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Development of immunotherapy for small cell lung cancer using iso-aspartylated antigen

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Development of immunotherapy for small cell lung cancer using iso-aspartylated antigen

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Development of Immunotherapy for Small Cell Lung Cancer
Using Iso-aspartylated Antigen.

By
Madhura Sachindra Lotlikar

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial Fulfillment of the
Requirements for
Degree MASTER OF SCIENCE
(Biochemistry and Molecular Medicine)

MAY 2019

1

Table of Contents

Chapter 1: INTRODUCTION ................................................................................................. 3
1.1 Small Cell Lung Cancer ............................................................................................... 3
1.2 Molecular Aspects of SCLC ......................................................................................... 4
1.2.1. Cell Line Studies .................................................................................................. 4
1.2.2. SCLC Genetically Engineered Mouse Models (GEMMs) ..................................... 4
1.3 SCLC Cell of Origin ...................................................................................................... 6
1.4 Tumor Heterogeneity: An Integral Aspect of SCLC .................................................... 9
1.5 SCLC and the Immune System ................................................................................. 10
1.5.1. SCLC: An Immune Eliciting Cancer .................................................................... 10
1.5.2. The cause of Anti-HuD/ELAVL4 Immunogenicity ............................................. 12
1.6 A rationale for DNA-Based Immunization ................................................................ 16
1.7 Rationale for Different Prime-Boost Strategies:
Homologous and Heterologous Regimens .............................................................. 19

Chapter 2: METHODS ........................................................................................................ 21
2.1 Methods: From DNA Construct to DNA Vaccine ..................................................... 21
2.1.1. The Region of the ELAVL4 Protein Used for the Experiments .......................... 21
2.1.2. Preparation of DNA Constructs ........................................................................ 21
2.1.3. In Vitro Testing of Secretion by IL2 SS-ELAVL4 Constructs ............................... 22
2.1.4. DNA Delivery by Gene Gun ............................................................................... 26
2.2 Methods: From Protein Construct to Protein Vaccine ............................................ 31
2.2.1. Protein Production and Purification ................................................................. 31
2.2.2. Isoaspartylation of ELAVL4 ............................................................................... 34
2.2.3. Dialysis of protein in PBS .................................................................................. 34
2.2.4. Filter Sterilization .............................................................................................. 35
2.2.5. Emulsion Preparation by the Syringe-Extrusion Method ................................. 35
2.2.6. Subcutaneous Injection of Emulsions ............................................................... 37
2.3 Mouse Groups and Immunisation Regimen ............................................................ 38
2.4 Mouse Sample Collection ......................................................................................... 39
2

2.4.1. Blood collection and plasma preparation ........................................................ 39
2.4.2. Spleen collection ............................................................................................... 39
2.5 Analysis of Samples .................................................................................................. 40
2.5.1. Plasma analysis ................................................................................................. 40
2.5.2. ELISPOT Assay ................................................................................................... 40
2.5.3. T-Cell Proliferation Assay .................................................................................. 41
2.6 Data Analysis ............................................................................................................ 42

Chapter 3: RESULTS ........................................................................................................... 43
3.1 Development and testing of DNA immunization vectors
for ELAVL4 1-117 secretion ......................................................................................... 43
3.2 Immunization strategy and antibody reactivity ....................................................... 46
3.3 T cell Proliferation Assay Results ............................................................................. 54
3.4 ELISPOT Assay Results .............................................................................................. 56
3.5 Interpretation of results ........................................................................................... 61

Chapter 4: FUTURE DIRECTIONS ....................................................................................... 63

REFERENCES ...................................................................................................................... 66

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Chapter 1: INTRODUCTION
1.1 Small Cell Lung Cancer

Lung cancer is a devastating disease-the second most common cancer and by far
the leading cause of cancer death among both men and women (causing more deaths
than cancer of the colon, breast, and prostate combined) (1).
Small-cell lung carcinoma (SCLC) accounts for 10-15% of new lung cancer cases. (American
Cancer Society).
The overall 5-year survival of SCLC is about 6% and depends on the stage; it is less
than 2% for extensive disease (ED) SCLC. SCLC is the second most aggressive cancer in
humans and is a high-grade, neuroendocrine (NE), recalcitrant lung malignancy (1). This
cancer type occurs almost exclusively in smokers. SCLC cells have been shown to have a
high mutational burden associated with tobacco smoking (2). The risk of SCLC
development increases with the increase in duration and intensity of smoking in a dose-
dependent manner (3). SCLC shows early metastasis; two thirds of SCLC cases have
already spread to other areas of the lung and other parts of the body (i.e. ED) at the time
of diagnosis (4,5,6) (seer.cancer.gov). Early diagnosis of SCLC is very challenging due to
lack of early symptoms, the aggressive nature of this disease, and the difficulty in
obtaining an accurate diagnostic needle aspiration (7). Paradoxically, SCLC shows a high
initial responsiveness to the standard chemotherapy of cisplatin/carboplatin plus
etoposide. However, most patients undergo drug-refractory relapse within months of
completing therapy. After approval of Topotecan in 1996, no new drugs were approved
by the US Food and Drug Administration (FDA) for recurrent SCLC (7,8) in over 30 years,
illustrating the need for new therapies. Very recently, the use of immune checkpoint
inhibitors has shown promise in treating SCLC, emphasizing the importance of studying
the immune system in cancer, and the potential that immune modulation might have in
SCLC treatment (9).
The fact that most SCLC patients exhibit advanced disease at diagnosis, receive
standard chemotherapy in their local clinics, and do not come to academic centers, has
limited availability of tumor tissue for study, and is one of the major hurdles in SCLC
research. This, and the complexity of this tumor type are reasons that SCLC was excluded
from The Cancer Genome Atlas (TCGA) efforts (7). Despite these limitations, insight has
been gained into the molecular characteristics and cell of origin of SCLC, as described in
the next sections.

4

1.2 Molecular Aspects of SCLC

1.2.1. Cell Line Studies

Due to the difficulty in obtaining biopsy tissue from SCLC patients, substantial
effort was devoted to the development of numerous SCLC cell lines, derived from various
sources like bone marrow, lymph nodes, pleural effusions or lung from treated and
untreated patients (10,11). These SCLC cell lines have been valuable - from the discovery
of importance of inactivation of RB1 and TP53 in the pathogenesis of SCLC, to the
identification of the frequently amplified LMYC gene, increased expression of MRP5,
activation of WNT pathway inhibitors, to the discovery of increased expression of P38
mitogen– activating protein kinase (12), the importance of the hedgehog and notch
pathways in SCLC (13), and distinguishing driver from passenger mutations. Human and
mouse SCLC cell line studies also facilitated identification of nuclear factor 1 B (NFIB)
transcription factor amplification and its role in regulation of apoptosis, senescence,
proliferation and anchorage-independent growth of fibroblasts (14). To overcome certain
limitations of cell line models, xenograft models have also been developed (15, 16, 17,
18, 19, 20) that contributed in understanding of SCLC pathology.
Both these types of models have certain limitations (12,18, 21,22,23). They do not
lead to sporadic, de novo tumorigenesis in an immunologically competent host (24) and
thus do not allow immunotherapy studies. For such studies, an intact host immune
system and vasculature are required. This can be provided by genetically engineered
mouse models.

1.2.2. SCLC Genetically Engineered Mouse Models (GEMMs)
Currently the study of multistage pathogenesis of SCLC and other high-grade NE
carcinomas of the lung is only provided by GEMMs for NE carcinoma (25,77).
A big leap in modelling SCLC in mice was achieved almost 2 decades ago, when the
laboratory of Anton Berns developed a mouse SCLC model. It was based on somatic
homozygous double knock out of homozygously floxed Rb1 and Tp53 genes by
intratracheal instillation of Adeno-Cre into the lungs (25). This is clinically relevant, since
>95% of human SCLCs have sustained mutations in P53 and RB1 (25,26). This model
showed the importance of Rb1 and Tp53 loss as a prerequisite for SCLC pathogenesis, and
thereby served to functionally validate the roles of these genes in SCLC. Most of the mice
in whom SCLC had been induced showed extrapulmonary metastases at sites such as the
bone, brain, ovaries, adrenal gland, liver, and histopathology and molecular markers
typical to human SCLC, thus mimicking the clinical situation well (27). The long period of
5

tumor latency (the time from Cre-induction to clinically detectable malignancy) permitted
in situ observation of pre-neoplastic lesions and premalignant stages at earlier time
points, something that is difficult in patients due to SCLCs late diagnosis. This model
shows spontaneous secondary mutations including amplifications of Mycl1, Mycn
and Nfib , and loss of Pten and Pik3ca, as seen in human SCLC (14, 28, 29, 30, 31, 32).
Many subsequent variations of the Berns SCLC model were made to expedite
tumor growth, such as a triple knock out that includes Nfib, encoding an oncogenic
transcription factor (29). Schaffer et al., in 2010 developed a conditional
Trp53
fl/fl
/Rb1
fl/fl
/p130
fl/fl
triple-mutant model that showed numerous detectable lesions
at 3 months after Ad-Cre infection targeted to lung epithelia. P130, a member of pocket
protein family, is a cell cycle regulator (inhibitor) reported to be lost in SCLC (33). Tumor
suppressor PTEN is another secondary mutation altered in ~20% of human SCLC (28,31).
Different zygosity of Pten loss achieved by intratracheal infection with Ad-Cre showed
different courses of lung cancer-like metastatic SCLC (in heterozygous Pten) or non-
metastatic lung adenocarcinomas with neuroendocrine differentiation (28). Co-existence
of various gene mutations mentioned above is seen in mouse and human SCLC (14, 30,
31, 32). And engineering these together intuitively will accelerate the tumor progression
and decrease latency (34). However, this may not reflect the clinical situation where these
mutations are the result of gradual selection and evolution and may not arise
simultaneously. Thus, to achieve the real complexity and heterogeneity of SCLC seen in
a stepwise cancer-genome evolution, a temporal model of tumorigenesis may need to be
developed which is proposed by McFadden (31).
The reduced mutational complexity in mouse SCLC compared to human SCLC
(likely due to absence of smoke exposure), in fact facilitates the identification of
secondary hits important for driving progression of SCLC due to decrease in mutational
“noise” in genome sequencing studies. Thus, GEMM are very informative in
understanding tumor pathway addictions and vulnerabilities to associated therapies
(28,31). One big benefit of the development of the Berns SCLC model was its use to
elucidate the cell of origin of SCLC as described in next section.

6

1.3 SCLC Cell of Origin

For a cell to give rise to cancer, it must have (or acquire through mutation) a high
proliferative potential and an intrinsic self-renewability (35). The identity of the cell of
origin is a key factor that determines the characteristics of each type of cancer. Indeed,
when the same mutation is induced into different target cells it can lead to profoundly
different types of tumors (35). Knowing which cell acquires the first genetic hit on the way
to a particular cancer type can be important for better prediction of tumour behaviour
and to tailor the therapeutic approach to a lung cancer subtype (35). Further, this might
also help to predict key molecular pathways altered in cancer and provide a better
understanding of the surrounding environment that might foster cancer growth (35).

SCLC predominately localizes to the midlevel bronchioles and airway junctions, and
commonly metastasizes to mediastinal lymph nodes, brain, liver, bone, and adrenal
glands (36). SCLC exhibits many differentiated properties of neuroendocrine cells, such
as neurosecretory-type dense core granules L-dopa decarboxylase (DDC), neuron-specific
enolase (NSE), bombesin-like immunoreactivity (BLI), and creatine kinase, brain
isoenzyme (CK-BB) (36). Thus, SCLCs are thought to originate from pulmonary
neuroendocrine cells (PNECs) or neuroendocrine progenitors (NEPs) who synthesize and
release these bioactive substances. PNECs are rare, innervated epithelial population of
cells in lungs which represent <1% of all lung cells. They act as airway sensors (37,38)
Various environmental cues are sensed, processed and responded to in the form of
alteration in
pulmonary blood
pressure, breathing
rhythm, regulation of
bronchomotor tone,
modulation of the
immune response,
regulation of lung
growth and
development due to
interaction with of
secretory substances
with nearby epithelial
cells, fibroblasts,
Fig 1: Neuroendocrine bodies (NEBs) of
the lung. Left: NEBs in the central
airway (arrows) in neonatal rat lung.
Below: NEBs show non-ciliated,
slender nuclei, and are found at airway
junctions in neonatal rabbit lung.
Adapted from Van Lommel A (2001)
7

