Genetic Engineering of T Lymphocytes for Cancer

Immunotherapy

Optimisation of Gene Transfer

Thesis for M. Sc. degree in laboratory science within the programme of Biomedical
Laboratory Sciences
June 2006

Camilla Lindqvist

Supervisor: Angelica Loskog, Ph.D.

Division of Clinical Immunology, Uppsala University
Abstract

T lymphocytes can be rendered specific against a wide range of antigens by the genetic
transfer of a chimeric receptor, a fusion between the antigen-binding domain of an antibody
and the signalling domain of a T cell receptor. The use of such chimeric T lymphocytes has
shown promising results for cancer therapy. Previous experiments in our laboratory have
shown low rates of gene transfer using retroviral vectors. In this study, investigations have
been done to increase the number of genetically modified cells. Different enhancers such as
PLL and polybrene have previously been used in combination with retroviral transduction.
The optimal retroviral protocol in this study showed to be the use of retrovectors produced
with twice the normal concentration of the plasmids encoding env and gag-pol rather than the
use of the enhancers. A 6-day pre stimulation of T lymphocytes prior transduction together
with a centrifugation step increased the rate of modified cells even further. Alternative
approaches of gene transfer were also investigated, including plasmid transfection and
adenoviral transduction. While transfection protocols yielded low numbers of modified cells,
adenoviral vectors showed the highest rate of gene transfer.

Keywords

Chimeric receptor, tumour, retroviral transduction, plasmid transfection, retroviral enhancers

Sammanfattning

Cancer r den sjukdom som idag, efter hjrt-krl-sjukdomar, krver flest ddsfall i i-lnder.
Som en alternativ behandlingsmetod mot cancer pgr just nu forskning om genetiskt
frbttrade immunceller, s.k. chimra T lymfocyter, skulle kunna anvndas fr att dda
tumrceller. De chimra cellerna r utrustade med en konstgjord receptor som r en fusion av
en antikropp och en signalkedja. Det gr att cellerna kan riktas mot ett brett urval av
cancertyper. Att f cellerna att ta upp generna som behvs fr den konstgjorda receptorn har
visats sig vara problematiskt. Den hr studien har drfr som ml att frbttra cellernas
frmga att ta upp gener. Fr detta har vi anvnt oss av retrovirus- och adenovirus-system
tillsammans med frsk att f cellerna att spontant ta upp generna, sk. plasmid-transfektion.
Studien har visat att de bda virussystemen ger hgst antal modifierade celler. Olika
substanser som tidigare har visat sig frhja graden av gentillfrsel har testats, men vr studie
har visat att tillverkningen av virusvektorerna har strre pverkan p resultaten n ngon av
de olika hjlpmedlen.

2
Abbreviations

APC antigen presenting cell

CTL cytotoxic T lymphocyte
FACS fluorescent-activated cell sorting
FBS foetal bovine serum
GFP green fluorescent protein
ITAM immunoreceptor tyrosine-based activation motif
LTR long terminal repeat
Luc Luciferase
MHC major histocompatibility complex
PBMC peripheral blood mononuclear cell
PBS phosphate buffered saline
PEST penicillin-streptomycin
PLL poly-L-lysine
Polybrene 1,5-dimethyl-1,5diazaundecamethylene polymethobromide
RN retronectin
TCR T cell receptor

Introduction

Cancer is one of todays most prevalent diseases with almost 50 000 cases being diagnosed in
Sweden every year 1. It is the second most common cause of death (after ischemic heart
disease) in developed countries, accounting for about 23% of all mortality2. The risk of
developing cancer increases by age and for individuals over 75 the risk is almost 30%
(statistics for Sweden)1. There are about 200 different types of cancers, where prostate cancer
and breast cancer is the most common in men and women, respectively3.

Cancer arises from the uncontrolled proliferation of transformed endogenous cells. The
growth of malignant tumours is largely dependent on the tumour cells proliferative capacity
and the ability of these cells to invade surrounding tissues and metastasise to distant sites.
Each neoplastic cell has an alteration in its genome that is responsible for its abnormal
growth. The four main genetic mechanisms that are believed to transform cells into neoplasms
are expression of oncogenes (stimulates growth), loss of activity of tumour suppressor genes
or anti-oncogenes (inhibit cell growth), over-expression of genes whose products prevent
normal cell death and/or loss of activity of genes that under normal circumstances repair
damaged DNA. Cells normally need to have more than one of these genetical errors to be
transformed into cancer cells.
Many tumour cells express antigens on their surface that triggers the immune system. They
can either be tumour specific, i.e. they are only expressed on the cancer cells, or are tumour
associated, i.e. the antigens are found on other cells than the tumour, but are over-expressed
by the cancer cells. These antigens enable the immune system to recognize the tumour while
ignoring adjacent normal cells. The principal mechanism of tumour immunity is killing of
tumour cells by CD8+ cytotoxic T lymphocytes (CTLs). CTLs recognize antigens presented
to them by the class I major histocompatibility complex (MHC) molecule on antigen
presenting cells (APCs) such as dentritic cells (DCs). However, the DCs need to be activated
in order to stimulate the CTLs. Upon DC activation they express MHC I together with co
stimulatory molecules. In absence of co stimulation, the CTLs become anergic or inactive.
Since the tumour cells may down regulate MHC I or have other defects in the antigen

3
presentation pathway, they may be poor targets for CTLs. Except down regulation of antigen
presentation they may secrete growth factors for T regulatory cells and/or secrete inhibitory
proteins. T regulatory cells and inhibitory proteins can directly suppress the antitumour
reactivity of CTLs.

