mice to be used for 3D imaging
studies to determine tumor growth. These 12 mice were divided into three groups: 4 mice were
inoculated with the 625 classical cell line, 4 mice with the 625 variant cell line and the remaining
4 with PBS as control. These three groups were further divided into sub groups based on the
euthanasia time point. Two mice in each group were euthanized at 2 weeks and 4 weeks after cell
line insertion, respectively (Table A1). The lungs were extracted and fixed from all the mice.
Because the imaging is expensive, the lungs of the mice at the 4-week time point were imaged
first. Since we did not observe any lesions, the shorter incubated and PBS control mice were not
imaged.
3.3.2 Anti-ELAVL41-117 antibody response
Blood was collected from each of the 12 mice at euthanasia. This blood was processed to
extract plasma. The plasma was used to check for antibody response generated against both native
ELAVL41-117 (D0) and aged or isoaspartylated ELAVL41-117 (D7) in these mice by western blot.
Comparatively strong antibody response was detected at 2-week time point in 2 out of 4 mice and
weak response was detected at 4-week time point in 2 out 4 mice. Substantial anti-ELAVL41-117
response was shown by 2 out of 8 mice or 25% of the mice. Reactivity was observed against both
32
monomeric and multimeric forms of both native and kinked peptide. We did not see any antibody
response in mice intubated with PBS (Figure 11).
3.3.3 Splenocyte and T-cell response
6 of the 18 mice were used for T cell assays. These 6 mice were divided into three groups:
2 mice were intubated with 625 classical cell line, 2 mice with 625 variant cell line and the
Figure 11. Anti-ELAVL4 auto-antibodies in mice with induced SCLC. Mice were instilled with two cell lines,
625C and 625V, for two different time periods, 2 weeks and 4 weeks. The first panel shows mice intubated with
625C for 2 and 4 weeks, second panel shows mice intubated with 625V and, the third panel shows mice intubated
with PBS as control. Antibody response was tested against native (D0) and isoaspartylated (D7) ELAVL4 1-117.
Response was seen in 25% of the mice and the antibody reacts with the multimeric complexes as well. No response
was observed in mice intubated with PBS. A very weak response was seen in mice euthanized at 4-week time point.
monomeric form of the polypeptide
* multimeric form of the polypeptide
33
remaining 2 with PBS. These three groups were further divided into sub groups based on
euthanasia time point. One mouse in each group was euthanized at 2 weeks and one at 4 weeks
(Table A2). One of the mice died in between experiments. Two T-cell assays were conducted: a
proliferation assay and an ELISPOT assay.
The proliferation assay was conducted to determine the growth of splenocytes in response
to native ELAVL4 1-117 and isoaspartylated ELAVL4 1-117 antigen as well as 625C and 625V cells.
Con A was used as a positive control. Positive control did not work properly as Con A was lower
than no stimulation. Hence, it is difficult to draw any conclusions from these results. One of the
reasons could be that Con A was added too early and the T-cells were exhausted by the time they
were harvested. No significant results were obtained from the experiment as the T-cell response
was no different between experimental and control mice. Growth of splenocytes, in all mice, was
inhibited in response to SCLC 625C and 625V cell lines (Figure 12).
34
The ELISPOT assay was used for determining reactivity of T lymphocytes against native
and aged ELAVL4 1-117 peptides and 625C and 625V cells. Con A was used as a positive control.
No significant trend was observed in T-cell activation in response to different stimulations. The T-
cell response was extremely low and variable. For the mouse intubated with 625C for 4 weeks, T-
cells seem activated in reaction to 625C cells and the same was observed in the mouse intubated
with 625V for 4 weeks. Response to cell lines was variable among both experimental and control
mice (Figure 13).
Figure 12. Proliferation assay. The T-cell activity was quite low and inconsistent across both experimental
and control group. The positive control didn’t work properly. But in all the cases, T-cell activity was
hampered in presence of the two SCLC cell lines.
35
Figure 13. T-cell ELISPOT assay. T-cell response was not consistent across both experimental and
control mice. The T-cell response was low and variable. T-cell activity was observed in 4 week intubated
mice in response to the cells they were intubated with, i.e., the mouse intubated with 625C for 4 weeks
showed increased T-cell activity in response to 625C cells. Same was the case for one mouse intubated
with 625V for 4 weeks.
36
3.4 Discussion
In this study, we conducted a 4-week pilot study to develop a novel SCLC syngeneic mouse
model using previously derived 625 classical cell line and newly derived 625 variant cell line. 625
classical cell line was chosen as it perfectly models the human classical cell lines and expressed
highest levels of ELAVL4 as compared to other cell lines. A variant cell line derived from the
classical was used to observe the difference between ELAVL41-117 response and T-cell response
between the two types of cell lines. The cell lines were implanted in the lungs of the mice by
intratracheal delivery. One problem was that no tumors were observed at the end of 4-week study.
However, we observed an anti-ELAVL41-117 antibody response as soon as two weeks, which
suggests that these cells trigger an immune response much before the actual emergence of the
lesion. We observed very low to no immune response in mice intubated with cell lines for 4 weeks.
It could be that the immune response is either inhibited or suppressed in these mice but we need
pre-bleeds as well as 2-week bleeds for these mice to conclude anything. Though this immune
response is not observed in all SCLC patients, it still might be a useful biomarker for detection of
SCLC. For T-cell assays, the proliferation data did suggest that T-cell expansion might be
suppressed by the cell lines, perhaps due to secretion of suppressive factors, but this might merely
be the result of debris from the added irradiated cells. In contrast, some T-cell activity was
observed in response to SCLC cells at 4 weeks as suggested by ELISPOT, which does not support
the idea that the 625 cell lines suppress proliferation. Thus, it is hard to conclude anything based
on these results.