endothelial cells, smooth muscle cells and nerve fibers (37,38). The uniqueness of these
endocrine cells lies in the tight aggregation of these cells in neuroepithelial bodies (NEB),
tactically located at the bronchoalveolar junctions, a location thought to maximize
contact with inhaled air (Fig. 1) (In contrast, solitary PNEC are found in the respiratory
epithelium of the nose, upper and lower airways (37,38)). PNEC have been implicated in
various lung developmental diseases and in the increased secretion of bioactive
substances upon lung damage. They also show hypertrophy, hyperplasia and increased
proliferation upon exposure to
nicotine from tobacco (38).
While tumor phenotype and
location suggest a PNE cell origin
for SCLC, it does not provide
convincing proof (35). There has
been an overlap of genetic,
peptide and hormone markers
between SCLC and NSCLC
(36,39). Moreover, 10% of SCLCs
show areas of squamous cell
carcinoma and adenocarcinoma
suggesting a possible origin from
a multi-/bipotent
stem/progenitor cells or that the
cancer results from the
phenotypic plasticity of
pulmonary non-neuroendocrine
cells (21). Elegant work by
Sutherland et al. however,
provided data supporting the
ability for PNE cells to become
SCLC. The authors used
adenovirus carrying Cre
recombinase (Adeno-Cre) driven
by different lung cell-specific
promoters to inactivate Tp53 and Rb1 in different lung cells in a mouse model in which
these genes were homozygously "floxed" (4). Instillation of Adeno-Cre driven by the
cytomegalovirus promoter or by a NE-specific promoter in lungs, gave rise to SCLC. When
using a surfactant protein C promoter, which deleted Tp53 and Rb1 in alveolar epithelial
AT2, Sutherland et al. observed a lower rate of neuroendocrine neoplasms (45 vs. 83%)
Fig. 2: Hypothetical Schematic of Trp53- and Rb1-Induced
SCLC.
Loss of Trp53 and Rb1 in different cell types by Ad5-Cre
viruses show that CGRP-positive NE cells (red) efficiently
gave rise to NE lung tumors, indicating NE cells are the
predominant cells of origin for SCLC.
SCLC was also detected in Ad5-SPC-Cre infected mice. (Can
be due to loss in a differentiated AT2 cell or rare SPC-
expressing progenitor cell type that has ability to
differentiate along the NE cell lineage.
Sutherland et al., 2011
8

and increased latency (4). These findings support a model in which SCLC arises from
neuroendocrine cells but does not exclude the possibility of other cell types as cell of
origin (Fig.2). In some cases, the cell of origin of SCLC might be a normal stem cell in the
lung, or an early progenitor cell or a restricted progenitor cell (35) or in some cases a
mature cell type that might acquire dedifferentiation.

9

1.4 Tumour Heterogeneity: An Integral Aspect of SCLC

Other than factors external to tumor, tumor behavior is also influenced by intra
tumoral cell variation and the cross-talk between cell types (21). A tumor can be
heterogeneous, containing cells of varied genetic and phenotypic characteristics,
proliferation rates, potential to metastasize, and differential capacity for therapeutic
response and tumor initiation/repopulation (21, 40).
Likewise, many cells are capable of limited replication in SCLC but only a very small
percentage of cells can give rise to progressive tumor colonies continuously (41). Another
evidence of heterogeneity is that admixtures of NE and non-NE cells, differing in
morphology and expression of mesenchymal markers, have been seen in primary mouse
SCLC and human SCLC tumor cell lines (21,42). Human SCLC can also show the presence
of and progression to a NSCLC phenotype after chemo-resistant recurrence of treated
SCLC or at the time of initial diagnosis (27).
The different subsets of intratumoral cells are thought to be clonally derived, divergent
subclones that are hypothesized to enhance the tumor's capacity to adapt with respect
to invasion, metastasis and resistance to therapies (21).
Given that heterogeneity appears to play a functional role in SCLC pathology with
suggested crosstalk between variant cell types, the study of the variety of pathways in
metastasizing majority cells and also in less predominant/rare variant cells is necessary.
SCLC morphological and functional heterogeneity suggests that combination therapeutic
approaches may be required to effectively treat this cancer type (42, 43). Given the poor
survival of patients and the fact that they often do not seek out academic centers, animal
models will be key to develop such multi-target drugs. Standard chemotherapy consists
of cisplatin + etoposide, but resistance usually develops soon after treatment is
completed. One promising alternative therapy is immunotherapy, based on the
observation that SCLC is associated with several autoimmune diseases and that certain
autoantibodies are associated with improved survival, as discussed in the next section.

10

1.5 SCLC and the Immune System

1.5.1. SCLC: An Immune Eliciting Cancer

SCLC has been shown to elicit T-cell as well as B-cell responses against various
antigens (Ags) expressed by the tumor. Mouse models of SCLC and human tumor biopsies
have shown T cell infiltrates and numerous different autoantibodies have been seen in
SCLC patients (43,44, Pulido thesis, unpublished results). Moreover, in a small fraction of
SCLC patients (less than 1%) a severe immune response is seen, called a Paraneoplastic
Neurologic Syndrome (PNS). This immune reaction (a non-metastatic systemic effect)
attacks parts of the brain, spinal cord, peripheral nerves or muscle, which naturally
express antigens that are ectopically expressed by tumors.
(https://www.mayoclinic.org/diseases conditions/paraneoplastic-
syndromes/symptoms-causes/syc-20355687 ). There are at least 20 known antigens in
SCLC-associated PNS and often patients show the presence of combinations of antibodies
(Abs) (44). The type of PNS depends upon the identity of the autoantigen, location of its
normal expression in the body, and its function. A few examples are: paraneoplastic
encephalomyelitis/ sensory neuronopathy (PEM/SN), in which Hu proteins (now called
ELAVL) are the target; Lambert-Eaton myasthenic syndrome (LEMS), in which voltage-
gated calcium channels (VGCC) and SOX proteins are the antigenic targets and cancer-
associated retinopathy (CAR), in which the eye protein recoverin is the antigenic target.
These cancer-associated autoimmune diseases are severely debilitating and are usually
the cause of death in PNS-affected SCLC patients (in whom SCLC is often a secondary
diagnosis) (44).
As mentioned, PEM/SN is the result of an anti-Hu/ELAVL autoimmunity in SCLC.
Hu/ELAVL proteins are homologous to the Drosophila melanogaster protein Elav
(embryonic lethal, abnormal vision) which has a role in eye and central nervous system
development of the fly (45). The mammalian ELAVL family consists of 4 RNA binding
proteins: ELAVL1, ELAVL2, ELAVL3, ELAVL4. Expression of 3 Hu/ELAVL proteins–HuB/Hel-
N1/ELAVL2, HuC/ELAVL3, and HuD/ELAVL4–is normally restricted to the nervous system
and gonads, while expression of HuR/ELAVL1 is ubiquitous (46).
All SCLC patients show ectopic expression of neuronal Hu/ELAVL proteins in their
tumors. They can show no, moderate or severe autoimmune responses to Hu/ELAVL
proteins (Fig. 3). While the majority of patients (~85%) do not carry anti-Hu/ELVAL
antibodies, ~15% carry moderate antibody levels and a small fraction (less than 1%) have
PNS and very high Ab titers. In 85% of PEM/SN patients, SCLC is present and is the likely
underlying cause for the disease.
11

The symptoms of PEM/SN often precede SCLC tumor diagnosis and most PNS patients
have small or undetectable SCLC lesions. (44, 47). If autoantibodies precede the diagnosis
of the tumor, it suggests that the cancer can still be very small when autoimmunity starts.
If true, it opens a window for improving the overall outcome in these patients, perhaps
by beginning antitumor treatment before metastasis occurs, and suggests that auto-Abs
might be used as potential molecular markers of SCLC (48). Examination of Tp53
flox/flox
;
Rb1
flox/flox
mice in which SCLC had been induced with intratracheal instillation of
Adenovirus-Cre showed an anti-HuD/ELAVL4 immune response arising while the tumor
was still small and asymptomatic (48). It is also possible that tumors are small in PEM/SN
patients due to regression of larger tumors. In that case, it would suggest the immune
system can combat SCLC. Many studies have suggested a better prognosis for SCLC
patients with PNS than with patients without one. Secondly, SCLC patients showing
moderate Ab responses can show indolent tumor growth, and presence of low-level
antibodies is correlated with improved survival (Fig. 4) and a high probability of complete
12

response to primary chemotherapy relative to HuD/ELAVL4 antibody-negative patients
(44,49). These observations are exciting because they suggest a SCLC-protective role of
an immune response against neuronal Hu/ELAVL proteins. This is the basis our hypothesis
that the anti-Hu/ELAVL response can be used to develop immunotherapy for SCLC. The
Berns GEMM model offers a promising platform to study the development and timing of
the anti-Hu/ELAVL response with respect to SCLC onset and progression as well as a
powerful tool for developing immunotherapy (44, 48, 50). Its utility has already been
demonstrated when it was used to discover the antigenic epitope of HuD/ELAVL4, as
discussed below.

1.5.2. The cause of Anti-HuD/ELAVL4 Immunogenicity

Ectopic expression of an autoantigen is usually not sufficient to elicit immune
reaction and autoimmunity. The antigen should be presented in a way that the immune
system is unaccustomed to – like abnormally increased expression levels, altered
localization, or altered post translational modification or structure. One possible
explanation for the anti-HuD/ELAVL4 response could be that the genes might carry
mutations. This was deemed unlikely when genes of the Hu/ELAVL family were examined
in SCLC tumors, including those from PEM/SN patients, and lacked mutations (51).
Another possibility is that the self-protein might carry abnormal post-translational
modifications, thereby becoming a neo-self Ag. This could initiate an immune response
which may then cross react with native protein in cells naturally expressing these (nervous
system) (44). The intracellular autoantigens in native or altered form can be exposed to
the immune system via necrosis, which is extensively seen in SCLC. A hint to a possible
post-translational modification of Hu/ELAVL proteins came from the observation that the
short unstructured N-terminal region that precedes the first of three RNA recognition
motifs (RRMs) was rich in asparagines (N) and aspartates (D). Such sequences can be
prone to isoaspartylation.
Isoaspartylation is a spontaneous protein damage that occurs under physiological
conditions (Fig. 5). It introduces kink in the polypeptide chain at asparagines or aspartates
when they are followed by small residues (Xaa) such as glycine, serine, or histidine and
are located in flexible protein regions. Isoaspartylation is enzymatically repaired by the
enzyme PCMT1 (protein-L-isoaspartate O-methyltransferase, previously known as PIMT),
which is ubiquitously expressed but is most highly expressed in the brain (52). Indeed,
mice in which this repair enzyme has been knocked out show a great increase of protein
isoaspartylation in the brain and die of severe seizures within 2 weeks of birth (78,79).
13

This suggests that isoaspartylation is particularly problematic in the brain or in neuronal
tissues.

Isoaspartylation has been linked to immune responses to self-proteins. For example,
induction of strong B-cell and T-cell responses were seen in mice immunized with an
isoaspartylated-peptide of an autoantigen of human systemic lupus erythematosus, while
the mice-maintained tolerance to the peptide in normal conformation (53). It was
demonstrated that otherwise immunologically tolerated melanoma antigen tyrosinase
related protein (TRP-2) antigen became immunogenic upon isoaspartylation. This
suggests that isoaspartylation could be a universal trigger explaining autoimmunity
initiation in a fraction of self-antigens or that isoaspartylated-prone proteins could be
used as inducers for cancer-specific immunity (54).
Fig.5: Mechanism of spontaneous isoaspartate formation and enzymatic repair in proteins.
The formation of isoaspartyl sites occurs by spontaneous dehydration of Asp-Xaa linkage or
deamidation of an Asn-Xaa linkage (dashed arrows) to produce a metastable succinimide that has a
half-life of 2–4 h at physiological pH and temperature. Succinimide undergoes a spontaneous
hydrolysis to generate an unequal mixture of two products: 15–30% of the product is a normal l-
aspartyl linkage (left solid arrow) and 70-85% forming an abnormal l-isoaspartyl-Xaa linkage (right
dashed arrow). In presence of PIMT (PCMT), isoaspartyl sites are converted to α-carboxyl-O-methyl
esters (downward pointing solid arrow). At physiological pH and temperature, the α-carboxyl-O-
methyl esters have a half-life of only a few minutes, undergoing spontaneous demethylation to
reform the same succinimide from which they originated. Repair occurs when a portion of the
succinimide spontaneously hydrolyses to generate a normal l-aspartyl site. This repair pathway
requires multiple cycles for substantial net repair, the reaction rate of repair is very high compared
with the damage reactions that generate isoaspartyl sites.
Reissner KJ et al, 2006.
14

Based on these observations, the Offringa lab hypothesized that HuD/ELAVL4 could
undergo isoaspartylation and that this could trigger the autoimmune response. Indeed,
incubation of HuD/ELAVL4 under physiological conditions was shown to induce
isoaspartylation (50, 55). An N-terminal fragment of recombinant human HuD/ELAVL4
was incubated at pH 7.4 and 37°C for up to seven days in PBS or HEPES buffer. This N-
terminal domain (the first 117 amino acids including RRM1) of HuD/ELAVL4, contains
three potential canonical isoaspartyl conversion sites (N7, N15, and D36) and five
additional N or D residues (Fig. 6).