To defeat cancer, all the malignant tumour cells need to be eliminated. There are curative
treatments for some of the different cancer types, providing the disease is detected early. The
relative five-year-survival in Sweden are approximately 60%3. Surgery is the most commonly
used therapy against solid tumours. It can be used as a single therapy or in combination with
radiation therapy and/or chemotherapy. Most patients that are cured from cancer disease are
so by this method. Even though surgery treats many patients, it is an invasive procedure that
always involves a risk. Furthermore, healthy tissues surrounding the tumour usually need to
be removed as well.
Radiation therapy uses a method were X-rays creates free radicals in the irradiated tissue.
This damages the DNA of cells and hence their ability to replicate. Tumour cells have a less
efficient repair system than healthy cells, which often results in the death of the tumour, while
healthy cells may survive. Even though this therapy seems to be efficient for killing cancer
cells, it requires high specificity. Furthermore, cancer therapy gives severe side effects, some
of which are alopecia (hair loss), skin reactions and nausea (early side effects). Among some
of the later side effects are secondary malignancies and impaired fertility or sterility at worst.
Chemotherapy functions by disturbing the cell replication by targeting the DNA and/or
transport systems within the cell. It is most efficient on rapidly dividing cells, which is well
concordant with cancer cells. Unfortunately, many of the bodies own cells are also rapidly
dividing, e.g. blood cells, hair and epithelia, and the chemotherapeutic drugs cannot
distinguish between healthy cells and cancer cells. This results in side effects such as
alopecia, nausea, anaemia, thrombocytopenia and also an increased sensitivity for infections
due to the decrease of leukocytes.

One of the biggest problems with the current therapies against cancer is that they are not
specific enough. Both radiation therapy and chemotherapy affects healthy cells as well as
cancer cells, which leads to the many side effects that can be seen within these therapies.
An alternative approach for cancer therapy is to re-educate the bodys own immune system
to defeat cancer cells. Immunotherapy may decrease the side effects due to its higher
specificity.
Several strategies have been evaluated within this field and there are several novel therapies
that are currently being clinically explored. Among these are e.g. adoptive T cell therapy
where a biopsy from the patients tumour are excised and the tumour infiltrating lymphocytes
(TILs) are extracted. Cells with high avidity to the tumour are selected and expanded. For the
tumour specific cells to be effective, the patient need to undergo immunosupression before
being re-infused with high numbers of the tumour specific cells4. The immunosupression
creates a space in the lymphocyte population that allows the transferred cells to proliferate4.
Furthermore, it has been shown that there are a subpopulation of CD4+ cells, CD4+ CD25+
(and according to more recent findings FOXp3+ 5) that are the so called T regulatory cells and
these are believed to protect the body against self reactive immune responses6. The adoptive
transfer of expanded autologous tumour reactive T lymphocytes has been successful in the
regression of e.g. metastatic melanoma. To enhance these effects the T regulatory cells need
to be depleted prior therapy. This can be done by a general immunosupression drug, but as a
side effect of this the patients developed different autoimmune diseases7. For this treatment to
be effective, the patient also needs to receive high doses of IL-2, the principal growth factor
for T cells, which is known to be toxic in high doses8. Furthermore, T regulatory cells also

4
thrive upon IL-2 administration9. Instead of finding means to deplete T regulatory cells the
cytotoxic T lymphocytes can be genetically engineered to resist the negative actions of the T
regulatory cells.
The first signal for T lymphocyte activation is provided to the cell by antigen recognition
by the T cell receptor (TCR) binding to the MHC molecule. The second signal is from
binding of costimulatory molecules which function together with the TCR to activate the T
cell. The best-characterized costimulatory molecule is CD28, which is located at the T cell
surface and binds to B7-1 and B7-2 on the APCs. Binding of CD28 enhances the production
of cytokines such as IL-2 that is the principal growth factor for T lymphocytes. Co
stimulation is also necessary for inhibiting T cell anergy.

CD8 TCR

CD28

CD3 ITAM

The T cell receptor (TCR) for CD8 consists of the and chain of TCR and the , , and chain of the CD3
molecule, here shown together with the surface molecule CD8 and the costimulatory molecule CD28.

When a T cell receives both antigen recognition and costimulation several surface receptors
and intracellular signal molecules form an immunological synapse around the TCR. Lck is a
tyrosine kinase that is associated with the cytoplasmatic tail of the CD4 or CD8 molecule.
When the immunological synapse forms, Lck will be brought into place near the
cytoplasmatic tail of the CD3 molecule. CD3 consists of 3 chains, of which the chain
contains the most immunoreceptor tyrosine-based activation motifs (ITAMs). Lck
phosphorylates the tyrosines in the ITAMs, creating a docking site for another tyrosine kinase
called ZAP-70. ZAP-70 becomes phosphorylated by Lck and can then phosphorylate a
number of other cytoplasmic signalling molecules including linker of activation of T
lymphocytes (LAT). This is the beginning of the activation of T lymphocytes that ultimately
leads to activation of transcription factors that regulate gene expression of e.g. IL-2.