The shortcomings of the project were that no tumors were observed by the end of 4 weeks,
loss of one experimental mouse in between experiments, the fact that no pre-bleed and in between
bleeds were collected from the mice and that there were large variations between mice immune
37
responses. To overcome these problems, the project should be repeated with a larger sample size
and allow tumor longer growth period to generate significant data. Also, we should use a
previously derived and studied variant cell line rather than classical cell line and its derived variant
line, since the 625 cell line had not been tested as thoroughly. The mice should be bled more
frequently to correctly map the immune response generated in the mice. We should also collect
pre-bleeds of the mice to ensure that the response that we observe is due to cancer cells. Also, as
we used assay conditions normally used to detect immune responses to foreign agents like viruses
which gives high responses they may not detect these auto-antibody responses well enough. We
should also plate irradiated 100,000 cells for both 625C and 625V cells to make sure that the
irradiated cells are not generating the proliferation signal. We can also plate fewer number of
cancer cells as these are much bigger than splenocytes and could be hampering their ability to
proliferate. The syngeneic model appears to be promising for the study of anti-ELAVL41-117
antibody response but will require further development.
38
Chapter 4: Immunization studies with native and isoaspartylated
ELAVL41-117.
4.1 Introduction
As mentioned earlier, SCLC is accompanied by autoimmune syndromes due to abnormal
expression of neuronal proteins, including paraneoplastic encephalomyelitis/sensory
neuronopathy (PEM/SN). Although all SCLC express neuronal ELAVL antigens, only about 1%
of SCLC patients suffer from PEM/SN (Szabo et al., 1991, Dalmau et al., 1995). These patients
exhibit high titer antibodies against neuronal ELAVL proteins in their tumors and some patients
can show tumor regression. But these patients pay a heavy price as these autoimmune diseases can
be lethal. However, a substantial fraction of SCLC patients, who do not exhibit autoimmune
symptoms can nevertheless express anti-ELAVL antibodies although at lower titers, and can show
a significantly better survival (Dalmau et al., 1990, Dalmau et al., 1992, Kazarian et al., 2011,
Pignolet et al., 2013). Thus, its crucial to understand the mechanism involved in SCLC-triggered
autoimmune disease so as to develop strategies to harness this immune response for therapy.
There are four members of the ELAVL family (also known as “Hu” proteins), out of which
SCLC tumors most commonly express ELAVL4 (HuD) (Manley et al., 1995). ELAVL proteins
contain an unstructured N-terminus followed by three RNA recognition domains, RRM1, RRM2
and RRM3. A hinge region separates RRM2 and RRM3 domains (Szabo et al., 1991, Good et al.,
1995). ELAVL deletion and mutation constructs have shown that the immune epitope resides in
the N-terminal and RRM1 region (Pulido et al., 2016). A recent study from our laboratory has
shown that these proteins undergo a specific spontaneous post-translational modification known
as isoaspartylation that render them immunogenic. Isoaspartylation occurs at physiological
conditions and leads to kinks in the polypeptide backbone at asparagines (N) or aspartates (D) and
39
occurs when these residues are found in flexible regions of the protein and are followed by small
residues (Xaa) such as glycine, serine, or histidine. A beta-linked Asp-Xaa peptide bond is formed
when an succinimide intermediate that can spontaneously form under physiological conditions
undergoes dissociation (Figure 14). This modification is repaired by an enzyme, known as PIMT
(Protein-L-isoaspartate O-methyltransferase) (Kim et al., 1997, Pulido et al., 2016). When not
properly repaired, isoaspartylation can induce a strong B-cell and T-cell responses against self-
antigens (Doyle et al., 2006)
To better study the nature of the immune response against isoaspartylated ELAVL4, with
the ultimate goal to determine if it might be developed into SCLC immunotherapy, we conducted
an immunization study in which the mice were immunized with both native and isoaspartylated
(“aged”) N-terminal ELAVL4 fragments. These consisted of amino acids 1-117, including the
Figure 14. Isoaspartylation conversion and repair. The diagram describes the process of
conversion of aspartates to isoaspartates and its repair by PIMT (Pulido et al., 2016).
40
flexible extreme N-terminal region and the RRM1 domain. We determined the B and T cell
responses generated against these antigens.
4.2 Materials and Methods.
4.2.1 Recombinant ELAVL41-117 production
ELAVL41-117 was generated using a bacterial expression and purification system as
described in Section 3.2.4, and aliquots were aged in PBS at 37° C as described. For each mouse,
100 µg of the produced protein was added to appropriate volume of PBS to make the final volume
up to 50 µl. The native samples were stored at -80° C to minimize spontaneous isoaspartylation.
4.2.2 Mouse immunization
Four out of twelve FVB/N mice were immunized subcutaneously with 100 µg of aged and
four with native recombinant protein, emulsified 1:1 with incomplete Freund’s incomplete
adjuvant. The vaccine emulsions were formed by using syringe-extrusion technique. The PBS-IFA
mixture was pushed through a reinforced 22-gauge connector between two glass syringes on ice
until it become extremely hard to push. The emulsion should have a toothpaste like consistency.
Viscosity can also be checked by droplet test in which when a drop of mixture is added in the water
it stays on the surface. Four of the mice were immunized with PBS. The injections were made with
Freund’s incomplete adjuvant with the total volume of 100 µl. The mice were injected at Day 0, a
booster dose was given at Day 7 and, ultimately the mice were euthanized at Day 17 (Figure 15).