Two experimental approaches were used to test whether this protein fragment becomes
isoaspartylated in vitro under physiological conditions: on blot labelling with PCMT, which
adds a methyl group to isoaspartyl residues, and the use of an isoaspartyl -HuD/ELAVL4-
specific affinity-purified rabbit antiserum. Both showed isoaspartylation, as well as the
accumulation of higher molecular weight complexes that are very reactive with the
isoaspartyl-HuD/ELAVL4 antisera (50). Thus, HuD/ELAVL4 appears to undergo
isoaspartylation in vitro (55).
Next, PCMT knockout mice were used to test whether isoaspartylation would occur in
vivo in the absence of the repair enzyme. Brain tissue was examined, as HuD/ELAVL4 is
naturally expressed there. The PCMT knockout mice but not wildtype mice showed strong
isoaspartylation in the brain, showing that ELAVL4 is prone to isoaspartylation in vivo (50).
To check if isoaspartylation renders the HuD/ELAVL4 N-terminal region immunogenic,
mice were immunized with isoaspartyl and native HuD/ELAVL41-117 and B- and T-cell
responses were tested. The immunisation strategy was a homologous prime-boost
regimen using native or isoaspartyl-HuD/ELAVL4 polypeptide or phosphate-buffered
saline (PBS). The boost was performed a week after priming and spleens were collected
10 days later on the day of euthanasia. The results suggested that isoaspartyl-
HuD/ELAVL4 is more immunogenic than native HuD/ELAVL4 as the former mice showed
early and strong B- and T-cell responses even before delivery of the booster dose. In vitro
Fig.6: Primary structure representation of HuD/ELAVL4 showing N terminal- RRM1-RRM2-RRM3
domains.
Canonical isoaspartyl sites (N or D followed by S, G or H in N terminal-unstructured region) are
boxed, all N and D with potential for isoaspartylation are bolded.
15

exposure of human peripheral blood cells also suggested that human T-cells respond
more strongly to isoaspartylated than native HuD/ELAVL4 (50). In subsequent work, a
former Master’s student, Prerna Sehgal carried out an immunogenicity assay with a
similar immunisation regimen but, for T-cell assays, no significant difference in T-cell
proliferation assay was observed between native versus isoaspartyl antigen immunised
mice- thus induction of immunity at the T-cell level was not strong and reproducible. The
conditions used to prepare protein and concentrations used to stimulate T-cells were
different between the two sets of experiments. Nonetheless, the trend of data suggested
that mice immunized with isoaspartylated HuD/ELAVL41-117 showed more T-cell
proliferation than other groups.
Thus, based on these previous experiments by PhD student Mario Pulido (50, 55) and
master’s student Prerna Segal (MS thesis), the aim of my thesis project was to develop an
optimized immunization protocol using healthy mice to reproduce strong B cell response
and to elicit specific T cell immunity as well, so that this could later be elaborated upon
to test this approach therapeutically in mice with SCLC. Before embarking on a specific
immunization strategy, it was important to consider different immunization regimens, as
discussed in the next section.

16

1.6 A Rationale for DNA-Based Immunization

Vaccines work by triggering 1) Pathogen Associated Molecular Patterns (PAMPs)
described as the conserved molecules or patterns present in pathogens but absent from
host cells and 2) Damage Associated Molecular Patterns (DAMPs), the endogenous
‘danger’ signals that indicate damage to host cells and tissues (also released by injection
at a site that causes tissue damage or by aluminum salt-based adjuvants) (56). Both of
these can trigger the innate immune system that further activates the adaptive immune
system (56). It is seen that DNA vaccination has the capacity to elicit antigen (Ag)-specific
cell-mediated immune response and addition of CpG ligands (DNA) with protein
immunization was shown to enhance the immune responses (57). The importance of DNA
vaccination was boosted by the realization of presence of Toll Like Receptors (TLR) on
macrophages (56).
With DNA priming, CD8+ T cells in lymphoid organs show a high frequency of
lymphatic migration marker CD62L (central/lymphoid-memory) and these cells are more
resistant to inhibitory signals (56). CD62L is a cell adhesion molecule for T cells which
when present on the cells, will slow cells down due to binding of ligands on endothelial
cells. Central memory lymphoid cells are positive for this receptor, while peripheral
memory T cells are negative for it, so they no longer stay in the lymphoid organ. CD26L
thus acts a migration marker. With DNA-based priming, CD8+ T cells in lymphoid organs
showed long term persistence and a high expansion potential in vivo. Upon optimal re-
exposure of these primed cells with peptide Ag, they lose their migration marker (i.e.
become CD62L negative), acquire inhibitory receptors, expand, and differentiate. These
peripheral memory/effector cells then survey the peripheral organs for cognate Ag.
Effector cells are prone to exhaustion due to high PD1 expression. PD1,
programmed cell death protein 1, is a surface receptor constituting an immune
checkpoint that downregulates the T cell inflammatory response by promoting apoptosis
and promoting self-tolerance. This prevents autoimmune diseases, but it can also prevent
the immune system from killing cancer cells, since many cancer cells express PDL1 -the
ligand for PD1. Hence, one can choose to do iterative priming with DNA to replenish the
central lymphoid memory T cells (56).
The transcriptome of CD8+ T cells from peptide-immunized mice showed significant
upregulation of PD-1, CTLA-4, Lag-3 and the prostaglandin receptor Ptger2 (all inhibitory
receptor genes) compared to cells from DNA-primed T cells (57). CTLA4, cytotoxic T-
lymphocyte-associated protein 4, also acts as an immune checkpoint and is upregulated
on activated T cells that transmit inhibitory signals to T cells.
A mechanism underlying the ability of a DNA vaccine to evoke both adaptive and
innate immune responses has been proposed. Nucleic acid (DNA from a DNA vaccine)
17

plays a role at the interface of the innate and adaptive immune responses to vaccines.
The innate immune system should be triggered to elicit protective adaptive immune
responses and Toll-like receptor (TLR) signaling is an emerging nucleic acid sensing
interface for this trigger (56). TLR is one of the many molecules of the innate immune
system belonging to the pattern recognition receptors (PRR) found in the blood stream
or on the cell membranes of macrophages, neutrophils, dendritic cells (Kuby
Immunology,7
th
edition). These receptors, as the group name suggests, recognize the
pattern of the combination of protein/ nucleic acid or lipid molecules like microbial
molecules never found in multicellular organisms, such as lipopolysaccharide (LPS),
double stranded RNA, flagellin, unmethylated CpGs, apoptotic host cells. Out of 10
discovered human TLRs, four TLRs (TLR3, TLR7, TLR8 and TLR9) are nucleic acid sensors
that recognize pathogen-derived nucleic acids (56). They recognize these specific foreign
patterns and activate the transcription, synthesis and secretion of cytokines that promote
inflammatory responses by bringing in more macrophages, natural killer (NK) cells, and
neutrophils that play key role in Ag presentation to T-cells thereby promoting adaptive
immune activation (56, Kuby Immunology,7
th
edition).
For DNA vaccination, virally or bacterially-derived vectors are used as the carrier
of the Ag DNA sequence. These plasmids contain CpGs peculiar to bacteria (PAMP) which
are recognized by TLRs (TLR 9) on cells as part of innate immune system (56). Thus, the
plasmid vector acts as a built-in adjuvant. The PAMPs and DAMPs can also be recognized
by PRRs on resident bystander tissues or the dendritic cells (56, 57). Bystander cells may
induce the release of tissue-derived factors like cytokines that help in dendritic cell
activation. Furthermore, some cytokines necessary for T-cell differentiation are also
provided by bystander cells which co-operate with activated dendritic cell in the secreted
cytokine milieu of the lymph nodes (56). PRR-mediated detection directly by dendritic
cells leads to their activation, maturation and migration to lymph nodes, where they
present Ag and provide co-stimulatory signals and cytokine signatures to T-cells for their
activation. The downstream effectors of DNA recognition ultimately lead to type 1
interferon (IFN) production (56). Type I IFN expression also appears to be important for
cross-presentation activity of dendritic cells (DCs), as well as for the differentiation of
T helper 1 (TH1) cells and the promotion of TH1-type isotype switching in B cells (56).
These T-cells, depending upon interaction with major histocompatibility complex (MHC)
class I or II molecules on DCs and cytokine secretions will differentiate into various sub-
classes, also activating B-cells specific for Ags (56).
The Ags expressed from the plasmid transfected into stromal cells is acquired by
DCs via the endosomal pathway and presented to TH cells via MHC II Ag-presentation:
cross presentation. While in DCs, these antigens may be directly processed and presented
on MHC class I molecules to naive CD8+ T cells (56).
18

Altogether, direct presentation, cross-presentation and bystander (stromal/ damaged
cell) cytokine production all seem to play a role in the adaptive immune response to
DNA vaccines. However, DNA vaccination alone gave modest immune responses in
various clinical trials (57) and may need an immunization boost as discussed below. Now
that the PRR and TLR roles in nucleic acid sensing and the downstream impact on the
adaptive immune system are understood, novel TLR agonists are being developed and are
currently in preclinical or clinical stages. Our studies are not aimed at investigating the
mechanism of the DNA prime + protein boost regimen but are aimed at testing its utility
in triggering an anti-HuD/ELAVL4 response. Thus, we proposed to test DNA immunization
as one of our anti-HuD/ELAVL4 immune priming strategies and later combining it with a
boost regimen as discussed below.

19

1.7 Rationale for Different Prime-Boost Strategies:
Homologous and Heterologous Regimens

Having decided that a DNA-based priming system would be worth testing, the
next consideration is the nature of the boosting step. Usually, an effective vaccine is
administered more than once spaced by a period of certain days or months or years in
the form of a prime-boost strategy. When an antigen is delivered in the same vector
for the prime and boost steps, it is called a homologous prime-boost strategy. In
contrast, a prime-boost given with unmatched vectors using the same antigen is called
a heterologous prime-boost strategy (56,57, 58).
For many cancer vaccines, homologous prime boost approaches showed
suboptimal immune responses (57). Some vaccines elicit only Ab responses, which are
not enough to eradicate cancer cells (57). Some vaccinations show temporary immunity
against a cancer antigen, but that soon dies off. Or, they give rise to non-specific
immune responses (57). Not only the quantity of Tumor Associated Antigen (TAA)-
recognizing T cells is important, but their quality and capability of eradicating cancer in
an immune-evasive environment is important, in other words, their avidity (57). Many
findings have shown that a heterologous prime-boost regimen is more immunogenic
and effective than a homologous prime-boost one (57). The full mechanism by which
this might be the case is yet to be discovered. However, there are certain hypotheses
based on experimental evidence.
A heterologous prime-boost regimen has the potential for different vectors to
work synergistically, eliciting humoral as well as cellular immune responses.
It also has diminished anti-vector immunity that prevents target epitope clearance and
immune responses against the vector, rather than the target antigen and generates
high “quality” antigen-specific CD8+ T cell responses rather than a high number, with
less co-inhibitory receptors (57). It has been hypothesized that the different priming
and boosting vectors synergize to mount an improved immune response against a
single epitope (57). Furthermore, the sequence of vector administration has been
shown to be important, with respect to specific T cell phenotype activation (Fig.7) (57).
The highest immunity was seen in a DNA prime-protein boost regimen in an experiment
that compared it with heterologous protein prime-DNA boost, homologous protein and
homologous DNA immunization (57). It appears that in heterologous DNA prime-
protein boost, the DNA-primed cells, rather than becoming exhausted by mechanism
mentioned in section 1.6, become effector T cells upon boosting with protein. Timing
and sequence is important due to differential capabilities of immune responses and the
activation of negative regulatory mechanisms based on the regimen used (57).

20

To summarize our strategy: To prime immune system by DNA vaccination is to awaken
and “train” the cells of immune system above background with full vigor (low negative
regulatory mechanisms), localize/group them in lymphoid organs (through presence of
the CD62L marker) and make them aware of a specific potential threat (an Ag). On re-
exposure by boosting, these cells now with high potential, expand and differentiate into
effector surveillors, and migrate outside the lymphoid organ (low CD62L marker) into the
systemic circulation to search for the cognate Ag elsewhere in the body (including on
cancer cells) to kill them. At some point, these effector cells can become exhausted (high
negative regulatory mechanism) and thus need to be replaced by new potential, trained
soldiers (by iterative DNA vaccination priming) (57).
Thus, based on the literature, the aim of my thesis is to test whether a
heterologous DNA-prime-protein boost immunization regimen might provide an optimal
way to generate an immune response to HuD/ELAVL4.

For the following chapters, HuD/ELAVL4 will be referred as ELAVL4.

Fig. 7: Different intensity of immune reaction providing temporal perspective on the synergy for
different sequence of prime boost regimens. It explains why the exact prime-boost sequence is
important based on the differential capability of vectors or regimens to elicit T cells with different
properties such as susceptibility to negative regulatory mechanisms (PD-1 low or PD-1 high T cells),
see text. Highest and persistent immunity is seen in a heterologous- DNA prime-protein boost
system.

Bot et al. 2010
21

Chapter 2: METHODS
2.1 Methods: From DNA Construct to DNA Vaccine

2.1.1. The Region of the ELAVL4 Protein Used for the Experiments

We chose to clone the first 117 amino acids which encompass the flexible N-
terminal region and RRM1 (Fig.8). As described earlier, ELAVL4 has 3 RRM domains with
a hinge between RRM 2 and 3. The N-terminal region is presumed to be unstructured, has
been shown to become isoaspartylated in vitro and in vivo and has been shown to be
immunogenic (50). Full-length ELAVL4 was seen to precipitate under isoaspartylation
conditions (unpublished data Offringa lab) and expression of the short N terminal region
alone (without RRM1) was not successful (unpublished data, Offringa lab). Production of
the N-terminal region fused to the globular RRM domain is the best option.