5
 APC

MHC I
peptide
CD 8
TCR

Lck
P
P ZAP-70
P
T cell P

Activation of the T cell receptor is initiated by binding of the TCR to MCH which leads to phosphorylation of
CD3 molecule. This leads to further phosphorylation and finally results in regulation of transcription of e.g. the
growth factor IL-2.

By using artificial receptors, so called chimeric receptors, a T cell can be rendered specific for
a wide range of antigens. A chimeric receptor is a fusion between an antibody and a TCR,
where the light and heavy chain from the variable region of an antibody, or the single chain
variable fragment (ScFv), is used for antigen-recognition. The ScFv is fused to a hinge, also
derived from an antibody. This provides the receptor with flexibility and by this improving
antigen recognition. The hinge is then connected to the intracellular signalling domain CD3-.
A signal sequence also needs to be added, to ensure that the receptor is transported to the
plasma membrane after it is synthesised. This signal sequence is spliced off after the receptor
is in place.
After studies showing that chimeric T cells was poorly efficient and failed to expand in
vivo10 several studies have been done using the addition of different parts of the costimulatory
surface molecule CD28. This have been shown to enhance the chimeric T cells proliferation
and effectiveness in vitro11,12 and in vivo11,13,14. By adding the intracellular part of the CD28,
chimeric T cells have also shown less sensitivity for inhibition by T regulatory cells
(unpublished results, Loskog et al, Leukaemia 2006). Furthermore, studies have shown that
the costimulation provided by CD28 is sufficient for IL-2 production by the cell itself upon
antigen-recognition12. Patients receiving chimeric T lymphocytes may not have to be
immunosupressed or administered with high doses of IL-2 for the therapy to be effective. This
will probably diminish the occurrence of autoimmune diseases, since the T regulatory cells
are left active.

6
 VH Antibody domain (ScFv)

VL
Transmembrane domain (hinge)

TCR domain (CD3-)

iCD28 domain
The chimeric receptor with the antigen recognising ScFv connected to a hinge, both derived from an antibody.
CD3- and the intracellular part of CD28 originate from the TCR.

The genetic engineering of T lymphocytes has proven to be difficult. The method of choice
for transducing T lymphocytes is currently by retroviral transduction12,13.
Retroviruses are enveloped, positive-stranded RNA viruses that integrates into the hosts
genome. The genome of a simple retrovirus contains three major genes that encodes for
structure and enzymatic proteins. These involve gag (group-specific antigen) that encodes
capside proteins, pol that are the genes for polymerase, protease and integrase and finally env
(envelope) that encode glycoproteins. At each end of the genome there are long terminal
repeat (LTR) sequences that contain promoters, enhancers and genes for binding different
cellular transcription factors.
The polyprotein encoded by the env gene are after translation cleaved into glycoproteins.
The glycoproteins form spikes of different sizes that are located on the capside. The larger
glycoproteins binds to cell surface receptors and the smaller ones promote cell-cell fusion.
The virus infects the host cell by binding of the larger spikes to the CD4 or CD8 surface
receptor protein and chemokine receptors. The envelope then fuses with the plasma
membrane of the cell. Once inside the cell, a copy of the RNA genome is made in the form of
a negative-strand DNA that is later transcripted into complementary DNA (cDNA). The
RNA genome is degraded by reverse transcriptase that also acts as a ribonuclease. During the
synthesis of the provirus (cDNA), sequences from each end of the genome are duplicated,
bringing together both LTRs, which is necessary for integration. This also creates a sequence
with promoters and enhancers that regulate the transcription. The DNA is then delivered into
the nucleus of the host cell where it is spliced into the chromosome with the aid of integrase.
Once inside the chromosome, the viral DNA will be transcribed as a cellular gene.
In this report, a three-plasmid system has been used, to produce retroviral vectors. One
plasmid contains the genes for gag and pol, the second plasmid the gene for env and the third
plasmid carries the retroviral backbone (LTR) and the gene(s) that are going to be
incorporated into the genome of the T lymphocyte. They will be transfected into cell lines,
which will express the different proteins needed for virus production (env, gag and pol) and
ensemble nonreplication competent retroviral vectors containing the chimeric gene. Since the
retroviral genes are delivered in three separate plasmids, risk of replication competent
recombinants is minimised. The viral vectors obtained can therefore be used to integrate
desired genes into the cell genome without facing the risk of retroviral infection.