41
4.2.3 Plasma Collection
Blood was collected at three different time points for plasma extraction: before
immunization, just prior to boosting and at euthanasia. Blood was extracted by heating the tail in
warm water to dilate the vein. The incision was made using a lancet along the vein. Around 50-
100 µl of blood was collected. At euthanasia, blood was collected by cardiac puncture. Plasma was
extracted as described in section 3.2.3.
4.2.4 Anti-ELAVL41-117 plasma reactivity testing
Mouse plasma samples obtained were tested at a 1:200 dilution factor as described in
Section 3.2.4.
4.2.5 Splenocyte and T-cell response
Spleens were harvested from all the mice on Day 17 at euthanasia. T-cell responses against
both the native and aged ELAVL4 (1-117) polypeptides as well as against 625C and 625V SCLC
cell lines were measured using a proliferation assay and an ELISPOT assay as described in Section
3.2.6.
Figure 15. Schematic describing the strategy implemented to assess the
immunogenic potential of native and isoaspartylated-ELAVL4 1-117 in mice
(Pulido et al., 2016).
42
4.2.6 Statistical Analysis
Statistical Software Prism 5 was used to generate the graphs. One way ANOVA test and
unpaired T-test were used to study responses of control and experimental mice in response to
different stimulations.
4.3 Results
4.3.1. Immunogenicity of isoaspartylated ELAVL41-117.
12 mice were included in this immunization study. These 12 mice were divided into three
groups: 4 mice vaccinated with native ELAVL41-117 fragment, 4 mice with isoaspartylated
ELAVL41-117 fragment and the remaining 4 with PBS (Table A3). The mice were immunized
subcutaneously to test the effect of isoaspartylation on the immune response of the mice (Figure
16).
4.3.2 Anti-ELAVL4 1-117 Antibody response
Blood was collected from each of the 12 mice at 3 different time points: before injection,
prior to booster dose and at euthanasia. This blood was processed to extract plasma to determine
Figure 16. Schematic to show the different groups of mice used in the immunization study. Out of 12 of
the mice, 4 were immunized with native ELAVL41-117, 4 with aged ELAVL41-117 and 4 with PBS as
control. Plasma and spleens were extracted for studying antibody responses and T-cell responses.
43
B cell response. The plasma was tested for the presence of antibodies against both native
ELAVL41-117 (D0) and aged or isoaspartylated ELAVL41-117 (D7) in these mice by western blot.
We saw a strong immune response in all the four mice immunized with isoaspartylated peptide
with both monomeric and multimeric complexes at euthanasia. We saw reactivity in native mice
too, but only 3 out of 4 mice reacted with both monomeric and multimeric forms of the protein.
The sample size is too small to make any speculations about native vs. isoasp differences. One
drawback of native peptide immunizations is that the protein might age during the experiment in
vivo which is hard to control for. Some background reactivity against the higher molecular weight
complexes was noted in pre-bleeds of the mice and in the control mice which could be due to high
dilution of primary antibody that we use (Figure 17).
44
4.3.3 Splenocyte and T-cell response
Spleens of the mice were harvested at euthanasia to carry out T cell based experiments.
Two T cell assays were conducted: a proliferation assay and ELISPOT assays. The proliferation
assay was conducted to determine splenocyte proliferation in response to incubation with ELAVL4
Figure 17. Humoral response to native and aged ELAVL4 1-117 vaccination. To examine the antibody
response, 4 mice each were immunized with native and aged recombinant peptide and 4 with PBS as control. They
were given booster dose at day 7 and finally euthanized at day 17. Mice were bled at 3 different time points: pre-
immunization, pre-boost and euthanasia. The above figure shows 3 panels. The first panel shows mice immunized
with the native ELAVL41-117, the second panel shows mice immunized with aged ELAVL41-117 and, the third
panel shows mice immunized with PBS as control. The antibody response was tested against both native
ELAVL41-117 (D0) and isoaspartylated ELAL41-117 (D7). We see some background activity in pre-bleeds of some
mice and also, in mice vaccinated with PBS. But a response against monomeric ELAVL4 is seen only in protein-
immunized mice. The antibodies of the mice immunized with ELAVL4 fragment react strongly with both
monomeric and multimeric complexes when tested with plasma samples collected at euthanasia.
monomeric form of the polypeptide
* multimeric form of the polypeptide
45
1-117 and isoaspartylated ELAVL4 1-117 antigens. We also tested responses to SCLC cell lines 625C
and 625V, which express ELAVL4. Con A was used as a positive control. No significant difference
was observed between experimental and control mice. A slight trend was hinted in response to
3µg/ml concentration of aged peptide among different experimental groups. Though the trend
tested was not significant the results are suggestive and would require a larger sample size to test
it. In case of stimulation with cell lines, 2 mice immunized with aged peptide appeared to show
high activity in response to 625C cells (Figure 18).
Figure 18. Proliferation assay data. No significant trend was observed but the data was suggestive. Mice
immunized with aged peptide showed the most proliferation in response to highest concentration of
isoaspartylated peptide. 2 mice immunized with aged peptide showed increased activity in response to
625C SCLC cell line.
46
The ELISPOT assay was used for determining T lymphocyte activity in response to
different treatments of ELAVL4 1-117 and isoaspartylated ELAVL4 1-117 antigen as well as 625C
and 625V cells. Con A was used as a positive control. The highest concentration of native and
aged peptide (3µg/ml) appeared to elicit response. The same 2 aged immunized mice who
responded to aged peptide in the proliferation assay responded in the ELISPOT assay. Little or no
T cell activity was observed for mice immunized with aged peptide in response to 625C cells but
the sample size is too low to make any speculations (Figure 19).
Figure 19. T-cell ELISPOT data for immunized mice. A response was observed at highest concentration of
native and aged peptide for mice immunized with aged polypeptide. The same 2 mice who responded to highest
concentration of aged antigen in proliferation assay respond in ELISPOT.
