Lebanese University

Faculty of Sciences I
Department of Biochemistry
(2007-2008)

5T4 oncofetal antigen: Janus molecule in life and

a novel target against tumors

Hilal Bakri
hllbakri@hotmail.com
 Table of Contents

Abstract:...........................................................................................2
I. Introduction: ..................................................................................2
II. Generation & structure: ...............................................................3
III. Function:.......................................................................................3
IV. A positive role in biological development:...................................4
V. A negative role in pathological events:..........................................5
VI. A novel promising target 5T4 to immunotherapy for cancers......5
VII. Passive immunotherapy:.............................................................5
VII.I. Monoclonal antibody...........................................................6
VII.I.I Scfv monoclonal antibody............................................................................6
VII.I.II Fab monoclonal antibody............................................................................7
VIII.I. Viral vector vaccines..........................................................8
VIII.II. Dentritic cell based vaccines............................................9
VIII.III. T reg cell and active DC therapy:................................11
IX. Conclusion:.................................................................................11

Abstract:

Two decades ago, scientific studies and researches were done on the embryonic cells,
where scientists first defined a highly glycosylated modified antigen on the surface of
these cells and were given the name of 5T4 Oncotrophoblast Glycoprotein or simply
Oncofetal Antigen. 5T4 is a transmembrane protein expressed on the surface of
embryonic cells & many different malignant tumor cells. Studies showed that this antigen
is involved in many biological and pathogenic processes, such as cell migration, invasion,
and implantation (embryogenesis), and neoplastic metastasis in series of tumorigenesis. It
is considered as tumor associated antigen due to its restricted expression profile, which
makes it a contender for cancer immunotherapy. This helped explain the molecular
function for discovering how tumor s diffuses and magnify, and understand the successful
strategies of cancer therapy.

I. Introduction:

2
5T4 Oncofetal antigen is a type of heavily glycosylated transmembrane protein
recognized by monoclonal antibodies secreted from human syncytiotrophoblast
microvillus projections of the plasma membrane (StMPM), rose against wheat germ
agglutinin-glycoprotein. It is isolated from the placenta, and chemically characterized as
a 72 KDa product encoded by the trophoblast glycoprotein gene (TBGP gene).It is also
expressed by tumor cells associated with metastasis and low cancer formation.

II. Generation & structure:

5T4 Oncofetal antigen is found on a variety of carcinomas and weakly on specialized

epithelial cells. It is produced after the hatching of the blastocyst from the zona pellucida,
due to the over expression of the TBGP gene. The gene-not like others-don’t have a TATA
nor CAAT boxes, but a number of potential sp1 binding sites to help initiate the
transcription of the gene to later produce the protein (or antigen). The 72 KDa product is
encoded by the opening reading frame, having a 420 a.a, 46 KDa protein core-with 2
leucine rich repeat domains-separated by a hydrophilic stretch of amino acids with 8
potential N-glycosylation sites, and a 44 a.a cytoplasmic tail. The N-terminal act as a
putative signal and the C-terminal acts as the anchorage sequence.

Fig: 1 Generation of 5T4 on the trophoblast from the zona pellucida

III. Function:

5T4 Oncofetal antigen plays a role in many physiological & pathological cell events, and
that is done through modulating the cells migration, adhesion, mobility, shape and
morphology. We can be more perceive by stating that it increases cell mobility and

3
migration (due to the increase in contact between actin & cadherin on the surface of the
cell), enhances cell adhesion, and changes the cells shape and the morphology & integrity
of its membrane. Changing in the morphology of the membrane includes the change of E-
Cadherin on the cell surface of epithelial cell (important for embryo development after
the blastocyst stage & formation of cell-cell adherence junctions) to N- Cadherin
promotes migration and invasion of cells).