In this report, both transfection and retroviral transduction were used for gene transfer. To
ease the analysis of transduction/transfection rate, the T lymphocytes were genetically
engineered with a Luciferase/ green fluorescent protein (GFP) bicistronic vector instead of the
genes for the chimeric receptor. GFP gene expression can easily be analysed using

7
fluorescence-activated cell sorting (FACS). Different transduction enhancers were also
investigated for an optimal transfer rate. Polybrene and retronectin (RN) are both widely
accepted retrovector transduction enhancers. Polybrene has previously been used to transduce
adherently growing cells13 while RN is evaluated in T cells that grow in suspension. However,
RN is expensive and a new technique is warranted. Poly-L-lysine (PLL) is a poly-cation that
glue negatively charged agents. PLL is currently used in experimental murine models to
help adhesion of adenoviral vectors to epithelial cell in vivo15. In this project, we examined if
it could be used to enhance the retroviral transduction of T lymphocytes.
Other factors investigated in this report were the affect of pre-stimulation time before
transduction, if the retrovector could be further improved by changing the production protocol
and if multiple transductions could increase the levels of transduced cells.
Finally it was investigated if different cell types are equally sensitive for transfection and if
different concentration of plasmids had an effect on the transfection rate.

Methods

Cell culture

Human embryonic kidney cells, 293T (ATCC, Manassas, VA USA), were cultured in DMEM
medium supplemented with 10% foetal bovine serum (FBS), 1% Penicillin-Streptomycin
(PEST) and 0.1% sodium pyruvate (all from Invitrogen, Paisley, Scotland) in 37C, 5.0%
CO2.

Retroviral production

Retroviruses were produced using a three-plasmid system. The MLV-Luc/GFP plasmid

(3.75g) containing luciferase and GFP (gift from Gianpietro Dotti, Baylor College of
Medicine, Houston, TX, USA) was co-transfected into 293T cells with a Peg Pam plasmid
(2.5g) containing the viral gag-pol (gift from Elio Vanin, Baylor College of Medicine,
Houston, TX, USA) and a RDF plasmid (3.75g) containing the viral envelope RD114 (gift
from Mary Collins, London, UK) together with Gene Juice (Novagen Inc., WI, USA). Cells
were cultured in supplemented DMEM and the virus-containing supernatant was harvested
after 48 and 72 hours and stored in 80C.

Isolation of human PBMC

Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats,
provided by the Uppsala University Hospital blood central, by ficoll-paque gradient
centrifugation (Amersham Biosciences, Uppsala, Sweden). Cells were stored in RPMI-
medium supplemented with 20% FBS and 10% DMSO (all from Invitrogen, Paisley,
Scotland) in 80C.

Stimulation of human T lymphocytes

PBMCs (1x107) from healthy donors were stimulated with 10g OKT-3 (Cilag A6 Int., Zug,
Switzerland) and 720U IL-2 (Apoteket, Uppsala University Hospital, Uppsala, Sweden) in

8
10ml RPMI-medium supplemented with 10% FBS, 1% PEST, 1% -mercaptoethanol and
0.1% sodium pyruvate (all from Invitrogen, Paisley, Scotland). Cells were cultured for 3 days
before being used for transduction/transfection.

Retroviral transduction of human T lymphocytes

Different strategies were used to enhance the transduction of stimulated PBMCs. Standard
protocol: 25g Retronectin (RN) (Takara Shuzo CO., Japan) in 1ml PBS was coated onto a
plate the day before transduction and incubated at 4C overnight. On the day of transduction
the RN was replaced with 1ml retroviral vector (retrovector) and incubated for 45 minutes in
37C. The vector was then replaced with 2ml vector containing 0.7 million stimulated T
lymphocytes, 1ml RPMI-medium and 300U IL-2. The cells were analyzed by FACS at day 4.
Soluble RN: 0.7 million cells were mixed with 3 ml retrovector in a plate. RN and 300U of
IL-2 was added and cells were cultured for 4 days before being analyzed by FACS.
Polybrene: 0.7 million cells were mixed with 3ml retrovirus, 10g polybrene (SIGMA-
ALDRICH Inc, Saint Louis, MO, USA) and 300U IL-2 in a tube. The sample was centrifuged
at 100 x g for 5 minutes and cultured overnight in 37C before being transferred to a plate.
Cells were analyzed as above. Poly-L-lysine (PLL) plate: Cells were transduced as in the
standard protocol except PLL (SIGMA-ALDRICH Inc, Saint Louis, MO, USA) was used
to coat the plate instead of RN. Soluble PLL: 0.7 million cells were incubated in 0.1mg PLL
for 5 minutes. The sample was then centrifuged at 100 x g for 5 minutes and the PLL was
replaced with 3ml retrovirus and 300U IL-2. The sample was centrifuged again and incubated
overnight in 37C before being transferred to a plate. Cells were analyzed by FACS as above.
env,gagpol x2: Conducted as the standard protocol with the difference that the retrovectors
were produced with twice as much of the retroviral plasmids env and gag-pol.
Duration of stimulation prior transduction: Stimulated T lymphocytes were cultured
various days before retroviral transduction. At every second day the stimulated T cells got
150U IL-2 and at day 8 they received 1ml fresh medium. Cells were transduced on either day
2, 4, 6, 8, 10 or 12 after stimulation. Cell proliferation and viability were analyzed on the day
of transduction. The env,gagpol x2 protocol was used, with the exception that the cells were
incubated with 2ml virus for approximately 20 minutes before being transferred onto the pre-
coated plate. Cells were analyzed by FACS on day 4 post transduction.
Optimization of plasmid quantities: Human T lymphocytes were stimulated for 6 days
before being transduced with retroviruses manufactured with different concentrations of env
and gag-pol. Retroviruses were produced to contain 1x, 2 x, 4x or 8x env and gag-pol in
relation to MLV-Luc/GFP. Cells were transduced according to the standard protocol but
with the extra 20 minutes incubation of the cells with the retrovirus. The experiment was done
in duplicate with one of the plates being centrifuged for 1000 x g for one hour. Cells were
analyzed by FACS on day 4.
Multiple transductions: Cells were divided into 4 groups and transduced according to the
standard protocol. Group 1 was transduced once (day 0), group 2 twice, group 3 three times
and group 4 four times. Cells were analyzed by FACS on day 4 post transduction.