47
We decided to look at the two aged immunized mice who responded in proliferation assay
and ELISPOT. Mouse 192 and Mouse 193 showed slightly higher activity in response to highest
concentration of aged peptide and 625C cells for proliferation assay but this response was near
background when compared to no stimulation. In case of ELISPOT, mouse 192 and mouse 193
respond to highest concentration of native and aged peptide, though the difference wasn’t
significant between the native and the aged stimulation. The difference between no stimulation
and aged peptide stimulation was significant for both mouse 192 (p=0.0387) and mouse 193
(p=0.0011). The difference between no stimulation and native wasn’t significant for mouse 192
but significant for mouse 193 (p=0.0475) (Figure 20).
Figure 20. Proliferation and ELISPOT assay response in mouse 192 and mouse 193. The response in proliferation assay
was not above background but we do see some T-cell activity in case of ELISPOT assay. The response was more significant
in response to highest concentration of isoaspartylated peptide for both mouse 192 (p=0.0387) and mouse 193 (p=0.0011).
Native vs no stimulation wasn’t significant for mouse 192 but did reach significance for mouse 193 (p=0.0475). There was
no significant difference in the native and isoaspartylated peptide stimulation in both the mice.
48
4.4 Discussion
Previous studies from other laboratories had shown that anti-ELAVL4 autoantibodies
expressed in patients are directed towards N-terminal portion of the protein which contains the
unstructured N-terminus and the first RRM domain (Manley et al., 1995, Sillevis Smitt et al.,
(1996), Kazarian et al., 2009). However, none of these studies tested the reactivity of RRM1 region
alone. Our recent analysis showed that patients antisera activity against RRM1 alone was weak or
absent (Pulido et al., 2016) and suggested the activity lies in the unstructured N-terminus. Our lab
previously generated a rabbit anti-isoasp ELAVL4 antiserum. Mutants in the regions
corresponding to the peptide used for immunization showed decreased or no reactivity with the
affinity purified rabbit antiserum, suggesting that antiserum is extremely specific for this post-
translational modification (Pulido et al., 2016). This indicates that an isoasp-specific antibody
response is possible.
We carried out an immunization study to examine the immune responses generated in
healthy mice upon vaccination with both the native and isoaspartylated peptide. One of the initial
setbacks of our project was that during sample preparation for our first injection some of our
needles broke and we lost a few of the immunization samples during preparation. We compensated
by immunizing all animals with less protein. There appeared to be an immune response in mice
immunized both with native and isoaspartylated polypeptide at euthanasia though the results were
more consistent with isoaspartylated peptide. But we do require a larger sample size to draw
conclusions. In ELISPOT, T-cell activity in response to highest concentration of both native and
aged polypeptide is suggested.
The shortcoming of this project was that due to loss of samples the mice were not
immunized properly and there was a fluctuation in B- and T-cell response. To overcome these
49
issues, the project should be repeated with a larger sample size, and we should also test reactivity
against the RRM1 domain both with and without N-terminus regions, to show the difference in
immunogenicity and also, employ a different vaccination method to compare the results. These
studies can help in confirming the role of isoaspartylated protein in triggering the immune system
and help develop a model for SCLC treatment.
50
Chapter 5: A Cohort Study for SCLC mouse model based on the
previous study
5.1 Introduction
A preliminary study carried out by previous graduate student Mario Pulido was aimed at
developing a syngeneic mouse model for SCLC using cell lines instilled into the lung. In his
experiment, mice were intubated with 4 cell lines: 2 classical cell lines (625 and 486) and 2 variant
cell lines (477 and 494). 12 mice for each cell line treatment were exposed to 100,000 cells and 1
million cells. The results suggested that classical cell lines grow much slower than variant cell
lines in vitro and in vivo. 477 variant cell line showed the fastest tumor propagation and most
frequent immune response.
One of the drawbacks that we suffered in SCLC mouse model pilot study using the 625C
and 625V cell lines (Chapter 3) was that on 3D Imaging of the lungs at 4 weeks we didn’t observe
any tumors. However, we did observe an immune response in 25% of the mice, which suggested
that this response occurs even before the emergence of the tumor and if harnessed properly might
be able to become a useful therapeutic. Thus, we decided to replicate the preliminary study
conducted by Mario who saw much quicker results. In this new study, as we did not see tumors
when using 625C and 625V cell lines, we decided to use the 477 variant cell line to further develop
a SCLC cell line-based mouse model and follow the mice until they showed tumor-associated
distress, which is assessed by using a rubric designed by Paster et al., or if the weight of the mice
dropped below 20%.
51
5.2 Materials and Methods
5.2.1 Generation of 477V SCLC cell line
The 477 variant cell line was cultured in modified HITES (mHITES) for eight to ten
passages as described in Section 3.2.1. The cell lines were tested for mycoplasma before
administration.
5.2.2 Intratracheal Intubations of 477V SCLC cell line in healthy mice
Twenty 14-16-week-old FVB/N Tp53fl/fl; Rbfl/fl transgenic mice were used in this study.
SCLC was induced by intratracheal intubation of the 477 variant cell line as described in Section
3.2.2. 10 of the mice were instilled with 100,000 cells and the rest of the 10 mice, were instilled
with 1 million cells (Figure 21).
5.2.3 Plasma Collection
Blood was collected one week before the mice were intubated with SCLC cells (“pre-
bleed”). The next blood collection was done three weeks after 477 variant cell were instilled and
subsequently, every two weeks after that. Plasma was processed as described in Section 3.2.3.