IV. A positive role in biological development:

Studies showed that the type I of the protein has the highest level of expression. It plays a
positive role in the body due to its structural components. The extra cellular domain,
having the domain of 7 leucine-rich repeat regions (LRRs), flanked with cysteine-rich
region, helps in the intervention of certain protein-protein interactions. The cytoplasmic,
having a PDZ domain-binding motif (containing: Ser/Asp/Val), is concerned in the course
of embryogenesis.
Concerning metastasis, during murine post-implantation, 5T4 Oncofetal antigen displays
restricted expressions to diverse epithelial cell types formed from all 3 germ layers. This
however is companied with the invasion of the trophoblast cells into the maternal host
cells, in the uterus wall to aid in the adhesion and fixation of the embryo, in addition to
the formation of the placenta.
The antigen can be used in marking of the differentiation of premature embryonic stem
cells. 5T4 positive epithelial cells can express formation of chimera more efficiently than
5T4 negative cells (7 times more).
5T4 also plays an important role in the nervous system. It can help in the development of
the epithelia of the nervous system. Many of the (LRRs) domains of the protein, present
in the central nervous system, are involved in the formation of neural circuits.
A novel function we can mention about the 5T4 Oncofetal antigen is that it is involved in
the procedure of forming dendrodendritic synapse between granule cells (GCs) &
mitral/tuftal cells. An example of this process can be located in the olfactory bulb, where
a major population of interneurons-5T4 positive GCs-extend their peripheral dendrites
preferentially toward the superficial stratum of external plexiform layer (EPL).As a
summary we can say that the expression of the 5T4 antigen comes parallel to the
formation of dendrodendritic synapse between GCs & mitral/tufted cells.

Fig: 2 5T4 right and control left

4
V. A negative role in pathological events:

The over expression of 5T4 antigen in both malignant and embryonic stem cells explains
the acquirement of invasive and migratory phenotype. Placentation may share aspects of
such mechanisms where foetal trophoblast cells, these aspect similarities was observed as
avoiding host immunity and surveillancespindle-like (dendritic) morphology, increased
motility, reduced cell-cell adhesion and proliferation rate, and loss of pluripotency of
metastatic spread epithelia.
Microvillus projections present in tumor cell (B16 F10 melanoma cells and A9 L
fibroblasts) membranes which over expresses the cDNA encoding the human gene TBPG
results the diversity of E-cadherin structure that influences the protection of the antigen
against proteolytic attack and promotion of the interaction with other molecules
responsible for invasion and migration and increased motility, that permits cancerous
cells metastasis where the it interact with the N-linked glycosylated domain which
contains PDZ binding domain that interacts with the PDZ domain protein and LRR motif
which plays the role of cell signaling assemblies and remodulation the cytoskeleton thus
alters the integrity of the cell membrane and cell adhesion. 5T4 antigen strongly it is
over-expressed on many different types of human carcinoma such as gastric, colorectal
and ovarian, cervical, renal, gastrial and breast cancer.
VI. A novel promising target 5T4 to immunotherapy for cancers

Cancer immunotherapy is the use of the immune system to reject cancer the main premise
is stimulating the patient's immune system to attack the malignant tumor cell that are
responsible for the disease .Tumor antigen is a substance produced in tumor cells that
triggers an immune response in the host. Tumor antigens are useful in identifying tumor
cell and are potential candidates for use in cancer therapy. Mutation of other genes
unrelated to the tumor formation may lead to synthesis of abnormal proteins which are
called tumor associated antigens.
A number of oncofoetal or tumor-associated antigens (TAAs) have been identified and
characterized in human and animal tumors. Tumor cells have been observed to resume
expression of TAAs, and the application of TAAs for tumor diagnosis, targeting and
immunotherapy has therefore been suggested. 5T4 oncofetal antigen is a 72 kDa
glycoprotein defined by a monoclonal antibody raised against human placental
trophoblast and is expressed widely in carcinomas, but having a highly restricted
expression pattern in normal adult tissues. It appears to be strongly correlated to
metastasis in colorectal and gastric cancer. 5T4 satisfies the criteria for TAA and could
prove to be effective antigens for immunotherapy against cancer that lead to cancer cell
death depend on the relative immune mechanism included antibody-dependent cell-
mediated cytoxicity (ADCC) and complement-dependent cytoxicity (CDC)

VII. Passive immunotherapy:

5
Passive immunotherapy is transfer of active humoral immunity in the form of ready made
antibodies, from on individual to another. It can occur naturally, when maternal
antibodies are transferred to the fetus through the placenta, and can also be induced
artificially, when high levels of human antibodies specific for pathogen or toxins are
transferred to non immune person, it may involve antigen specific T cell and cytokines .It
is used when there is a high risk of infection and insufficient time for the body to develop
its own immune response, or to reduce the effect of immunosuppressive diseases.