Transfection of human T lymphocytes

Purified CD8+ cells (1x106) from human PBMCs (purified by the separation columns and
CD8 beads from Miltenyi Biotec, GMVH Bergisch Gladbach, Germany) and OKT-3/IL-2
stimulated PBMCs (1x106) were transfected with 3.75g MLV-Luc/GFP with Gene Juice at

9
37C. The cells were cultured for 4 days in RPMI-medium with 240U IL-2 (OKT-3/IL-2
stimulated cells) or 3g OKT-3 and 240U IL-2 (CD8+ cells) before FACS analysis.
Transfection with different concentrations of MLV-Luc/GFP: OKT-3/IL-2 stimulated
PBMCs (1x106) was transfected as above but with different concentrations of the MLV-
Luc/GFP plasmid; 1x (3.75g), 2x (7.50g), 4x (15.00g) and 8x (30.00g). The cells were
cultured for 4 days before analysis by FACS.

Results

Transduction enhancers

The results show that different aids such as PLL and polybrene did not have effect on the
transduction rate of T lymphocytes. RN was used both coated on the plate and soluble in the
cell/virus mixture, as was PLL. Treating cells with soluble PLL resulted in a slight increase of
the transduction rate, but as a consequence the cell viability was under 4% (compared to
approximately 80% of the other groups). By increasing the rate of env and gag-pol plasmids
in relation to the MLV-Luc/GFP plasmid a better transduction level was obtained (Figure 1).

12

10
% GFP+ cells

8
soluble
6
plate
4

0
RN polybrene PLL env,gagpol
x2

Figure 1 RN, polybrene and PLL were used to aid the transduction of the stimulated T cells. RN and PLL were
used both as soluble and coated on plates. env,gagpol x2 shows that a retrovector that is produced with twice as
much of the gag-pol and envelop plasmid is far better than any of the aids.

Pre-coating of plates with RN were continued to be used, since no better results were obtained
with any of the other aids. The retrovectors containing twice the concentration of env and
gag-pol showed an impressive increase of GFP+ cells.
Experiments were also done using all of the three viral plasmids in twice their normal
concentration. However, these retrovectors resulted in having an even lower transduction rate
than the initial ones.

The role of stimulation duration prior transduction

The number of days that OKT-3/IL-2 stimulated PBMCs was cultured before transduction
showed to have effect on transduction capacity. Here the cells were cultured for 2, 4, 6, 8 and
12 days prior transduction. Considering time, transduction rate and viability, the most

10
efficient day for transduction was day 6 post stimulation, even though similar transduction
levels were seen at day 8 and 10 (see Figure 2).

a) total number of living cells

20

15
(millions)

10

0
d2 d4 d6 d8 d10 d12
Time (days)

b)

74
72
70
viability (%)

68
66
64
62
60
d2 d4 d6 d8 d10 d12
Time (days)

c)

16
14
12
% GFP+ cells

10
8
6
4
2
0
d2 d4 d6 d8 d10 d12
Time (days)

Figure 2 Stimulated T lymphocytes were transduced at various days after stimulation. Figure 2a shows the
proliferation, 2b the viability of cells prior transduction and 2c the transduction rate of the cells. At day 8 the
cells received fresh medium

11
For the following experiments, stimulated T lymphocytes were cultured for 6 days before
transduction. The cell viability at the day of analysis (4 days after transduction) was
approximately the same (results not shown), disregarding duration of prior stimulation.

Transduction with retrovectors containing different concentrations of env, gag-pol

Experiments were done to see whether increasing the amount of the viral plasmids env and
gag-pol in relation to the MLV-Luc/GFP plasmid could elevate the transduction rate. The
transduction rate was modestly increased by elevated concentration of these plasmids,
however the addition of a centrifugation step had a much greater effect (Figure 3).
Previous experiments using only 10 minutes centrifugation resulted in no increase of GFP+
cells (results not shown). However, in this experiment a 1 hour centrifugation step proved to
be effective.
Experiments have also been done using higher titers of virus compared to cells. This
approach did not result in any increase of GFP+ cells (results not shown).

35
30
25
% GFP+ cells

20 not centrifugated
15 centrifugated

10
5
0
env,gagpol env,gagpol env,gagpol
x2 x4 x8

Figure 3 Virus were produced using different concentrations of the viral plasmids for envelope and gag-pol.
Using an additional centrifugation step can enhance the transduction of stimulated T cells further.

Retrovectors have also been produced using ultra-low IgG FBS and different batches of FBS
to see whether proteins in the serum could affect the transduction of cells. However, no
significant differences could be obtained by this approach (results not shown).