Figure 21. Schematic to show the different groups of mice used in the intubation study. 20 mice were
intubated with 477V cells. 10 of the mice were intubated with 0.1 million cells and the remaining 10 mice
with 1 million cells. Plasma was extracted for studying antibody responses and lungs were extracted for
potential imaging.
52
5.2.4 Anti-ELAVL41-117 plasma reactivity testing
Mouse plasma samples obtained were tested for anti-ELAVL41-117 immune response at a
1:200 dilution factor using Western blots carrying recombinant native and isoaspartylated
ELAVL41-117 as described in Section 3.2.4.
5.2.5 3D Imaging
The lungs of the mice were extracted for 3D imaging as described in Section 3.2.5.
5.2.6 Weight and Paster score records
The health of the mice was assessed as well as their weights were recorded every week
after intubations. If a mouse depicted any signs of abnormal behavior, the particular mouse was
monitored for its health by the Paster score every day.
5.2.7 Statistical Analysis
Statistical Software Prism 5 was used to generate the graphs by carrying out Kaplan-Meier
survival analysis.
5.3 Results
5.3.1 Culturing of SCLC cell lines and intratracheal intubations of mice
The 477 variant cell line was cultured in modified HITES for eight to ten passages. These
cells were tested for mycoplasma before the intubations. Twenty mice FVB/N Tp53fl/fl; Rbfl/fl
transgenic mice were used for this study. The mice were divided into two groups based on the
number of cells injected. 10 of the mice were intratracheally instilled with 100,000 cells and the
remaining 10 with 1 million cells (Table A4).
53
5.3.2 Survival Analysis
A rubric designed by Paster et al. was used for health assessment of mice with internal
tumors (Figure A1). Mice showing a score lower than 5 were euthanized. The mice started showing
signs of sickness around one and a half month to two months, much later than observed in the
study conducted by Mario. There was no significant difference between the median survival of
mice intubated with 0.1 million cells and 1 million cells. The median survival for mice with the
lower number of cells was 80 days and for higher number, it was 63 days. 4 of the mice were
euthanized for reasons other than cancer load; 2 male mice fought and had injuries and 2 mice got
necrotic tails due to excessive bleeding. The study was stopped at 84 days and the remaining four
mice were euthanized, but didn’t look sick (Figure 22). Out of these four, two were in the group
of mice with low number of cells and the two in the group with high number of cells.
54
5.3.3 Paster score and Weight curve
Most of the mice started showing signs of tumor burden around 40-50 days after
instillation. The health of the mice started deteriorating 2-3 weeks before their Paster score dropped
to 5 or below. Some mice died before their Paster score dropped to 5. As mentioned in Chapter 2,
Mario observed fluctuations in the health of the mice. The mice would look sick one week and
then recover the next week. We didn’t observe any such variations in the health of the mice (Figure
23).
Figure 22. Kaplan-Meier analysis of mice instilled with classical and variant SCLC-cells. Though there
was no statistical difference in their survival, mice injected with 100,000 cells lived slightly longer than
mice with 1 million cells as indicated by their median survival.
55
The weight of the mice was monitored over the period of the study. Mice showing 20% or
more decline in their weights were euthanized. Fluctuations in the weight was observed for some
mice, others showed relatively consistent weight. Some mice showed sudden decline in their
weights at which point they were euthanized. Some of the mice didn’t show any weight changes.
This is very different from Mario’s study in which he observed oscillation in the weights of the
mice which was in conjunction with ELAVL4 antibody response in these mice. (Figure 24).
Figure 23. Paster score of 477V SCLC cell line intubated mice. Paster score was plotted for the 12 mice.
8 of the mice were not included as they were not euthanized for tumor-associated distress. Mice showed
declining Paster score and were euthanized if the Paster score reached 5 or below. A few mice died before
they reached a score of 5.
Figure 24. Weight distribution of 477V SCLC cell line intubated mice. Weights were plotted for the 12 mice.
8 of the mice were not included as they were not euthanized for tumor-associated distress. Some mice showed
fluctuations in their weights, others showed relatively constant weights. Some mice showed sudden decline in
their weights at which point they were euthanized but some mice showed no weight changes.
56
5.3.4 Lung fixation
Lungs of these mice were extracted and fixed at euthanasia. But some lungs of the mice
looked healthy and we couldn’t observe any lesions on them.
5.3.5 Anti-ELAVL4 1-117 Antibody response
Blood was collected from all the mice after every two weeks. This blood was processed to
extract plasma which was stored at -80° C. The plasma was used to for checking antibody response
generated against both unaged or native ELAVL4 1-117 (D0) and aged or isoaspartylated ELAVL4
1-117 (D7) in these mice by western blot. No strong immune response was observed in these mice,
few of the mice showed reactivity to only higher molecular weight bands but even less showed
activity to the monomer. Background reactivity was seen in some pre-bleeds (Figure 25).
57
Figure 25. Antibody response in 3 mice with 477V cells. Mouse 268 (intubated with 1 million cells) showed no
ELAVL4 antibody response but did develop tumors. Mouse 272 (intubated with 1 million cells) showed slight activity
against multimeric form of the polypeptide at 3-week time point but no response at other time points. The lungs did not
show lesions. Mouse 243 (intubated with 0.1 million cells) showed antibody response at pre-bleed but we see dwindling
response at lower time points. The lungs showed no tumors.
monomeric form of the polypeptide
* multimeric form of the polypeptide
58
5.4 Discussion
From preliminary studies conducted in our lab by Mario Pulido, it was suggested that
variant cell lines are more aggressive and express a more frequent immune response against
ELAVL4. He instilled the cell lines in the mice both by intratracheal instillation and by tail vein
injections. 24 mice each were used in the study with triplicates for both 0.1 and 1 million cells for
each of the 4 cell lines. The tail-vein injection study was not promising as the tails of the mice
became necrotic which was lethal to the mice. According to his results, mice instilled with variant
cells developed cancer faster. The mice with variant cell lines showed much more frequent anti-
ELAVL4 response. Mice transplanted with the 477 variant cell line developed cancers faster than
classical cell lines and the other variant cell line. The median survival observed for the mice in
which this cell line was instilled in the lung was 60 days, with one mouse dying as soon as the 25
th
day post implantation. A fluctuation in the weight and the health of the mice was observed which
was concurrent with the oscillating anti-ELAVL4 response observed in these mice. A decrease or
increase in the weight of the mice showed poor health of the mice and correspondingly low anti-
ELAVL4 responses (Pulido thesis, 2015).