VII.I. Monoclonal antibody

Monoclonal antibody therapy is a form of passive immunotherapy because it uses

antibodies made in large numbers outside the body (in the lab) rather than by a person's
own immune system so it don't require the person's immune system to take an "active"
role in fighting the cancer. When used as a treatment for cancer it may bind to the cancer
cells having the tumor antigens on their surface, the immune system will see the cancer
cells marked with bound antibodies as foreign and destroy them. A second strategy is to
use the antibodies to block the binding of cytokines or other proteins that are needed by
the cancerous cells to maintain their uncontrolled growth. Both strategies may help rid
the body of the tumor cells.

Fig: 3 Monoclonal antibodies for cancer

VII.I.I Scfv monoclonal antibody

The most well known example of monoclonal antibody is single chain antibody such as
single chain antibody Fv (scfv) and the conjugated engineered antibody. Although several
clinical trials have shown beneficial effects by targeting tumor-associated antigens
(TAAs) with monoclonal antibodies, a number of issues, including poor penetration of

6
the tumor mass and human antimouse antibody responses, remain. The use of
recombinant single-chain Fv (scFv) fragments has the potential to address these and other
issues while allowing the addition of different effectors functions.

VII.I.II Fab monoclonal antibody

A phase 2 study of a tumor –targeted super antigen (TTS) (ABR-214936) recognizing the
5T4 oncofetal antigen in patients with advanced renal cell carcinoma shows that this
tumor targeted super antigens activates and recruits T cells to tumors expressing the target
antigen. This drug (ABR-214936) is a recombinant fusion of a fab of a monoclonal
antibody fragment recognizing the oncofetal antigen 5T4 and the modified super Antigen
SEA (D227A) .The cytotoxic effect is mediated by direct tumor cell killing (perfroin) and
secretion of cytokines. This lead to tumor cell death, inflammation, vascular permeability
and further recruitment of immune cells to the tumor site.

Fig: 4 Action of ABR-214936 on tumor cell

VII.II. Antigen specific T cell infusion

Another effective strategy for passive immunotherapy is the infusion of tumor antigen-
specific T cells since it plays a role in host response to a tumor. To prevent tumor cell
from escaping T-cell killing genetical modification to T cell ex vivo to posses McAB
specificity for protein epitope with a chimeric T-cell receptor is applied. Chimeric
immune receptors (CIR) are engineered molecule encompass tumor- or virus-specific
ligands or antibodies fused to the signaling domains of either the T cell receptor or Fc
receptor.

7
 Fig: 5 Antigen specific T cell infusion mechanisms

VIII. Active immunotherapy:

A type of immunotherapy that attempts to stimulate the host's intrinsic immune response
to the tumor, either nonspecifically or specifically. Active immunotherapy, also called
therapeutic vaccines, aims to induce a therapeutic immune response through the patient
own immune system. In particular, the use of active immunotherapy in cancer treatment
is being evaluated. Most of the cancer vaccines currently under development aim at
inducing a killer-cell response, whilst others attempt to generate antibody responses. It's
characterized by long term specificity, long term efficacy and giving body protection
against tumor genesis. There are two types of vaccines: viral vector vaccine and dentritic
cell (DC) vaccines involved in active immunotherapy.