2.1.2. Preparation of DNA Constructs

DNA constructs were made using the pcDNA3.1(-) backbone which is a mammalian
expression vector in which inserted genes are expressed from the cytomegalovirus (CMV)
promoter. Since the DNA must be transcribed and translated in a eukaryote, we added
a Kozak consensus sequence upstream of the start codon: 5’-ACCATG-3’. A Kozak
sequence occurs on eukaryotic mRNA and plays a role in the initiation of translation. It is
recognized by the ribosome and the strength of Kozak sequence affects the amount of
protein produced. The Kozak sequence we used includes an ATG as initiation codon for
proper initiation of translation (59,60,61). G or A at position -3 and G at position +4 are
most common in sequences with a strong Kozak consensus ((G/A) NNATGG). The ATG was
in frame with a wildtype (WT) or modified ((mod)-which showed better secretion for
proteins chosen by Zhang et.al) IL2 signal sequence (SS) (which is 60 bp) which in turn was
M E P Q V S N G P T S N T S N G P S S N N R N C P S P M Q T G A A T D D S K T N
L I V N Y L P Q N M T Q E E F R S L F G S I G E I E S C K L V R D K I T G Q S L G Y G
F V N Y I D P K D A E K A I N T L N G L R L Q T K T I K V S Y A R P

Fig.8: Amino acid sequence of mouse ELAVL4 1-117. This includes N terminal region and RRM1. The N-
terminal region is underlined which is first 36 amino acids.

22

in frame with ELAVL4 1-117. WT and mod IL2 SS was based on experiments conducted by
Zhang et al. in 2005. Details and significance of IL2 SS is mentioned in section 3.1.
It is noted that the mouse and human ELAVL4 have a difference of 1 nucleotide at
97
th
position (in RRM1 domain) resulting in a single amino acid difference. To avoid any
effects of this difference we performed site-directed mutagenesis on our human ELAVL4 1-
117 cDNA using the NEB kit for Site Directed Mutagenesis to substitute A to G, changing a
ACC codon, encoding threonine in humans to a GCC codon, encoding alanine in mice.

2.1.3. In Vitro Testing of Secretion by IL2 SS-ELAVL4 Constructs

Our aim was to achieve secretion of ELAVL4 1-117 from cells that were transfected
by gene gun delivery of DNA in vitro. To test the ability of the DNA constructs to result in
secretion, we first developed an in vitro assay, prior to in vivo immunization. To do this,
we transfected the DNA constructs into a mammalian cell line, lung adenocarcinoma cell
line PC9 (which does not express ELAVL4) and measured protein expression and secretion.

2.1.3. a. Rationale for the In Vitro Assay Workflow
Following transfection with a construct of interest, detection of the secreted
proteins can be challenging. Different ways of detecting secretory proteins in media are
outlined (62). Secreted proteins are diluted since they enter the media that surrounds
secreting cells. Protein recovery and concentrating it can be challenging due to the
presence of salts, chemical compounds and proteins from serum in medium. In addition,
some intracellular proteins are released by dead cells which might create a false positive
result suggesting secretion. Ultrafiltration, phenol extraction, trichloro acetic acid (TCA)
precipitation are some common methods of protein concentration, as described (63). We
chose ultrafiltration for the experiment since it is theoretically suitable for my purpose
and comparatively simple. In ultrafiltration the media over the cells having secreted
protein is spun at high speed in a filter which is characterized by a molecular weight cut-
off of membrane. The protein of interest is captured in the retentate and the media and
other smaller proteins pass through the filter as flow through. This process
simultaneously concentrates and purifies the protein. Protein was quantified using Biorad
DC Assay since it is a Detergent Compatible (DC) protein detection assay. In the presence
of protein, resulting blue color is measured at 750 nm due to the chemistry between 3
solutions (A, B, C) used in the detection kit, which has Alkaline Copper Tartarate , Folin C
reagent and surfactant solution respectively (73). Because we intended to use Western
blots to detect the protein of interest we wanted to reduce the protein load in media.
Therefore, we incubated the cells in a low serum medium that had 50% less FBS than full
23

media. This dramatically decreased BSA in the media as we observed using a Ponceau
stain (data not shown here). Prior to using low serum media for transfection, we had
tested my PC9 cells (a human lung adenocarcinoma cell line) in this media for any visible
suboptimal growth rate or drastic change in cell morphology that might affect expression
and secretion of our protein. However, the cells looked normal and their growth rate was
comparable to the same cells grown in full media. Confluency was reached on the same
day as with full media grown cells, with same number of cells seeded.

2.1.3. b. Assay Workflow
A) Transfection of PC9 cells with DNA constructs in RPMI 1640 full media.
B) Change the media to OPTIMEM (low serum media) the next day.
C) Harvest media and cells at 52-53 hours after changing the media. (The best time to
harvest was determined empirically).
D) Ultrafiltration to concentrate the protein in media (from 4ml to 75 – 90 ul, a volume
that had been determined would work for detection).
E) Quantitate using the Biorad DC Assay.

12x10
4
PC9 cells in full media were plated in each well of a 6-well plate. Cells were
transfected in triplicate with each construct on the 2nd day of cell seeding in full media
(10% FBS) using FuGENE® HD Transfection Reagent: DNA ratio (2.4 ul: 1200 ng).
Instructions were followed as per the FuGENE® HD Transfection Reagent manual. After
an overnight incubation in 37
o
C, 5% CO 2 incubator, media over the cells was changed to
low serum media (OPTIMEM, which has 50% reduced serum). The transfection efficiency
was 60-70% as determined by a transfected plasmid encoding green fluorescent protein
(GFP) on a well from each plate as a positive control. Each construct was transfected in
triplicate and were carried in 6 well plate. The transfection schema is outlined in Table 1.

24

Media and cell lysates were harvested approximately after 2 and half days of
changing media. The fractions from triplicate transfections were pooled. First, media was
collected and centrifuged at 1000 rpm (Centra CL2 benchtop centrifuge) for 3 min to
separate any detached cells or cell debris to prevent clogging of the ultrafiltration
membrane and false positive signals in media from cell debris. Cells were then trypsinised
and washed with cold PBS for 3 times and the pelleted. The cell pellet obtained after the
last wash and media were stored in a -80
o
Celsius freezer until they were used.
The media was concentrated using Amicon Ultra-4 centrifugal filter 3KMWCO. The
filter was rinsed with Milli-Q water as instructed in their manual. A maximum 3.5 ml was
ultra-filtered at a time at 4
o
C at 7500 g until the volume reached 75-90ul. The retentate
Fraction of PC9
analysed
Transfected with Purpose

A] Cell lysate
1) WT SS- ELAVL41-117

2) Mod SS- ELAVL41-117

3) NO SS- ELAVL41-117

4) Untransfected
Expression (less than no SS-
ELAVL41-117 , since secreted (?))

Same/ More expression (?)

Expression more inside cells (since
not secreted)

No or only background ELAVL4
expression.
Ab validation for lysate.

B] Media from
the PC9 cells
1)WT SS- ELAVL41-117

2) Mod SS- ELAVL41-117

3) NO SS- ELAVL41-117

4) Untransfected
Secretion

Secretion (compare with WT SS-
ELAVL4)

No secretion or leaky ELAVL4 1-117 ?

Negative control for expression
and secretion. No ELAVL4
expected.
Ab Validation for media.
Table 1: Transfection and cell fraction analysis schema for in vitro testing of
secretion by IL2 SS-ELAVL4 constructs. WT IL2 SS: Wild type IL2 signal sequence.
Mod IL2 SS- Modified IL2 signal sequence.
25

was recovered by aspirating it from the tube, as instructed by the manufacturer. Cells
from cell pellet were lysed using RIPA buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS ) and filter sterilized before use. 1:1000 of
protease inhibitor cocktail in DMSO (Sigma, P8340) to buffer volume was added.
The amount of protein in cell lysate and concentrated media were quantified using
the Biorad DC Assay and 40 ug of each sample was used for Western blot analysis. Proteins
were denatured at 100
o
C for 10 minutes with an equal volume of 2X home-made Laemmli
buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and
0.125 M Tris HCl, pH 6.8). 20 ul of total sample volume was resolved on 14% SDS-PAGE
gel run at 80 V through the stacking gel and at 90 V until the ladder is resolved completely
(Approx-1.5 hours). Proteins were transferred to methanol activated Immun-Blot PVDF
membranes (Bio-Rad, Hercules, CA, USA), that had been equilibrated in cold 1X Towbin
transfer buffer (25 mM Tris, 192 mM glycine, 20 % methanol). The gel and transfer
sandwich were equilibrated in 1X cold Towbin buffer before transfer at 4
o
Celsius for 1
hour. The blots were checked for protein transfer using Ponceau S staining and later were
blocked for 1 hour in 5% milk dissolved in 1X TBST (20 mM Tris and 150 mM NaCl, 0.1%
Tween 20).
Blots were probed with anti-ELAVL4 Ab (sc-48421, Santa Cruz Biotechnology, Inc)
at a 1:1000 dilution (diluted in 5% milk dissolved in 1X TBST), incubated at 4
o
Celsius
overnight on shaker and was washed next day with 1X TBST, 3 times for 15 minutes each.
It was incubated with HRP-Conjugated anti-mouse Ab at a 1: 5000 dilution (diluted in 3%
milk dissolved in 1X TBST) for 1.5 hours at room temperature on shaker, washed with 1X
TBST, 3 times for 5 minutes each. Each blot was submerged in Super Signal West Femto
Maximum Sensitivity Substrate (Fisher Scientific) and chemiluminescence was visualized
using a Biorad ChemiDoc XRS+ at different exposures up to 2 minutes. The same blot was
stripped using stripping buffer (Thermo Scientific, catalog 21059) and re-probed with anti-
actin rabbit polyclonal Ab at a 1:5000 dilution (AAN01-A, Cytoskeleton) to check for equal
loading of cell lysate and as a negative control to check for cell debris in media. For
efficient stripping, the blots were incubated with stripping buffer for 10 min at room
temperature and then for 20 min at 37
o
C, then washed with 1X TBST 3 times, 5 minutes
each. The membrane was then blocked with 5% milk in 1X TBST and probed with anti-
actin Ab (1:1000) at 4
o
Celsius overnight and washed again as described above and
detected with HRP-Conjugated anti-rabbit Ab at a 1: 5000 dilution as described above.

26

2.1.4. DNA Delivery by Gene Gun

We chose pcDNA3.1- WT IL2 SS -N Term -RRM1 and pcDNA3.1 constructs for
immunizations-based on the results shown and discussed in section 3.1. These DNA
constructs were thus used for DNA bullet preparation.
2.1.4. a. Rationale for Choosing Epidermal DNA Delivery by Biolistic Technology
Biolistic technology/ particle bombardment has evolved over various decades,
starting from 1987, when it was first described by Sanford
and co-workers. It has the widest range of tissue type
application amongst all the available transformation or
transfection methods. A prototype included a gun
powder-driven dual-chambered device; current gene guns
are helium-driven (64).
DNA can be delivered via various routes: intra
muscular; intra venous, intra-nasal, intraperitoneal,
subcutaneous and epidermal. Each route has associated
lymphoid tissues that provide specialized and active
immune surveillance. The route and vaccination delivery
system are critical since the nature of the immune
response generated depends on several key factors, such
as the route, method, and vaccination schedule employed (65). A study compared
protective immunity triggered by DNA vaccination by delivering it via various routes (65).
Epidermal delivery by gene gun was found to be a more efficient immunization strategy
than others, due to both efficient transfection and efficient antigen presentation and
recognition (65). DNA shot into cells via a gene gun reaches the epidermis which has a
dense network of antigen presenting cells: Langerhans cells and expressed Ag are subject
to immune surveillance by the skin-associated lymphoid tissue (65). Moreover, the skin’s
easy accessibility and visualization makes it an attractive target (65,66). Another
advantage of gene gun delivery is that it requires 250–2,500 times less DNA than direct
inoculation of DNA in saline (65, 66). Gene gun delivery has been found to consistently
produce a higher antibody response rate than the intramuscular route of vaccination (Fig.
10) (66).
Fig. 9:
Gene Gun Helios
TM
by
Biorad
27

The gene gun delivers
genes without the need
for virus-based delivery
systems or transfection
via chemically based
methods which can be
toxic. Repetitive
treatments are also
feasible (64, 66). The
Biolistic Helios Gene
gun (kindly provided by
the Kast lab) is a
convenient, portable,
hand-held instrument.
This helium-driven
bombardment of DNA-
coated gold microparticles (bullets) targets a smaller area (2cm
2
) and requires lower He
pressure than its predecessor PDS-1000/He system, which used a vacuum chamber (67).
Hence, the Bio-Rad Helios gene gun system appears to be an appropriate choice to deliver
DNA into the epidermis.
We do recognize that no single delivery system will be superior over others in all
situations. Each delivery system and route lead to different expression levels, time of
continued expression, transfection efficiency, microparticle cellular penetration ability,
DNA uptake by cells or direct intracellular penetration, the purpose in question, animal
strains used, and their natural immune system (e.g. their MHC haplotype).