Thus, to build on our 4-week pilot study, stated in Chapter 3, to develop a syngeneic SCLC
mouse model, we decided to repeat the study with twenty mice. Based on the Dr. Pulido’s data,
we chose 477 variant cell line. We decided to follow the tumor progression in these mice and,
collect bleeds bi-weekly to observe the anti-ELAVL4 response. 10 out of 20 mice were implanted
with 100,000 cells and the remaining 10 with 1 million cells. These mice were followed for 84
days after which the study was discontinued as the mice weren’t developing cancer as fast as they
developed in Dr. Pulido’s study.
59
Mario observed fluctuations in the health of the mice. The mice would look sick one week
and then recover the next week. No oscillation was observed in the health of the mice but some of
the mice did show fluctuations in their weights. The mice started showing signs of sickness around
7-9 weeks’ post-instillation and were sick for two to three weeks before we observed a sudden
weight drop in the mice at which point they were euthanized. These mice started developing cancer
around one and a half months to two months. The median survival for mice with 100,000 cells was
80.5 days and for mice with 1 million cells was 63 days. The data of the mice who died due to
reasons other than tumor burden as well as the data of the last 4 remaining mice was truncated.
However, these mice did develop cancer much more quickly than what we observe by
Adeno-Cre delivery. Thus, this model is much more powerful for studying SCLC for carrying out
rapid studies for developing treatments.
We see little antibody reactivity against ELAVL41-117 in these mice. Some mice showed
activity against higher molecular weight bands and some showed no antibody response. These
upper bands could be aligned in such a way that they might be showing avidity effect at low
concentration. Two-three of the mice showed activity at pre-bleed which could be background due
to high amount of protein used (1-2 g) or high dilution of plasma used. In the highly reactive pre-
bleed mouse it is possible a sample switch occurred, but we cannot repeat the blot as too little
plasma was obtained. Since then our bleeding technique has improved so that this should not be a
problem in the future.
The shortcomings of this project were that we could not fully replicate the results obtained
from Mario’s preliminary data, in the sense that the mice took much longer to develop cancer than
expected. Mario saw mice getting sick around 20 days whereas our mice started getting sick around
one and a half months to two months. In lungs extracted from some of the mice, tumors were not
60
detectable by naked eye and looked healthy. One cause of these differences was speculated to be
the different genetic background of the mice used in this study to those used for generating our
preliminary data. Some of the mice were backcrossed to FVB/N mice as we lost the original strain
of the mice which had become too old to breed. To overcome this, we have re-derived the original
strain from the embryos that were frozen down. Another reason could be that the SCLC cell lines
may have undergone genetic or epigenetic alterations due to serial passaging in vitro. We can
derive new mouse SCLC cell lines from mice that develop cancer and use those cell lines for
building our SCLC syngeneic model.
61
Chapter 6: Future Directions
This section discusses further studies that can be done to improve upon the preliminary
data generated and mitigate any drawbacks encountered in our previous studies. I also outline the
follow-up studies that can be built upon our results.
As mentioned in the previous section, due to genetic variability in the mouse SCLC cell
lines and the current mice derived from back crossing with FVB/N mice, we have re-derived the
original Berns lab transgenic line from the previously frozen embryos. These mice will be
administered with the autologous mouse SCLC cell lines, originally derived by Mario,
intratracheally, so as to replicate the results generated in his study. We would like to pursue with
477 variant cells based on the results generated by Mario but we would also like to genotype the
cancer cell lines to avoid additional immune response generated by H-Y antigen from cell lines
derived from male mice. According to Mario’s notes, mouse 477 was a male so for future studies
we would like to instill the cells in male mice only to avoid any further immune response. Also, in
our cohort study we saw the cancer develop much later than Mario’s preliminary study but we still
got tumors in a pretty decent time span with 1 million cells in the lung. We would also like to use
625 classical and variant cell lines, as with those we saw an immune response as early as 2 weeks
in our pilot study, something which we failed to see in the cohort study. We can use 625 cell lines
for both sexes of mice as 625 was a female mouse and wouldn’t result in an added immune
response. This cell line should still be genotyped to confirm this information. Once we pick a good
model, we will want to stick with that and expand upon it by using cell lines. We are considering
introducing luciferase into them to allow easy following of tumor development in the mice by
imaging as well as Paster score and weighing, drawing blood to look at antibodies but also
62
euthanizing a subset of mice in this cohort at certain time points to study their immune cells and
to image their lungs in 3D. However, Dr. Kast cautioned that luciferase can cause an immune
response, so it may not be ideal. Based on the fact that 25% of the mice showed antibodies in
Chapter two, it would be good to use 40 mice with 1 million cells instilled into each, and 2 random
mice (1 male and one female) euthanized at 4 weeks, 6 weeks, 8 weeks, 10 weeks, 2 weeks for
lung imaging and T-cell assays, leaving 30 mice to be followed completely until they get really
sick (survival curve). 10 additional mice can be instilled with PBS as control. The effect of these
tumor cells will be monitored by taking into account the weight of the mice, body metric
assessment (“Paster score”) and ELAVL4 antibody levels. The sample size has been chosen by
taking into account the previous studies conducted, our own preliminary data and also, our
approved protocol.