Multiple transductions

This experiment was conducted prior to the previous experiments e.g. env,gagpol x2 and
effect of pre-stimulation; thereby the low % of GFP+ cells.

12
 4,5
4
3,5
% GFP+ cells 3 d0
2,5 d0+d4
2 d0+d4+d8
1,5 d0+d4+d8+d12
1
0,5
0
d4 d8 d12 d16
Time (days)

Figure 4 Stimulated PBMCs were transduced with retroviruses at several occasions. d0 were transduced only
the first day. do+d4 were transduced both on the first day and then again 4 days after, and so on. The Figure
shows that by transducing the cells several subsequent times, the amount of Luc/GFP+ cells increases.

By transducing the stimulated T lymphocytes more than once, the number of GFP+ cells
could be increased even further. Results show that the number of transduced cells increased
with multiple transductions (Figure 4). At day 16 the viability for the group transduced four
times was 50%, which was the same as for the groups that had received either one or two
additional transductions.

Transfection of human T lymphocytes

Transfecting cells can be an advantage over transducing them considering the fact that there is
always a biological risk using retroviruses since they integrate into the host genome. There
are also several other advantages of plasmids. The transfection procedure is less time
consuming, plasmids are easier and less expensive to produce and they are easy to store and
handle. Therefore we evaluated the possibility to transfect T lymphocytes using the
transfection agent Gene Juice.
Furthermore, we wanted to investigate whether there was a difference in transfecting
purified CD8+ cells from OKT-3/IL-2 stimulated PBMCs, since the CD8+ cells might be our
future goal for transfection. CD8+ cells are easier to transfect than OKT-3/IL-2 stimulated
PBMCs as can be seen in Figure 5.

13
 7

% GFP+ cells 5

2
1

0
CD8+ cells OKT-3/IL-2 stimulated cells

Figure 5 Cells were transfected with the MLV-Luc/GFP plasmid after purification of CD8+ cells or after
stimulation of PBMCs with OKT-3 and IL-2.

Even though there was a slightly better transfection rate of CD8+ cells, the following
experiments were done using OKT-3/IL-2 stimulated PBMCs because they are easier
accessed. Enhanced transfection levels seen in the following experiments will later be
investigated in CD8+ T lymphocytes.

Transfection with different concentrations of MLV-Luc/GFP

To try to further enhance the transfection rate, stimulated T lymphocytes were transfected
using different concentrations of the MLV-Luc/GFP plasmid. As can be seen in Figure 6,
MLV-Luc/GFP x2 and x4 gave the best transfection levels.

16
14
12
% GFP+ cells

10
8
6
4
2
0
MLV-Luc/GFP MLV-Luc/GFP MLV-Luc/GFP MLV-Luc/GFP
x1 x2 x4 x8

Figure 6 Stimulated PBMCs transfected with various concentrations of the MLV-Luc/GFP plasmid.

Another approach for gene delivery is by using an adenoviral vector system. The ordinary
Ad5 serotype vector has been modified with the Ad35 knob to enable infection of T cells.

14
Preliminary results in our laboratory demonstrated high transgene expression of green
fluorescence protein (GFP) in T cells transduced with Ad5/35-GFP vectors (Figure 7).

0pfu/cell 200pfu/cell 1000pfu/cell 2000pfu/cell

0.3% 0.9% 45% 53%

Figure 7 OKT-3/IL2 stimulated T cells transduced with Ad5/35-GFP vector efficiently express GFP 5 days post
transduction. For efficient transduction (45-53%), 1000-2000 plaque-forming units (pfu) per cell are needed.

Discussion

The need for a new efficient cancer therapy is evident considering over 20% of all the
mortality in developed countries are due to cancer2. Current methods are curative for some
cancers but are also associated with many side effects due to their lack of specificity. A new
approach is to use immunotherapy by creating chimeric T lymphocytes specific for tumour
associated or tumour specific antigens. Since chimeric receptors are fusions between
antibodies and TCRs, they can be rendered specific for a wide range of antigens.
Numerous studies have shown that chimeric T lymphocytes prove to have cytolytic activity
against their antigens12,13,16,17. Many of these studies have also shown to have a tumour
rejection effect when injected in mice12,18,19,20.
There are several advantages of using chimeric T lymphocytes. The receptor specificity is
easily generated as mouse monoclonal antibodies against a variety of antigens already exist.
Once a chimeric receptor is generated, it can be used in patients, irrespective of their MHC
genotype, since chimeric receptors are non-MHC restricted. Furthermore, these receptors can
activate both CD4+ and CD8+ cells21, something that have been shown to increase the
chimeric T lymphocytes proliferation and activation in vivo19. By using both the CD4+ and
CD8+ cells, it has also been shown that the chimeric cells can reject tumours upon subsequent
tumour challenges19. Another advantages of using these receptors are that they are not limited
to recognising proteins, but can also react against carbohydrate and glycolipid antigens.
There are, however, some limitations involved with using chimeric cells. First of all the
targeted antigen must be a cell-surface molecule. Secondly, the signalling of the chimeric
receptor is not as complex as a normal signalling by the TCR, which involves e.g. structural
conformation, transmembrane association and the ability to interact with other cell-surface
complexes. Therefore, every newly developed chimeric receptor may need modification of
their signalling pathway, which may be very time consuming. A third limitation is that the
monoclonal mouse antibody fragment is potentially immunogenic, which may lead to the
clearance of chimeric cells in vivo. Humanized antibodies can be used to potentially overcome
this problem21.