Fig.10:
ELISA to show the IgG antibody subclasses following pCMV-S
vaccination by intramuscular and gene gun delivery. IgG1 subclass in
green, and IgG2a subclass in grey. Helios gene gun vaccination
resulted in a predominantly IgG1 response, indicative of immune
response mediated by T helper class 2 pathway (Th2).
Andrew Conn et al. Bio-Rad Laboratories
28

2.1.4. b. Basics of Gene Gun Operation
A gene gun is used for particle bombardment-mediated gene transfer using sub-
cellular sized particles
accelerated to high velocity.
Here, the sub-cellular sized
particles were gold
microcarriers coated with DNA
(68). The DNA is coated by CaCl 2
precipitation onto the inert gold
microcarriers. Other inert
microparticles like silver,
platinum or tungsten can also
be used. To increase the stability
of DNA in this form, an
encapsulating agent,
spermidine (a polyamine), is
used. The DNA-coated gold
microparticles are the "bullets" which are coated onto tubing cut into cartridges that feed
gene gun (Fig.11) (68)
The mechanism of DNA delivery via Helios Gene Gun is based on the inertia
principle (Fig.12) (66,67, 69). The gene gun is fired by pressing the trigger button and a
high velocity stream of compressed helium gas sufficient to accelerate the microcarriers
on the inner surface of the tube. The microcarriers pass through the acceleration channel
and finally penetrate through tissue and transform the cells (69). The low atomic weight
of helium results in maximum gas expansion and its pulse acts as a particle propellant.
Past the acceleration channel, the barrel is cone shaped and thus the pressure of helium
carrying particles decreases by expansion, also making the helium shock wave less
intense. Microcarriers spread from their original 1/16" diameter to an area approximately
1/2" in diameter at the target site. The spacer also vents the helium away from target (67,
69).

Fig. 11: Gene Gun Bullet Preparation and Delivery: Bio-Rad
Process.
29

2.1.4. c. DNA Bullet Preparation
DNA Bullets were prepared according to Biorad Gene Gun Manual Instructions
(69). A mixture of 100 l of 0.05M spermidine and 25 mg gold microcarriers was vortexed
and sonicated on full power using a sonicator
for 5 s. 50 l of a 2 mg/ml plasmid solution
was added to this. 100 l 1M CaCl2 was added
dropwise while vortexing on a low setting.
The suspension was let stand for 10 min, then
spun down with a 10 s pulse in quick spin
centrifuge. Unbound DNA was washed out 3x
by adding 1 ml ethanol and pipetting out the
supernatant. After the last wash, 3 ml
polyvinylpyrrolidone (PVP)/ethanol solution
(made by adding 8.75 ul of 20 mg PVP
dissolved in 1ml 100% ethanol to 3.5 ml
ethanol) was used to resuspend the gold
microcarriers and slowly applied to the Tefzel
tubing. PVP acts as an adhesive to the tube.
Before applying gold microcarriers, Tefzel
tubing was slid into the bar holder and dried by applying 20-30 psi nitrogen at 0.5 LPM
AIR rate for 15-30 min. After gold carrier application, gold was allowed to settle (3-5 min)
and ethanol was sucked off slowly by syringe. An even distribution of gold and the
Fig.13: Components of Helios Gene gun
system
A) Tubing cutter
B) Tubing prep station
C) Helios gene gun
Fig. 12: Gene gun-operating principle O'Brien JA et.al, 2007.
Helios (Bio-Rad) gene gun uses helium to accelerate microparticles to velocities sufficient to
penetrate cells. Microprojectiles sweep down the accelerating channel once helium gas
is forced through the cartridge and spread to a larger area as it leaves the gun thus
decreasing the shock wave that may damage the tissue.

30

absence of air bubbles was checked visually before allowing the suspension to settle. The
bar holding the tube was autorotated for 5 to 10 min under nitrogen gas flow at 0.3 LPM
to dry the tube. At the end of 10 min, the tube was removed and cut into 0.5-inch
cartridges each carrying approximately 2ug DNA, assuming equal distribution. The
cartridges can be stored at 4 C for up to 8 months with a desiccator capsule in a plastic
vial.
The amount of DNA in the cartridges was checked by dissolving the DNA from 3-4
randomly sampled cartridges by adding 500 ul TE buffer and vigorously vortexing so that
gold microcarriers sweep out of the cartridge. The solution was spun down for 5 min at
high speed in a table top microcentrifuge and the DNA concentration was checked in the
supernatant by Nanodrop spectrometer Thermo Fisher, which was 2 ± 0.4 ug/cartridge.

2.1.4. d. Microcarrier Delivery into Mice
Mice were put on chemical anesthesia with an intraperitoneal injection of 100 l
of anesthesia cocktail of xylazine and ketamine containing 10 mg/kg bodyweight of
xylazine and 100 mg/kg ketamine. The abdominal area of the mice was shaved with a
small electric shaver. The barrel liner of the Helios gene gun was then held directly against
the abdominal skin, and a single DNA/microcarrier shot was delivered per mouse using a
helium pressure of 400 psi.

31

2.2 Methods: From Protein Construct to Protein Vaccine

2.2.1. Protein Production and Purification

2.2.1. a. Rationale for the Method Chosen
For the DNA immunization, we used the first 117 amino acids of ELAVL4 (the
unstructured N-terminal region and the first globular RNA recognition motif (RRM1))
cloned into a bacterial expression vector. A difference with the protein encoded on the
DNA used for the gold bullets is the presence of a C-terminal hexahistidine tag used for
protein purification. The purified recombinant protein was aged/ isoaspartylated under
physiologic conditions in vitro or frozen immediately until use.
Since our aim was to inject the recombinant protein in vivo, the antigen should be
devoid of contaminants to reduce any non-specific immune response. One such
contaminant is lipopolysaccharide (LPS), an endotoxin present in the membranes of
bacteria. It is an agonist for Toll Like Receptor 4 - myeloid differentiation factor 2 pathway
which causes the production of proinflammatory cytokines by Nuclear Factor Kappa
B (NFK-B) prevent this response in mice, we eliminated this endotoxin at the source by
using a mutant form of E. coli called Clear coli BL21(DE3) cells (Lucigen). This strain has 7
deletion mutations that result in the production of an LPS variant not recognized by TLR4
(70). Other possible contaminants include chemicals from buffers (hence protein was
dialyzed into PBS). Possible endotoxin from other sources was avoided by using fresh
unopened packs of every plastic ware and filter sterilizing the protein prep.
The different protein constructs are listed in Table 2.

32

2.2.1. b. Recombinant Protein Production
The plasmids carrying the first 117 amino acids of ELAVL4 with a C-terminal
hexahistidine tag (M409) or the hexahistidine tag alone (P85) were transformed into
chemically competent Clear Coli BL21 (DE3) cells. The bacteria already contained a
plasmid (J57) carrying 2 tRNAs rare in E. coli but common in human genes (to improve
protein yield), a p15A origin of replication (compatible with the pET plasmid) and a
chloramphenicol (Cam) resistance gene. The transformed colonies were selected on LB
agar plates containing 100 ug/ml ampicillin 34 ug/ml Cam. Colonies were picked up after
2 days (Clear coli grow slowly (70)). 100 ml of LB starter culture with same final
concentration of Amp and Cam was inoculated with 1 colony of transformed cells and
cultured overnight at 37
o
C in an incubator with vigorous shaking. This was made up to 1
L the next day with selective LB media and cultured under same conditions until an optical
density OD at 600 nm was reached between 0.6-0.7. Protein production was induced by
adding 2 ml of 0.5 M IPTG (final concentration: 1 mM) and shaking vigorously for 4 hours
at 37
o
C. The culture was centrifuged at 6000 rpm (Sorvall S-3000 rotor), 4
o
C for 10
minutes. The pellet was resuspended in 10 ml sonication buffer (10 mM Tris-HCl pH 8.0,
350 mM NaCl, 0.5% Triton X-100, 10 mM imidazole) with 1 protease inhibitor tablet
(cOmplete™, Mini, EDTA-free (Protease Inhibitor Cocktail Tablets) Sigma- 4693159001)
and sonicated (15” followed by 30” pause. Repeat 5x, total sonication time 1 min 15 sec)
Protein Construct Purpose of construct
1) Native N Term -RRM1-6X His

2) Iso-Asp N Term -RRM1- 6X His

3) RRM1-6X His

4) 6X His

Iso-Asp negative control: less
immunogenic than Iso-Asp?

Early and robust response expected in
mice than native.

Immunogen negative control: No immune
reaction expected.

ELAVL4-Negative control. Control for
possible immune reaction against histidine
tag.

Table 2: Recombinant protein constructs prepared from Clear coli cells. Eventually
constructs 1,2, 4 were used for immunisations and for initial in vivo pilot experiment,
construct 3 was not used. 6X His stands for Histidine tag added to purify protein by
IMAC principle (see below).
33

and sheared with an 18G needle (10 times) as an extra measure. The sample was then
centrifuged at 13,000 rpm in Sorvall SS34 rotor for 10 minutes at 4° Celsius and the
supernatant was saved. 400 µl of Ni-NTA Agarose beads (QIAGEN, Cat. No. 30210) were
equilibrated thrice with an equal volume of sonication buffer for 10 minutes on a rotator
at 4
o
C. After the final equilibration, the beads were centrifuged for 2 minutes at 5000
rpm (Eppendorf table top microcentrifuge) and resuspended in 200ul sonication buffer.
This was added to the saved supernatant, which was incubated for 2 hours at 4
o
C on a
rotator.
To purify the protein, the beads were washed thrice with wash buffer (sonication
buffer containing 10 Mm Imidazole and 10% glycerol) by rotating for 3 minutes during
each wash. Bound protein was then eluted for 4 times in 300ul 500mM imidazole wash
buffer by rotating with buffer for 3 minutes during each elution. All the washes and
elutions were individually collected to compare the amount of ELAVL4 protein present in
each fraction and analyzed on PAGE (Fig. 14 ). Protein quantification was determined by
Bradford assay and confirmed with SDS-PAGE and G250 Coomassie stain quantification.
Highly concentrated elutions from different preps were pooled and resolved again on 14%
SDS-PAGE gel to check for degradation (Fig. 15).

Fig.14: A representative 14% SDS-PAGE of
G250 Coomassie stained fractions from His
tagged-ELAVL4 1-117 protein purification process.
From left SN: fraction before any washes. W1-
3: washes 1-3 in wash buffer. E1-4: Elutions 1-4
in elution buffer.
34

2.2.2. Isoaspartylation of ELAVL4

Isoaspartylation of ELAVL4 1-117 was achieved by incubation of a portion of the
native ELAVL4 1-117 in Phosphate buffered saline (PBS) (Fisher(Gibco)) at 37
o
C for 7 days
and the rest of the native ELAVL4 1-117 was stored at -80
o
C. Based on previous experiments
in the lab, isoaspartylation was indicated by a slight upward shift of the band relative to
native ELAVL4 1-117 on 14% SDS-PAGE gel stained with Coomassie Brilliant Blue G250
(Fig.15).

2.2.3. Dialysis of Protein in PBS

Batches of 3 ml protein preps were dialyzed into PBS using Slide-A-Lyzer G2 dialysis
cassettes (Thermo Scientific, Cat. No. 66380) 10,000 kDa molecular weight cut-off). The
dialysis cassette membrane was hydrated in buffer before sample application and dialysis.
The instructions in the manual were followed during dialysis. Samples were dialyzed at 4
o

C in 900 ml PBS (pH 7.4) for 2 hours. This was repeated once after a change of buffer and
one more time overnight. Dialysis buffer should be at least 300 times the volume of
sample; hence 900 ml buffer was used per buffer exchange. After dialysis, the protein was
checked on 14% SDS-PAGE gel, (run at 80 V in the stacking gel and 100 V in the resolving
gel). Gels were stained with Coomassie Brilliant Blue G250 to check protein integrity and
Fig.15: A representative 14% SDS-PAGE of
G250 Coomassie stained pooled eluted
fractions of Native and IsoAsp ELAVL4
1-117
compared to different amounts of BSA.
From the gel, 10X diluted 10ul Nat.
ELAVL4
1-117
~4.8ug. Thus 1x Nat. ELAVL4
1-
117
~4.8ug/ul. 10X diluted 10ul IsoAsp
ELAVL4
1-117
~3.5ug. Thus 1X IsoAsp
ELAVL4
1-117
~3.5ug/ul.
35

the concentration was checked by the Bradford assay. Proper functioning of dialysis was
checked apriori using a chemical test on these cassettes.
2.2.4. Filter Sterilization

The dialyzed protein was filter sterilized by passing it through a 0.20 um syringe
filter of 3mm diameter (ADVENTEC disposable membrane filter of cellulose acetate) that
has less filter retention volume and thus less sample loss. Protein concentration was
checked by Bradford assay and by SDS-PAGE and also compared with known
concentrations of bovine serum albumin (BSA).