We are planning on transducing our SCLC cell lines with NanoLuc (a much stronger
luciferase reporter) by lentiviral-based delivery (Stacer et al., 2013), which would improve our
mouse imaging technique as well as enable us to monitor tumor development in real time. Our
future mouse model would involve administering SCLC cells containing NanoLuc intratracheally
in the transgenic mice. At different tumor progression status, the lungs can be harvested for 3D
imaging and spleens for determining T-cell mediated response. However, as mentioned above, we
should also take into account that luciferase can also trigger an immune response.
Also, to avoid any background activity for the antibody results we can freshly prepare a
biotinylated piece of ELAVL41-117 which can be bound to streptavidin beads and can be used to
pre-clear specific antibody from mouse plasma. We can test that against our recombinant peptides
for an isoasp-ELAVL4 specific immune response.
63
Once we develop a syngeneic mouse model, we can start with a randomized control study
to further develop a cisplatin/etoposide treatment protocol for the mice. A pilot analysis was done
by Mario in which mice with SCLC were given two cycles of C/E, and it showed a marginal
improvement in survival. The next step would be to build upon this study by inducing SCLC in
the mice by intratracheal instillation of cells and give these mice up to four cycles of C/E, and
monitor survival. Later on, ELAVL4 immunizations can be combined with C/E treatment
depending on the result of the experiments.
There are certain drawbacks in peptide-based immunization studies. They require the usage
of Freund’s incomplete adjuvant which is highly irritating to the skin, and would not be an optimal
choice for patients. Such immunizations are also restricted in their ability to activate CD4
+
T helper
cells and generate long term memory response (Walters et al., 2017). Thus, we should also consider
different immunization techniques. One method is to use DNA-based immunization methods. We
have generated ELAVL4 plasmid constructs, containing the N-terminal region (amino acids 1-
117), with and without an IL-2 signal peptide to secrete the protein from the cells. Different
constructs will be used to determine the immunogenicity of ELAVL4 protein (Carpentier et al.,
1998, Zhang et al., 2004). These DNA constructs will be administered by gene-gun delivery
method which is much less invasive than protein immunization (Luz Garcia-Hernandez et al.,
2008).
A newer technology enables us to design anti-idiotypic antibodies with mimotopes of T-
cells, generated against specific antigen, expressed within the CDR3. This is processed by antigen
presenting cells to trigger a T-cell response (Metheringham et al., 2009, Xue et al., 2016). This
technique allows a much stronger immune response and is more efficient than simple
immunization with DNA encoding just the epitope (Metheringham et al., 2009).
64
Based on the previous studies conducted, our own preliminary data and also, our approved
protocol, it would be good to immunize 10 mice with native peptide, 10 mice with aged peptide
and 10 mice as control with our choice of immunization approach. We would also like to include
more boosters to get an ample immune response.
65
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71
APPENDIX
Table A1. A 4-week pilot study in developing syngeneic SCLC mouse model by
intratracheal delivery of SCLC cell line
Mouse
ID
Sex Experimental
group
First day of
experiment
Day of
Euthanasia
184 F Mice intubated
with 625C for 2
weeks
5/24/2016 6/7/2016
208 M Mice intubated
with 625C for 2
weeks
5/24/2016 6/7/2016
178 M Mice intubated
with 625V for 2
weeks
5/24/2016 6/7/2016
186 M Mice intubated
with 625V for 2
weeks
5/24/2016 6/7/2016
201 M Mice intubated
with PBS for 2
weeks
5/24/2016 6/7/2016
219 F Mice intubated
with PBS for 2
weeks
5/24/2016 6/7/2016
209 F Mice intubated
with 625C for 4
weeks
5/23/2016 6/21/2016
231 M Mice intubated
with 625C for 4
weeks
5/23/2016 6/21/2016
179 F Mice intubated
with 625V for 4
weeks
5/23/2016 6/21/2016
188 F Mice intubated
with 625V for 4
weeks
5/23/2016 6/21/2016
210 M Mice intubated
with PBS for 4
weeks
5/23/2016 6/21/2016
228 M Mice intubated
with PBS for 4
weeks
5/23/2016 6/21/2016
72
Table A2. Spleen extraction of mice intubated with cells for determining T-cell
responses.
Mouse
ID
Sex Experimental
group
First day of
experiment
Day of
Euthanasia
207 M Mice intubated with
625C for 2 weeks
6/9/2016 Died in
between
experiments
187 F Mice intubated with
625V for 2 weeks
6/9/2016 6/23/2016
190 F Mice intubated with
PBS for 2 weeks
6/9/2016 6/23/2016
189 F Mice intubated with
625C for 4 weeks
5/26/2016 6/23/2016
213 F Mice intubated with
625V for 4 weeks
5/26/2016 6/23/2016
217 M Mice intubated with
PBS for 4 weeks
5/26/2016 6/23/2016
Table A3. Mice utilized for immunization studies.