To genetically engineer T lymphocytes for cancer immunotherapy is difficult. Our laboratory

have previously used retroviral transduction but with poor results. The advantage of using
retroviruses is that the gene of interest will be incorporated into the genome of the host,

15
meaning that the expression of the chimeric receptor will not be lost if the T lymphocytes
proliferates. The genetic modification will persist throughout the life of the T lymphocyte.
However, integration into the genome may cause uncontrolled T lymphocyte proliferation
if the insert is integrated at an unfortunate location. This has been seen in a study on
immunodeficient children in Paris that received a genetically modified stem cell
transplantation22. This scenario has, however, not been demonstrated in hundreds of other
patients receiving genetically marked T lymphocytes19, suggesting the events in Paris might
be disease specific.
To be able to control the chimeric cells in case an unfortunate event like this should occur,
the gene for herpes simplex virus thymidine kinase (HSV tk) can be inserted together with the
other chimeric genes19,21,23. This gene renders the chimeric cells sensitive for the pro-drug
ganciclovir, which has been used to effectively deplete allogenic HSV-tk-expressing T
lymphocytes21,23.
If a retroviral system is used, a selection of transduced cells can be done by culturing the
chimeric T lymphocytes with its antigen. Provided that it contains the co stimulatory molecule
CD28, the chimeric cells have shown to proliferate without the addition of IL-2 since they had
endogenous production of that interleukin12. If the chimer is directed against CD19, the
surface receptor specific for B cells (to treat B-cell malignancies) the chimeric cells can easily
be selectively cultured with B cells. However, some tumour specific antigens are not as easily
obtained and cancer cells taken from patients are known to be difficult to culture. In these
cases a protocol for gene transfer that generate as many chimeric cells as possible are
important.

An alternative to retroviral transduction is plasmid transfection or other viral systems such

as adenoviral vectors. Both plasmid transfection and adenoviral vector transduction will give
rise to transient gene expression since these vectors do not integrate into the host genome.
However, provided that the expression lasts for at least 1 week in vivo we do not see
drawbacks using these vectors. In contrary, a transient expression could be an advantage if
unexpected events occur. Chimeric T cells are easily in vitro cultured and can be given to the
patients repeatedly if needed.
When using either one of these approaches an efficient protocol for genetic engineering is
important, since there are difficulties sorting the modified cells in such a way that they can
later be used in patients.

In this study we explored different strategies of enhancing gene transfer to T lymphocytes,

e.g. by using transduction enhancers when using retroviral transduction. Retroviruses are
transported to cells by diffusion, a slow process where 90% of viruses have lost their
bioactivity in time they reach the cell surface24. Different cationic polymers have shown to
increase the transduction rate, e.g. PLL25 and polybrene24,25, by overcoming the electrostatic
repulsion (due to both virus and cells being negatively charged). By this they enhance the rate
of sedimentation of the virus to the cell surface25. Our studies with these two agents did not
show any promising results. The reason for this is unknown, but can be due to the shorter
incubation time with the polymers compared to previous studies24,25. Polybrene has in some
studies also been used in combination with a centrifugation step13. However, by the time our
experiments with the enhancers were done, we did not have access to a centrifuge capable of
centrifuging plates. A retroviral transduction experiment was done in tubes with the addition
of a short centrifugation, but this did not show any increase of the gene transfer.
RN is a chimeric peptide of human fibronectin consisting of three functional domains: a
cell binding domain, a heparin-binding domain II and a CS1 site. When coated on the surface
of e.g. wells in culturing plates, RN theoretically enhances transduction of mammalian cells