2.2.5. Emulsion Preparation by the Syringe-Extrusion Method

2.2.5.a. Rationale for Using the Syringe-Extrusion Method
An emulsion is a thermodynamically unstable mixture. Water in Oil (W/O)
emulsion is achieved by fine dispersion of the aqueous phase (in this case containing the
peptide antigen in PBS) into very small droplets in the mineral oil phase (in this case
incomplete Freund’s adjuvant (IFA)) (71). IFA is complete Freund’s adjuvant (CFA) without
killed mycobacteria. The non-metabolizable oil component is paraffin oil and mannide
monooleate (72). IFA was chosen for our study because it has less side effects such as
granulomas, lesions and inflammation at the injection site as compared to CFA (72). Also,
its use is highly recommended for boosts (72).
Emulsions provide continuous slow release of antigen at the injection site by forming an
antigen
depot that
slows Ag
dispersion
into the
surrounding
environment
and immune
tolerance
and/or its
rapid
degradation
(71). The large multimolecular emulsion aggregates promote Ag uptake by macrophages
and dendritic cells, its transport to lymphoid organs and promote a persistent immune
Fig 16: Glass syringes with 22-gauge connector reinforced with a steel bar used for
emulsion preparation by syringe extrusion method.
36

response (71). Thus, preparing a stable emulsion is very critical for vaccine safety and
efficacy.
We chose the syringe-extrusion method (Fig.16) for emulsion preparation over
vortexing and homogenizing techniques. This is because firstly, it is cheap as compared to
homogenizing equipment (71). Secondly, the Ag-release profile of emulsions is more
reliable as compared to vortexing (71). Variability in immune response induction by
vaccine prepared by vortexing was found in other studies (71). Thirdly, statistically
significantly less IFNγ secreting cells were induced by vortex technique compared to
syringe- extrusion (71).

2.2.5. b. Emulsion Preparation
150 ug protein in PBS was emulsified with a same volume of IFA such that total
volume delivered to each mouse was 100ul. Native ELAVL4 1-117 -IFA, IsoAsp- ELAVL41-117 -
IFA, 6X His-only- IFA and PBS-IFA emulsions were prepared by the syringe-extrusion
method. Under sterile conditions, the mixture
was forced through a 22-gauge reinforced
connector between two glass syringes on ice
until it became extremely hard to push (this
takes approximately 20 minutes in my hands
per emulsion). The final product looks white
and should have a mayonnaise-like
consistency. Emulsion integrity was checked
by a water-drop test: A droplet of the protein
emulsion was dropped into a tube of sterile
distilled water and checked for its lack of
disintegration/ dispersion on the surface of
the water (Fig. 17). Preparations were made
fresh on the day of injections since they are
thermodynamically unstable, and the
components will separate over time. An
excess volume of each emulsion was made
since recovery from the syringe is 50- 70 %.

Fig.17: Water drop test: A stable
Antigen depot.
IsoAsp-ELAVL4 1-117-IFA emulsion in 1:1
ratio was prepared by syringe-extrusion
method.
It did not disintegrate or disperse in
water and was highly viscous.
37

2.2.6. Subcutaneous Injection of Emulsions

Emulsion were injected into mice subcutaneously at the scruff of the neck that is
surrounded by many draining lymph nodes. Mice were anesthetized using Isoflurane
inhalant anesthesia (Fluriso). 2.5-3.0 % isoflurane was delivered in oxygen at 2.5 l/min
flow rate using isoflurane vaporizer available in USC ZNI Vivarium. The area to be injected
was disinfected with alcohol and 100 ul of each emulsion was delivered with a 25G needle.
Mice were maintained on anesthesia (through nose cone for max. 45 seconds) during
injections and were warmed by completely covering them in my hands during their
recovery.

38

2.3 Mouse Groups and Immunisation Regimen

25 mice of both sexes were randomly distributed in groups of 5 and 5 mice each were
subjected to 5 different immunization regimens as shown in Table 3.

Immunizations lasted for 26 days. The day of immune priming was D0 and a boost
was given on D14. Blood was collected to extract plasma every week (D0: Pre-bleed; D07;
D14; D21; D26(terminal bleed)) (Fig.17) . One gene gun shot of 2ug DNA was delivered in
the epidermal layer of mice as described in 2.1.4 and 100 ul volume of 150 ug protein-IFA
emulsion in 1:1 ratio was subcutaneously injected as mentioned in section 2.2.6.

Group Regimen Prime Boost
1 Heterologous prime
boost
WT IL2 ELAVL4 1-117
DNA
IsoAsp ELAVL4 1-117
2 Homologous prime boost

Iso-Asp ELAVL4 1-117 IsoAsp ELAVL4 1-117
3 Homologous prime boost Iso-Asp ELAVL4 1-117 Native ELAVL4 1-117
4 Control Heterologous
immunisation
Empty Vector 6X His protein prep
5 Control Immunisation

PBS PBS
Table 3: Groups of mice for immunizations.
39

2.4 Mouse Sample Collection

2.4.1. Blood collection and Plasma Preparation

For blood collection, mice were warmed under heating lamp for 5 minutes and
then using a sterile surgical blade were nicked on the tail. EDTA-coated tubes (pre-chilled
on wet ice) were used to collect blood in a volume not exceeding 1% of the body weight
of mice. Blood was centrifuged at 13,200 rpm at 4
o
Celsius in a microcentrifuge for 10
minutes to separate the plasma. Plasma was stored at -80
o
Celsius until use. Plasma was
analysed for Ab response to native and isoaspartylated ELAVL4 1-117 by Western blot as
described in section 2.5.1.

2.4.2. Spleen Collection

On the last day of the study (i.e. day 26), mice were euthanised according to the
USC IACUC approval procedure (primary euthanasia in a CO 2 chamber followed by cervical
dislocation as a secondary measure). Spleens were collected in sterile conditions from all
24 mice in T cell media (TCM: RPMI-1640, 10% heat-inactivated FBS, 2mM Glutamine, 20
µg/mL Pen/Strep solution, 50 M 2-ME, 1 mM Sodium Pyruvate, 0.1 mM non-essential
amino acids. FBS source used: Gemini lot A64303D (heat inactivated). Splenocytes were
extracted from these spleens and used for assessment of cell mediated immune response
by T Cell Proliferation Assay and ELISPOT assay in Beckman Center for Immune Monitoring
at USC Norris Comprehensive Cancer Center.

40

2.5 Analysis of Samples

2.5.1. Plasma Analysis

100 ng native and isoaspartylated ELAVL41-117 were blotted on PVDF membranes
as described in section 2.1.3.b. The blots were checked for equal loading by Ponceau S
staining. Blots were probed overnight on a shaker at 4
o
C with plasma samples in a 1:250
dilution in 5% milk dissolved in 1X TBST. The next day, each blot was washed 3 times with
1X TBST, each for 15 minutes. A 1:5000 dilution in 3% milk in 1X TBST solution of
secondary goat anti-mouse IgG conjugated to horseradish peroxidase (PIERCE, 1858413)
was incubated at room temperature for 1 hour. The blots were washed 3 times with 1X
TBST, each for 5 minutes. Each blot was submerged in Super Signal West Femto Maximum
Sensitivity Substrate (Fisher Scientific) and chemiluminescence was visualized using a
Biorad ChemiDoc XRS+ at different exposures up to 2 minutes.

2.5.2. ELISPOT Assay

The significance of this assay is explained in section 3.4. Single cell suspensions of
splenocytes in T cell media were made by first mashing spleen through 70 um cell
strainers, then centrifuging to get a cell pellet, lysing the RBC in this cell mixture by ACK
(Ammonium-Chloride-Potassium) lysis buffer (Lonza Cat, No.10-548E) for 3 min on ice and
then again passing through 40 um cell strainers.
The pre-wet and ethanol activated Millipore Multi-screen HTS PVDF membrane in a 96-
well plate was coated with IFN Gamma Capture Ab and incubated overnight at 4 C. The
membrane was blocked with TCM for 1 hour at 37 C at 5% CO 2. After removing the
blocking solution, 100 l of TCM was added to unstimulated control wells, 100 L of 2X
Native/ IsoAsp ELAVL4 1-117 to stimulation wells (final concentration: 50 ug/ml and 10
ug/ml), and 100 L of 2X concanavalin A (ConA) (final concentration: 1 ug/ml) to positive
control wells. 500,000 isolated splenocytes were used for stimulated and unstimulated
wells or 100,000 cells for positive control were plated and incubated at 37 C at 5% CO 2
for 20 hours. The plates were washed 5 times with 0.05% PBST using a plate washer. The
biotinylated IFN  antibody was prepared and incubated for 2 hours at room temperature
away from light. The plates were washed 5 times with 0.05% PBST. 100 ul of streptavidin-
HRP conjugate of 1:4000 dilution in PBS/0.5% BSA solution was incubated for 1 hour at
room temperature, away from light. Plates were washed 6 times with 0.05% PBST and 3
times with PBS. 100 ul of 3-amino-9-ethyl-carbazole (AEC) which is the HRP substrate was
41

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 a Zeiss KS ELISPOT reader.

2.5.3. T-Cell Proliferation Assay

The assay was performed using native (Nat) and IsoAsp ELAVL41-117 as stimuli at
50ug/ml and 10ug/ml concentration, or only medium as a negative control for stimulus
and ConA at 1ug/ml as a positive control.
250,000 Splenocytes were plated in TCM. 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 media
was replaced after 4 days with 50 µl of fresh medium having 1 µCi 3H thymidine and
incubated overnight. The next morning, cells were harvested using a cell harvester to
collect intact DNA onto 96 well filter plates. The plate was dried overnight, and the
bottom was sealed . 25 µl of scintillation fluid was added and the data was collected as
counts per min (cpm) using a Packard Top Count NXT Scintillation and Luminescence
Counter to record 3H-thymidine (beta emission).

42

2.6 Data Analysis

Western blot signals were converted into mean grey value using ImageJ Software
after creating an image from the image process > image calculator function. These mean
values equivalent to the Ab responses were graphed using Prism 6 Software.
One-way ANOVA was performed for the T cell proliferation assay and ELISPOT test
using Statistical Software Prism 6 under the column statistics feature. Statistical
significance of variance was obtained, and Tukey's multiple comparison test was
performed to know which groups showed a significant difference.

43

Chapter 3: RESULTS
3.1 Development and Testing of DNA Immunization Vectors for
ELAVL4
1-117
Secretion

Significance:
As outlined in section 1.5.2, the N-terminal region of ELAVL4 is prone to
isoaspartylation, and this modification has been found to promote immunogenicity.
Because our goal was to test combined DNA and protein-based immunization, we
prepared recombinant ELAVL4 1-117 and “aged” it (isoaspartylated it in vitro as described
in the methods section) and sought to create a DNA-based vector that would express a
protein prone to isoaspartylation. Based on the hypothesis that isoaspartylation of
ELAVL4 might occur in SCLC patients due to the release of ELAVL4 from necrotic areas of
the tumour, we reasoned that a DNA vector that would result in secreted protein might
produce the antigen we required. Thus, a first aim of our experiments was to develop a
DNA vector that would yield secreted ELAVL4. To achieve secretion, a signal peptide is
required. Signal peptides function to direct proteins to the correct compartments in the
cell. Secretory proteins have N-terminal signal peptides (ranging in length from 16 to 45
amino acids) that direct them to the endoplasmic reticulum (ER) after being expressed
(63). A typical N-terminal signal peptide consists of a positively charged (basic) domain; a
central hydrophobic core and polar region followed by a C-terminal signal peptidase
cleavage site. The peptide is cleaved during translation and the protein is released into
the ER from where it is transported outside the cells (63).
Interleukin 2 (IL2) signal sequence (SS) is commonly used for commercial protein
production and gene therapy research and various papers described its use and
functionality (74). It was also shown that altering the basic charge and hydrophobicity of
this SS improved protein secretion in vitro and in vivo (74). We thus chose 2 types of IL2
SS: Wild Type IL2 SS (WT IL2 SS) and a modified IL2 SS (Mod IL2 SS) (in which 3 amino acids
had been changed from tyrosine to arginine in the basic domain of the IL2 signal peptide
and from serine, cystine to leucine, leucine respectively in the hydrophobic domain of the
IL2 signal peptide). In the L. Zhang et al. paper, of a variety of mutants were tried, but this
modified IL2 SS showed the highest secretion increase (3.7 fold) over WT IL2 SS (74). We
thus tested wild type and modified IL2 SS- ELAVL4 1-117 constructs to try to optimize the
efficiency of secretion in vitro. Table 4 describes the DNA constructs prepared. The
plasmids 1-3 from Table 4 were transfected into PC9 cells to test their ability to secrete
the ELAVL4 1-117 polypeptide. The plasmids 1-3 from Table 4 were transfected into PC9
44

cells to test their ability to secrete the ELAVL4 1-117 polypeptide. As previously mentioned,
PC9 is a lung adenocarcinoma cell line that does not express ELAVL4.