Mouse
ID
Sex Experimental group First day of
experiment
Day of
booster
injection
Day of
Euthanasia
196 M Mice immunized with
native ELAVL41-117
peptide
6/13/2016 6/20/2016 6/30/2016
215 M Mice immunized with
native ELAVL41-117
peptide
6/13/2016 6/20/2016 6/30/2016
227 M Mice immunized with
native ELAVL41-117
peptide
6/13/2016 6/20/2016 6/30/2016
233 M Mice immunized with
native ELAVL41-117
peptide
6/13/2016 6/20/2016 6/30/2016
192 M Mice immunized with
isoaspartylated
ELAVL41-117 peptide
6/13/2016 6/20/2016 6/30/2016
193 F Mice immunized with
isoaspartylated
ELAVL41-117 peptide
6/13/2016 6/20/2016 6/30/2016
73
214 M Mice immunized with
isoaspartylated
ELAVL41-117 peptide
6/13/2016 6/20/2016 6/30/2016
223 F Mice immunized with
isoaspartylated
ELAVL41-117 peptide
6/13/2016 6/20/2016 6/30/2016
172 F Mice immunized with
PBS as control
6/13/2016 6/20/2016 6/30/2016
177 F Mice immunized with
PBS as control
6/13/2016 6/20/2016 6/30/2016
191 F Mice immunized with
PBS as control
6/13/2016 6/20/2016 6/30/2016
206 M Mice immunized with
PBS as control
6/13/2016 6/20/2016 6/30/2016
Table A4. Mice intubated with 477V cells intratracheally for the cohort study
Mouse
ID
Sex Number of
injected
cells
First day
of
experiment
Day of
Euthanasia
238 F 0.1 million 9/12/2016 12/12/2016
239 F 0.1 million 9/12/2016 12/8/2016
240 F 0.1 million 9/12/2016 12/12/2016
241 M 0.1 million 9/12/2016 12/8/2016
242 M 0.1 million 9/12/2016 10/13/2016
243 M 0.1 million 9/12/2016 10/13/2016
244 M 0.1 million 9/12/2016 12/12/2016
245 M 0.1 million 9/12/2016 10/20/2106
246 M 0.1 million 9/12/2016 11/10/2016
247 M 0.1 million 9/12/2016 11/23/2016
248 M 1 million 9/13/2016 11/10/2016
254 F 1 million 9/13/2016 12/12/2016
255 F 1 million 9/13/2016 11/3/2016
267 M 1 million 9/13/2016 11/10/2016
268 M 1 million 9/13/2016 11/23/2016
269 F 1 million 9/13/2016 11/3/2016
270 F 1 million 9/13/2016 12/12/2106
271 F 1 million 9/13/2016 12/8/2016
272 M 1 million 9/13/2016 11/10/2016
273 F 1 million 9/13/2016 12/8/2016
74
Figure A1. Health Assessment rubric designed by Paster et al. to monitor the health
of the mice with internal tumors.
Abstract (if available)
Abstract
Small Cell Lung Cancer (SCLC) is an aggressive lung cancer subtype with a five-year survival rate of 7%. SCLC responds quite well to initial chemotherapy of Cisplatin/Etoposide but almost all patients experience a relapse and die within 1-2 years of initial diagnosis. One of the hallmarks of SCLC is the presence of cancer-associated neurological diseases, defined as Paraneoplastic neurologic syndromes (PNS). These diseases are associated with an auto-immune response caused by aberrant expression of neuronal proteins in the tumor. Paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/SN) is one of the various autoimmune diseases associated with SCLC. PEM/SN is characterized with abnormal expression of ELAVL proteins (previously known as Hu proteins) and occurs in ~1% of SCLC patients. As these proteins are ectopically expressed in SCLC tumors, they are classified as onconeuronal antigens. Due to abnormal expression of these onconeuronal antigens in SCLC, the immune system somehow identifies them as foreign, leading to generation of anti-ELAVL (anti-Hu) autoantibodies. These autoantibodies have been shown to be mainly directed against ELAVL4/HuD. Patients suffering from PEM/SN produce high titers of antibodies against ELAVL proteins which results in an immune system-mediated response against SCLC tumors. These patients can show spontaneous regression of their tumors. Interestingly, 15% of SCLC patients without PEM/SN show the presence of low titers of anti-ELAVL antibodies. Such patients have been shown to exhibit an improved survival, suggesting that even low levels of antibodies might inhibit tumor growth, and be protective against the tumor. But abnormal expression of self-antigen alone is not enough to generate an immune response. The immunogenicity of ELAVL proteins is defined by their ability to undergo spontaneous isoaspartylation under physiological conditions. This post-translational modification results in a kink in the polypeptide connectivity at asparagines (N) or aspartates (D). This modification is repaired in neuronal tissue by an enzyme called PIMT1 (Protein-L-isoaspartate O-methyltransferase) but the conditions in the tumor may prevent this repair. Thus, the goal of the project was to use this knowledge to develop a protocol to immunize mice with native and isoaspartylated ELAVL4 antigen to determine the therapeutic efficacy of each. Also, to design an improved SCLC mouse model in which SCLC is induced via introduction of autologous tumor cells intratracheally into the lungs. We hypothesized that this method of inducing SCLC will result in earlier formation of SCLC and will expedite further experiments. The syngeneic mouse model was successful and resulted in much faster tumor development than Adeno-Cre delivery. A pilot study conducted showed not much difference between the immune response generated by native and isoaspartylated peptide. Our observations warrant a much larger sample size, in which in-depth characterization of the humoral and cellular anti-ELAVL4 response is carried out at multiple time points. We conclude that our SCLC mouse model with instilled tumor cells and ELAVL4 immunization studies can be used to lay the groundwork for developing a novel SCLC immunotherapy based on the ELAVL4 antigen.
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Sehgal, Prerna
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Development of immunotherapy for small cell lung cancer using novel modified antigens
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Degree
Master of Science
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Biochemistry and Molecular Medicine
Publication Date
06/28/2017
Defense Date
05/31/2017
Publisher
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ELAVL4 immunizations
ELISPOT
immunotherapy
intratracheal intubations
isoaspartylated ELAVL4
isoaspartylation
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small cell lung cancer
splenocyte and T-cell response
syngeneic SCLC mouse model