16
by bringing the virus and the cell together (virus particles binding to molecules interacting
with the heparin binding domain II and the cells to CS1). As can be seen in our results, the
aid of RN resulted in no increase of transduced cells compared to the other aids. RN seems to
be milder to the cells, since cells pre-treated with PLL showed a cell viability of only 4%, and
polybrene is known to be toxic25. RN was therefore used in all of our following retroviral
transductions. Since the enhancers were tested before the experiments with retrovectors
containing twice the concentration of env and gag-pol and did not contain a centrifugation
step, it is possible that even greater transduction levels would have been obtained using the
addition of e.g. PLL or polybrene. However, because of the time limitation this was not
tested.
By producing the retrovectors with twice the concentration of env and gag-pol (env,gagpol
x2), the number of GFP+ cells increased. Producing the retrovectors with twice the
concentration of all three plasmids did not result in an increase of gene transfer, neither did
transduction with a higher virus: cell ratio. The viruses were also produced in cell lines
cultured in ultra low IgG FBS and different batches of normal FBS, to see whether the low
transduction rate was due to disturbing agents in the serum. However, this was not the case,
the initially used FBS batch was even slightly better than the other batches (results not
shown). The following experiments were therefore done using env,gagpol x2 retrovectors and
produced in the initial FBS batch.
By keeping record of cell proliferation and cell viability in previous studies, it was noted
that cell proliferation continued several days past day 3, which have previously been the
principal day of transduction. Hence, an experiment was conducted to investigate the
significance of duration of pre stimulation prior transduction. The results show that
transduction after day 6 post stimulation gave the best gene transfer. Even though transducing
the cells 8 or 12 days post stimulation gave a similar transduction rate, the cell viability is
more satisfactoring day 6. Furthermore, the method is more applicable in practice if a good
transduction rate is obtained in as short period of time as possible. Why it was optimal to
transduce the T lymphocytes day 6 post transduction is not known, but it might be due to the
proliferative state of the cells at that time. Retroviral vectors need proliferating/dividing cells
to integrate the genes into the host genome. It is possible that the cells are in a higher
proliferative state day 6 than e.g. day 3. In this experiment, the cells were incubated together
with the virus for approximately 20 minutes in 37C prior to being incubated in the plate.
Whether or not this step was important, we do not know, but it was continued to be used in
the following experiments.
Since env,gagpol x2 retrovectors were superior, we wanted to investigate whether
increasing the concentration of these genes further would give an even higher transduction
rate. Retrovectors were therefore produced containing twice, four times or eight times the env
and gag-pol plasmids compared to the GFP plasmid. The experiment were done in duplicate
where one of the plates were centrifuged while the other was not. As can be seen by the
results, the best approach for retroviral transduction seems to be using env,gagpol x2
retrovectors to transduce T lymphocytes 6 days post stimulation with the addition of one hour
centrifugation at 1000 x g.
Furthermore, we have shown that multiple transductions of T lymphocytes results in
elevated number of GFP+ cells for each transduction. However, since this experiment was
done prior the above mentioned experiments, only a low number of GFP+ cells were obtained.
Due to time limitation this approach could not be tested with the new improved protocol.
Future improvements for transduction enhancement can involve multiple transductions to
stimulated T lymphocytes cultured 6 days, with the retrovector containing env,gag-pol x2,
including the one hour centrifugation of the plate.

17
 Plasmid transfection experiments conducted in this study resulted in a lower number of
GFP+ cells than retroviral transduction. Initial results show that purified CD8+ cells are easier
to transfect than non-purified stimulated PBMCs (reason unknown). Following experiments
were done using stimulated PBMCs since they are easier accessed. Investigations of whether
increased plasmid concentrations could elevate the gene transfer were also conducted. Results
showed that the GFP plasmid in twice its normal concentration gave the highest transfection
rate.
Experiments with adenoviral vectors was done by our laboratory. Results show that a high
number of transduced cells could be obtained using this method.
The optimal system for gene transfer might be dependent on the target antigen of the
chimeric receptor. The most optimal system would be to use plasmid transfection, since these
experiment are easily conducted with no preparations needed. The plasmids are easy to store
and handle. It would also be the most economical considering the most expensive part of
laboratory work is most often the time of the employees. However, for this type of gene
transfer to be attractive, a higher transfection rate needs to be obtained.
By using an adenoviral vector system, a transduction rate of over 50% could be obtained,
which is far better than 14% for the plasmid transduction experiment. However, using the
adenoviral approach can be associated with several months of preparation, which is not very
cost efficient. The use of adenoviral vectors for cancer therapy is something that is still in its
infancy, but has shown promising results in preliminary experiments26. It offers the
advantages of a long-term gene expression without integrating into the host genome. Based on
this point adenoviral vectors would appear less mutagenic26. They also come with the benefit
that they deliver genes into both dividing and non-dividing cells.
The use of retroviral vectors offers many advantages, though they also involve a risk. By
transferring chimeric genes into both CD4+ and CD8+ cells, chimeric genes were not only
active on their antigen, but could also encounter a second challenge of tumour antigen,
involving their proliferation in vivo19. Retroviral transduction would also be preferable, as
mentioned before, for selection of chimeric cells since they could obtain endogenous IL-2
production by the addition of CD28. By not adding IL-2 to the medium, the chimeric cells
were able to proliferate while the native cells died12. The retroviral vectors can be produced
within 72 hours and are easily stored. However, the retroviral integration into the host genome
is random and the chimeric genes might be integrated close to an oncogenic promoter, in
which case uncontrolled proliferation of the chimeric cells could occur.
Studies may have to be done in vitro evaluating the advantages of each of the different
strategies. Different approaches for gene transfer may be needed in different cancer types.

18
Acknowledgement

First of all I would like to thank all the girls of the satellite lab (Angelica, Moa, Pella,
Roberta, Valeria and Chris) for being such a friendly, fun and competent gang! Thanks for all
of your advice and for the wonderful atmosphere you bring to the lab. Thanks to my
supervisor Angelica Loskog for being such a source of inspiration! I never thought that I
would find a research field that would capture my interest from the very start together with a
supervisor that I truly admire and feel that I have a lot to learn from. I have never met anyone
that have a sharp answer to all of my, sometimes far-fetched, questions! Thanks to Moa
Fransson for the all of the lunch conversations and all of the advice on my thesis. I wish you
the very best of luck with you Ph.D. studies, I know you will do well; you are both intelligent
and hard working! Finally I would like to thank all of the members of the GIG group for
being such a fun gang to work and party with!! I wish you the best of luck with all of your
research!

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