Results:
Western Blots were performed to detect ELAVL4 1-117 expression inside the cells (by
examining cell lysates) and secreted ELAVL4 1-117 (by examining cell medium from
transfected cells) as described in Chapter 2. The size of monomeric form of ELAVL4 1-117 is
12kDa and hence expected to run on gel between 11-17kDa of the protein marker. The
aggregates seen migrate above that size. Cell lysates from cells transfected with ELAVL4 1-
117 lacking a signal sequence showed ELAVL4 expression (Fig 18). Lysates from cells
transfected with ELAVL4 1-117 carrying a wild type or modified IL2 SS showed less or no
signal, which might indicate that these cells secrete most ELAVL4 outside the cell. The
WT-IL2 SS-ELAVL41-117 construct show higher molecular weight ladder-like faint signals in
cell lysate - which may indicate ELAVL4 aggregates (the protein has been found to
aggregate/multimerize in vitro (50,55)). The actin loading control indicates approximately
equal loading across the lanes.
Examination of cell media by Western blot shows a signal for ELAVL4 for both forms
of IL2-SS transfected cells (Fig. 19). However, the construct with WT SS seems more
efficient based on its stronger signal. It must be noted that most of the signal is from
ELAVL4 aggregates and less monomeric form is seen. Cells transfected with constructs
without a signal sequence show no detectable signal in media. In media, anti-actin acts
Constructs Purpose of construct
1) pcDNA3.1- N Term -RRM1

2) pcDNA3.1- WT IL2 SS -N Term -
RRM1

3) pcDNA3.1- Mod-IL2 SS-N Term
RRM1

4) pcDNA3.1

Negative control for secretion. No signal
sequence.

Should result in ELAVL4 secretion and
perhaps an improved immune response in
the mice due to isoaspartylation following
secretion.

The modified IL2 signal sequence has been
reported to improve secretion (see text).

Negative control; no ELAVL4 insert.
Table 4: DNA constructs prepared. N Term refers to the first 36 amino acids of ELAVL4
1-117

45

as an essential negative control: no detectable signal supports the idea that the signal
seen with anti-ELAVL4 blot is not due to cell death and release of intracellular ELAVL4 but
is indeed the secreted ELAVL4 .
Based on these results, we concluded that ELAVL4 is being expressed and that the WT SS
works best for secretion. We thus chose the WT SS-ELAVL4 construct for the DNA
component of in vivo immunogenicity assay.

Fig. 19: Western blot probed
with Anti-ELAVL4 1-117 (E 1-117
here) Ab showing secretion of
ELAVL4 1-117 in media over the
transfected cells.
Untransfected cells: negative
control for secretion.
Anti-Actin: control for false
positive secretion signal due to
release of intracellular ELAVL4
1-117 by cell death.
Fig. 18: Western blot probed
with Anti- ELAVL4 1-117 (E 1-117
here) Ab showing expression
of ELAVL4 1-117 in cell lysates
from transfected cells.
Untransfected cells: negative
control for expression.
Anti-Actin is equal loading
control for cell lysate.
100 ng of D7 (aged) and D0
(unaged) lanes: positive
controls. Positive controls were
run on same gel with lysates
but were cut and probed and
imaged with Anti-HuD/ELAVL4
Ab separately to avoid over-
Antibody consumption and
over-exposure than other
lanes.
46

3.2 Immunization Strategy and Antibody Reactivity

Hypothesizing that a heterologous prime/boost strategy using a DNA vector as the
priming step and isoaspartylated protein as the boosting step would give the best
immunization, 25 mice of both sexes were randomly distributed in groups of 5 and were
subjected to the 5 different immunization regimens, as outlined in Table 3. Five groups
including control immunizations were: Heterologous WT IL2-ELAVL4 prime-IsoAsp ELAVL4
boost, homologous IsoAsp ELAVL4 prime- IsoAsp ELAVL4 boost, homologous IsoAsp
ELAVL4 prime- native ELAVL4 boost, control heterologous pcDNA3.1 (empty vector)
prime- 6X His protein boost immunization and control PBS prime-PBS boost
immunization.
We harvested blood before (D0), during and after immunization (D07, D14, D21),
and the spleens at euthanasia (D26), as indicated in Fig.20. Mice were primed on D0 and
boosted on D14 and regimens were ended on D26. 2ug DNA was delivered via gene gun
as described in Chapter 2 and the protein-based boost was done using 100 ul of a 50%
suspension of incomplete Freund's adjuvant containing 150 ug recombinant protein that
had been "aged" (undergone natural isoaspartyl conversion) for 7 days.
Plasma from all the animals from different time points was tested for anti-ELAVL4
reactivity by Western blot analysis at 1:250 plasma dilution and quantified using ImageJ
software as mentioned in Methods. To compare the Ab reactivity, the same exposure
time (33.8 sec) for HRP conjugated secondary Ab was chosen for all the mice and for the
samples from different time points from the same mouse. However, as the number of
weeks post immunization increased, signal grew stronger and blots were over exposed
using the exposure time chosen. This, and the presence of anti-ELAVL4 aggregate signals
at later time points affected the quantitation of signals which under-represent the
increase seen at later time points.
47

Mice immunized with any regimen containing ELAVL41-117 protein showed strong
anti-ELAVL4 Ab reactivity post-immunization. The 2 control groups showed no Ab
reactivity against ELAVL4 (Fig: 24,25). Antibody responses (which indicate B cell-mediated
immune responses) were higher at any given time point in Isoaspartylated prime-
Isoaspartylated ELAVL4 1-117 boost regimen than in DNA prime-Isoaspartyl ELAVL4 boost
regimen. In DNA prime-Isoasp boost, Ab response after boost was higher than the Ab
response after priming, but the responses were elevated only towards the end of the
immunizations. From these results, Isoaspartylated ELAVL41-117 seems a better boost than
native ELAVL41-117 after isoaspartylated ELAVL41-117 priming to elicit Ab responses (Fig.
22,23).
Native ELAVL4 is not naturally immunogenic. Immunogenicity observed against
native ELAVL4 on the blot could be due to a phenomenon called epitope spreading. From
this western blot data, it seems that DNA prime and protein boost is slower in eliciting Ab
response- but since we euthanize mice at day 26, we cannot conclude if this regimen
might have shown a stronger and persistent response at later time points, despite its
slower initiation. Additional experiments extending the time of the regimen and/or
including additional boosting would be needed to test for the presence of a persistent
response. Ab response against ELAVL41-117 aggregates was also seen in some plasma
samples- as indicated from the western blot signals higher than 17-25 kDa.

Fig.20: Immunization Regimen.
48

Mice numbers 8,14, 16, 17 show Anti- ELAVL41-117 Ab response even in pre-bleeds. This
can be attributed to their high background reaction which was also seen by previous lab
members- Prerna Sehgal and Mario in their blots or can be due to mix up of samples from
post immunization weeks, which seems highly unlikely.
The interpretation of plasma analysis, T cell proliferation assay and ELISPOT assay will be
discussed in integrated fashion under section 3.5.
49

50

51

52

53

54

3.3 T Cell Proliferation Assay Results

250,000 splenocytes were stimulated for proliferation by native or Isoaspartylated
ELAVL4 at 50 ug/ml and 10 ug/ml concentrations. Concanavalin A (Con A) was used as
positive control and medium alone as a negative control. Every stimulation was
performed in quadruplicate. After incubating the cells with radioactive thymidine, signals
from 3H-thymidine (beta emission) incorporated into the DNA of these proliferating cells
were detected as counts per min (cpm) using Packard Top Count NXT Scintillation and
Luminescence Counter.
Raw data showed an at least 100-fold higher responses in Con A-stimulated cells as
compared to the other stimulations and in almost all cases, average counts per minute
(cpm) from unstimulated or medium-only treated cells was higher than Ag stimulated
cells. The quadruplicates showed some variability between for the same mouse sample.
Focusing on the test immunizations and negative controls, one-way ANOVA was
performed to test for statistically significant differences between the mean cpm across
different immunization regimens, for different concentrations of in vitro Ag stimulations.
The test was statistically significant with a P value of 0.0298 for 10 ug/ml IsoAsp ELAVL4
stimulated splenocytes. Further, multiple comparison test was performed to compare
pairs that show statistical significant difference. DNA prime-Isoasp Boost v/s Control DNA
prime- 6X His only boost showed a statistically significant difference with P value=0.0132
(Fig.26 a). Fig.26 b, c, d show one-way ANOVA results across different Immunization
regimens for rest of the concentrations used for in vitro splenocyte stimulation. Statistical
significance was achieved for none but was suggestive of a trend from highest to lowest:
DNA prime-IsoAsp boost > IsoAsp prime- IsoAsp boost > IsoAsp prime-Native boost >
Controls. Interpretation of T cell proliferation assay and ELISPOT assay are mentioned
together under the section 3.5.

55

56

3.4 ELISPOT Assay Results

Significance:
ELISPOT is enzyme-linked immunospot assay. It measures the cytokine released
from in vitro stimulated T cells. i.e. it measures vaccine efficacy by measuring capacity to
elicit potent T cell responses. Basically, it is the capture and detection of immune
molecules (like cytokines or Ab or growth factors) secreted by immune cells (like T and B
cells). This is achieved by coating a capture Ab specific for molecule of interest, then
plating the cells of interest which are stimulated by Ag addition. This leads to release of
the cytokine by cells and capture by coated Ab before diffusing into the culture medium
or undergoing proteolytic degradation and is further detected by a detection Ab specific
to the molecule of interest (75).
We did interferon-gamma (IFN-γ) specific ELISPOT. IFN-γ is secreted predominantly
by natural killer (NK) and natural killer T cells (NKT) as well as by antigen-specific Th1
CD4
+
and CD8
+
effector T cells. It has immuno-regulatory, and anti-tumour properties.
IFN-γ can also increase expression of both MHC class I on the surface of cancer cells, thus
bolstering granule-mediated and death ligand-mediated cytotoxic effects of CTL. There
are various ways to quantify IFN-γ secreted by cells- ELISA, flow cytometry based-assay,
and culture assays (75). However, these assays measure the total cytokine production or
intracellular cytokine – but do not inform if few cells produce a lot of cytokine or a large
number of cells secret little cytokine, if cells are immunologically active, or if the
intracellular cytokine protein is functional (75).
In Ag specific-ELISPOT assay, we can quantify the number of cells specific to Ag
with a sensitivity of single cell detection. This assay is considered as the most sensitive
cell-based assay available. It measures the frequency of cytokine-secreting cells at the
single-cell level since one spot corresponds to a single cell (75). The intensity of the spot
is equivalent to the cytokine-secreting ability of an individual cell. This helps to detect the
rare cells that are Ag specific cytokine secreting (75). Thus, using ELISPOT was found
appropriate for our purposes since
we wish to detect cell-mediated anti-tumour activity of immune system.

Results:
ELISPOT assays, aimed at detecting interferon (IFN) gamma secreted by the stimulated
splenocytes, were carried out. HTS PVDF membrane plate was coated with IFN Gamma
Capture Ab. 500,000 isolated splenocytes were plated on this membrane and incubated
for 20 hours with stimulants (50ug/ml, 10ug/ml native and Isoaspartylated ELAVL4). Con
A was used as a positive control stimulant and T cell medium-only on unstimulated cells
as a negative control. After stimulation and washing the cells, biotinylated IFN  antibody
57

was added and attached Ab was detected with streptavidin-HRP conjugate system and
the ELISPOTs were detected using Zeiss KS ELISPOT reader.
ELISPOTs from Con A were 400-500-fold higher than the ELISPOT in other
stimulations. Splenocytes from almost all mice across different immunization regimens
showed 0 ELISPOTs for 50ug/ml ELAVL4 in vitro stimulation. This raises certain questions
detailed in the interpretation of the results section. Signals from stimulants were
normalized to the background/ negative control signals and average spots per 500,000
cells were calculated. One Way ANOVA was performed to test for statistically
significant differences between the mean positive spots across different immunization
regimens (Fig. 27) and across different in vitro stimulations (Fig 28). This test showed no
statistically significant differences between the mean positive spots across different
immunization regimens for any in vitro stimulation. It showed significant difference across
different in vitro stimulations for IsoAsp prime-IsoAsp ELAVL4 boost immunization
regimen (Fig.28b). For simplicity of comparison of stimulations in graphs, data is plotted
with Con A separately in Fig.29.
Interpretation of ELISPOT and T cell proliferation assay are mentioned together under the
section 3.5

58

59

60

61

3.5 Interpretation of Results

We hypothesized that using a DNA prime-IsoAsp boost regimen, we would achieve
a persistent and higher cell-mediated immune response than using a homologous prime
with IsoAsp protein only. If true, we should observe stronger T cell proliferation and
higher ELISPOT counts.
T cell proliferation showed significant difference between immunization groups by
isoaspartylated ELAVL4 stimulation of splenocytes at 10ug/ml concentration (p= 0.0298).
DNA prime – Isoaspartylated ELAVL4 boost regimen showed statistically significantly
higher average CPM (T cell proliferation) than Empty DNA prime- 6X His only boost control
immunization group (p= 0.0132). However, no statistically significant difference was seen
across immunization regimens for IFN gamma secretion stimulated by antigen in ELISPOT
assay. This may suggest that the observed proliferation is by non-specific (non-IFN
gamma secreting) T cells or immunologically non-effector T cells. Since across all
immunization regimens, the average cpm showed responses equal to or lower than media
treated splenocytes, proliferation signals may possibly be represented by non-T cells in
splenocytes. Thus, it is important to understand the biological significance of T cell
proliferation assay even if statistical significance is achieved. The average cpm as well as
average spots in ELISPOT are too low for a non-viral antigen to suggest a strong T cell
mediated immunity elicited by the immunization, though significant.
Moreover, lower T cell proliferation and average ELISPOTs in in vitro stimulant-
treated than in media-only treated cells and the observation that average ELISPOTs are
very low or 0 in all splenocytes treated at 50ug/ml ELAVL4 in vitro raises questions about
possible toxicity of ELAVL4 for T-cell or accessory cell viability or function. ELAVL4
aggregates in vitro and such protein aggregates may be toxic to the cells. However, the
fact that 50ug/ml treated splenocytes show proliferation, might also suggest that higher
concentrations suppress the immune responses (in this case IFN gamma secretion from T
cells or suppressed antigen presentation from antigen presenting cells). If that is the case,
these results might not be accurate T cell mediated immunogenicity against our protein
of interest.
For B cell response, Ab responses were seen for isoaspartylated ELAVL4 1-117 as well
as native ELAVL4 1-117, possibly due to epitope spreading. At any given time, point, Ab
response was higher in Isoaspartylated prime-IsoAsp boost regimen than in DNA prime-
Isoasp boost regimen. However, it is important to determine the relevance of Ab response
with regards to immunogenicity to protect from SCLC and/or as an immunotherapy. Since
this response was measured at 1:250 dilution of plasma, further dilutions of plasma will
be necessary to accurately determine the Ab titre at later timepoints, and to better
62

compare the response. In addition, as mentioned it would be important in subsequent
experiments to determine the persistence of the antibody response and if the titre is
equivalent to expected human response.
Ab responses do exhibit the phenomenon of epitope spreading, but T cell
responses are specific to the modified Ag and usually retain tolerance for the normal
protein (76). However, here we observed similar responses of proliferation assay for both
native and isoaspartylated ELAVL4 in vitro stimulations. This and variability within the
replicates observed for certain mice might raise the question about the significance of
this assay to interpret and conclude results for cell mediated immunity for this
experiment.
Data from Ab responses, T cell proliferation and ELISPOT assay was tested for
correlation with each other considering all mice and also by considering separate groups.
But, this test was not significant (p>0.05 with Pearson’s r value ranging from -1 to 1).
Another speculation could be that the MHC haplotype of our mice strain is not
suitable for optimal presentation to T cells and thus no immunogenicity is detected. MHC
binding prediction test should be performed with FVB/N mouse strain MHC Haplotype
(H2Q). However, since the antigen is thought to be isoaspartylated, containing a kink in
the protein backbone, it is unclear if prediction algorithms would provide correct data.

63

Chapter 4: FUTURE DIRECTIONS

Mouse ELAVL41-117 DNA and ELAVL4 1-117 protein were shown to be immunogenic in
mice from our experiments. We see Ab responses in all the immunization regimens and a
statistical significant difference of T cell proliferation was seen for DNA prime- protein
boost v/s control immunization strategy. Though the T cell responses were very low, they
might suggest a trend from highest to lowest: DNA prime-IsoAsp boost > IsoAsp prime-
IsoAsp boost > IsoAsp prime-Native boost > Controls. However, further experimentation
is required before arriving at a definitive conclusion and fine tuning of immunization
regimens further is necessary. It is very important to understand the biological
significance of this result.
Though isoaspartyl ELAVL4 prime- isoaspartyl ELAVL4 boost regimen showed
higher Ab response than DNA prime- protein boost regimen for this cohort, T cell results
are not yet conclusive to choose between these 2 regimens. The next step should be to
repeat this pilot study by increasing the cohort size, tweaking assays and immunization
regimens using heterologous DNA prime-isoaspartylated ELAVL4 1-117 boost and
isoaspartylated ELAVL4 prime-isoaspartylated ELAVL4 boost regimens. A few possibilities
for lower immunogenicity must be considered and optimized before proceeding further.
Previous lab members had used up to 10ug/ml Ag for in vitro stimulation of cells
and had observed lower T cell immunity. Assuming the previous concentrations used
were too low for in vitro stimulations, this time we used up to 50ug/ml of Ag for in vitro
stimulations and still received low or no counts (for ELISPOT), showing low T cell
immunity. In fact, due to the observations mentioned in section 3.5, my first question
would be to understand experimentally and through literature review if recombinant
ELAVL4 or its aggregates are toxic to the T cells or non-T cells in splenocytes in vitro- which
might be the reason why T cells proliferation is inhibited, and/or suppressed in vitro
despite a probable in vivo cell mediated immune response. If ELAVL4 aggregates are toxic,
that could also be a cause of extensive SCLC necrosis seen in patients. We cannot exclude
the possibility that ELAVL4 or its isoaspartylated version is toxic in vivo and that
suppresses its response in vivo for the dose we used.
Apart from the reliability of the in vitro assay to test T cell immunity discussed in
earlier chapter, the immunogenicity also depends on the route of Ag administration, and
the vaccination schedule employed as detailed in chapter 2. To test the possibilities like
inefficient transfection via gene gun in mice or DNA bullets getting degraded over time or
inefficient gold microparticle coating on DNA, an in vitro assay of transfection of these
constructs via same gene gun system must be performed and checked for protein
secretion. Although this would not address the penetrability of gold-DNA microcarriers
64

(through skin in vivo), since the cells grown in tissue culture are in monolayer, it would
address other concerns mentioned above. Secondly, analysis of draining lymph nodes
along with spleen might be needed. Further, it might be possible that the end point of the
study is too far from the boost day so that the T cells might not be active against our
antigen of interest. An immunization regimen with an early end point should be tested in
the repeated study and tested for T cell immunity. At the same time, to test our
hypothesis that DNA Prime- Isoaspartylated ELAVL4 boost regimen, despite a slow initial
response will show persistent T cell and Ab responses, we might analyze Ab responses
from more frequently collected plasma or extend the study by iterative priming (which
will prevent primed T cell exhaustion).
The next goal of this project would be to perform immune-protection experiment
where mice are immunized first and then induced with SCLC.
Another factor that might affect the T cell responses in vivo are the MHC haplotype
of our mice strain, that may not be suitable for optimal presentation of ELAVL4 1-117 and/or
isoaspartylated ELAVL4 1-117 fragments to T cells. MHC binding prediction tests could be
performed with FVB/N mouse strain MHC Haplotype (H2Q), though as mentioned earlier,
these may not represent/predict isoaspartylated protein results. If the haplotype is not
optimal for immunogenicity studies, then we might need to change the mouse strain,
which due to the double homozygously floxed genes would require extensive crossing.
Though our published and unpublished lab studies have suggested that this model when
induced with SCLC mimics human anti-ELAVL41-117 reactivity in SCLC and that it is suitable
to study early Ab responses in SCLC patients, its suitability for cell mediated responses
remain unclear. This lab also had shown significant difference between T cell proliferation
count from mice immunized with native v/s isoaspartylated ELAVL4, but the counts were
very low.
Furthermore, it is important to understand if the Ab titer elicited by these
immunizations would provide any immune protection against SCLC or could be useful
therapeutically for SCLC. For this, serial dilutions of plasma samples from current study
must be analyzed for ELAVL4 1-117 reactivity and highest titer should be recorded. Next, to
test immune protection of this response, a new cohort of mice including controls should
be immunized at appropriate times and then induced with SCLC and checked for tumor
progression.
As mentioned earlier, the analysis of antibody response from human SCLC patients
has hinted towards the protective nature of anti-ELAVL4 Ab response (44, 47).
Understanding its mechanism will be critical is this observation is used as the basis for the
development of immunotherapy. PEM/SN patients can have very small SCLC tumors, but
it is unclear if their immune response have prevented tumor progression or whether it
causes tumor regression. This is difficult to study in humans but might be possible to study
65

in mice is large enough numbers were used. (In humans, less than 1% of SCLC patients
develop PEM/SN.)
One possibility that has not yet been mentioned is that ELAVL4 may play a
functional role in SCLC tumor growth or maintenance, and that the immune response may
antagonize this. In vitro experiments characterizing tumor properties of SCLC cell lines
expressing ELAVL4 and adding anti-ELAVL4 antibody/ inhibiting ELAVL4 expression can be
conducted. It would be interesting to connect dots between the facts that PNECs are
innervated cells and the cell of origin of SCLC and ELAVL4 is a neuronal RNA-binding
protein. Does anti-ELAVL4 directly act on tumor cells or PNECs or perhaps the nerves
innervating PNECs? Mixed primary cultures having innervated PNECs and SCLC could be
checked for same experiment mentioned earlier in this paragraph.
While the identification of isoaspartylation as the antigenic epitope of ELAVL4 in
SCLC-associated autoimmune response was a key observation, much work remains to be
done to elucidate the mechanisms of anti-ELAVL4 reactivity in SCLC and its therapeutic
applications. The very poor survival of SCLC patients and the better survival of anti-ELAV4
responsive patients provide a strong encouragement to continue investigating.

66

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Abstract (if available)

Abstract Small Cell Lung Cancer (SCLC) is a recalcitrant pulmonary neuroendocrine carcinoma that constitutes 15-20% of all lung cancer cases. It is the second most aggressive cancer that has already metastasized during diagnosis. Strikingly, SCLC shows high initial chemo-responsiveness to standard chemotherapy of Cisplatin/Etoposide in ∼60%-80% of cases, but eventually, show chemoresistance and relapse in almost all cases. The overall 5-year survival rate is less than 5%. Moreover, past four decades saw virtually no change in first-line treatment due to invariable hurdles in translational research like limited availability of patient biopsy material, exclusion from TCGA, lack of early detection method and tumor heterogeneity itself. A new approach to therapy is urgently needed. ❧ One of the hallmarks of SCLC is that it is an immune eliciting cancer and patients show combination of antibody (Ab) responses against various antigens (Ag) expressed on SCLC tumor. One such neuronal protein called ELAVL4 is ectopically expressed by all SCLC tumors and patients show anti-ELAVL4 Ab responses in various magnitudes. Less than one percent of all SCLC cases show very high titer anti-ELAVL4 response such that it attacks neurons, where the Ag is normally expressed and results in a neurologic autoimmune disease called Paraneoplastic encephalomyelitis/Sensory Neuronopathy (PEM/SN). Interestingly, patients suffering from PEM/SN have SCLC as a secondary diagnosis hinting towards a protective nature of anti-ELAVL4 Ab response against SCLC tumor growth. Many studies have shown that SCLC patients with moderate anti-ELAVL4 Ab responses were associated with indolent tumor growth, improved survival and a high probability of complete response to primary chemotherapy relative to anti-ELAVL4 Ab-negative patients. Furthermore, ectopic expression alone may not be the cause of immunogenic nature of ELAVL4. This protein undergoes spontaneous isoaspartylation at physiological conditions at its N terminal unstructured region which has canonical isoaspartylation sites (aspartate and asparagine residues) and that introduces a kink in protein. This common post-translational modification also found on other proteins is repaired in brain due to the presence of enzyme Protein-L-isoaspartate O-methyltransferase (PIMT) but is not repaired in these tumors which renders ELAVL4 immunogenic. ❧ Thus, the goal of this project was to develop an optimized immunization protocol using healthy mice to yield strong B cell and specific T cell response against isoaspartylated ELAVL4 by DNA and protein immunization, so that this could later be elaborated upon to test the approach therapeutically in mice with SCLC. Various immunizations that include isoaspartylated ELAVL4 in the form of protein and DNA were tested. We hypothesized that heterologous prime-boost regimen which delivers the same antigen in different vectors was more effective in eliciting B and T cell responses. In all our immunization regimens, we observed Anti-ELAVL4 Ab responses with homologous isoaspartylated ELAVL4 protein immunization showing highest response at any given time. But no significant T cell responses were observed. While not only ELAVL4 protein immunization but heterologous DNA prime-protein boost regimen was found to be immunogenic, results raise various questions and warrant further experiments. Much work remains to be done to elucidate the mechanistic role underlying protective nature of anti-ELAVL4 response and its therapeutic applications. The very poor survival of SCLC patients and the better survival of anti-ELAV4 responsive patients provide a strong encouragement to continue investigating.

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Asset Metadata

Creator Lotlikar, Madhura Sachindra (author)

Core Title Development of immunotherapy for small cell lung cancer using iso-aspartylated antigen

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 02/19/2019

Defense Date 01/22/2019

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

Tag DNA immunization,ELAVL4/HuD,Helios gene gun,heterologous prime-boost regimen,homologous prime-boost regimen,immunotherapy,isoaspartylation,OAI-PMH Harvest,protein immunization,small cell lung cancer

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Offringa, Ite A. (committee chair), Kast, Martin (committee member), Zhou, Beiyun (committee member)

Creator Email lotlikar@usc.edu,madhuralotlikar2@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-123328

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Legacy Identifier etd-LotlikarMa-7084.pdf

Dmrecord 123328

Document Type Thesis

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Rights Lotlikar, Madhura Sachindra

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

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