VIII.I. Viral vector vaccines

Many therapeutic viral vector are constructed based on TroVax virus which is a cancer
immunotherapy product produced by oxford biomedical. It is a novel cancer vaccine in
clinical development with potential application in most solid tumor types and delivers a
novel proprietary tumor associated antigen, 5T4, using a pox virus vector, which is
modified vaccinia virus Ankara (MVA) encoding the h5T4 protein .Vaccine viruses re
engineered to express foreign genes are robust vectors for production of recombinant
protein, the most common being a vaccine delivery system for antigens. Vaccination with
recombinant modified vaccinia Ankara-5T4 vaccine may stimulate the host immune
system to mount humoral and cytotoxic T lymphocyte (CTL) response against tumor
cells expressing 5T4 fetal oncoprotein antigen, resulting in tumor cell lyses. Studies had
shown that 5T4 immune response is boosted in the presence of MVA neutralizing
antibodies. Vaccination with TroVax is safe and well tolerated, so 5T4 immune response
can be induced without any evidence of autoimmune toxicity.

8
Fig: 6 (A) Production of the MVA-5T4 Constructs, (B) Mechanism of MVA strain encoding h5T4
protein.

Other viral vaccine vectors are based on either replication- defective adenovirus like
Adh5T4 or a lentivirial vectors, like the equine infectious anemia virus vector (EIVA),
murine leukemia virus (MLV), human immunodeficiency virus (HIV)

Other therapeutic strategy targeting 5T4 antigen for destroying tumor cells take place
through a viral vector encoding a fusion protein. To develop therapeutic strategies that
recruit both humoral and cellular arms of the immune response, we have constructed
chimeric proteins linking either the human IgG1 Fc domain or the extra cellular domain
of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4.The engineered
retroviral constructs can produce fusion proteins able to interact simultaneously with both
5T4-positive cells and with the receptor/ligands of the immune effectors moieties.
Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells
results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the
scFv−HIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell
lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene
therapy for human cancers. Its propose that the combination of engineered T cell
chimeric receptor with specific vaccination strategies can improve the active tumor
therapy through mechanisms of enhancing Ag-specific cellular immune response.

VIII.II. Dentritic cell based vaccines

Lack of efficient tumor antigen presentation in DCs in cancer patients has led to the use
of DC-based vaccines. As fig.5 shows the appropriate tumor antigen is bound antigen is
bound to the DC cell surface. The tumor antigens are taken up by dendritic cells; they are
processed and presented to the T cells along with the appropriate co stimulatory signal.
Once activated by the DCs the cytotoxic T cells recognize and destroy the tumor cells and
destroy the tumor cells expressing the tumor antigen.

9
 Fig: 7 DC based vaccine

Concerning the source of DC as fig.8 demonstrate they are collected from blood of the
patients own tumor cells. This DC s is then reintroduced into the patient and stimulates
the immune system.DC vaccines have been used in patients with metastatic melanoma,
renal carcinoma and prostate cancer.
.

Fig: 8 Origin of DC used in DC based vaccine therapy.

Active immunotherapy strategy for tumor is more effective than passive one, since it
increase the efficiency and improvement of the immune response against cancer cell also
it involve the whole immune response activation and breaking the immune tolerance to
tumor antigen involving different effecter cell interaction.

10
VIII.III. T reg cell and active DC therapy:

Regulatory T cell (sometimes known as suppressor T cells) is a specialized subpopulation

of T cell that acts to suppress activation of the immune system most common is
CD4+CD25+ regulatory T cells or "Tregs" and thereby maintain immune system
homeostasis and tolerance to self antigens. In the tumor-bearing host, tumor-associated
antigens (TAAs) could be taken up by tolerogenic dendritic cells present in the tumor
microenvironment, plasmacytoid (pDC). PDCs have recently been shown to induce
tolerance to an inhaled non-self-antigen. The allergen was taken up by pDCs, which
presented the antigen to allergen-specific T cells that subsequently became T regulatory.
These putative tumor-specific Treg cells may then circulate throughout the body and
respond to self-antigens shed by tumors in the tumor-draining lymph nodes.

Fig: 9 T reg cell and DC vaccine therapy

Subsequently, after activation, they may reside in the tumor-draining lymph node or
infiltrate the tumor bed and suppress any precursor antitumor effectors CD8+ T
lymphocytes from destroying their self-target, either through cell–cell contact by
inhibiting T-cell help and activation (e.g., IL-2 deprivation or other mechanisms) or
through release of immunosuppressive cytokines, such as IL-10 or TGF-β.TGF-β
secreted by most melanomas could play a role in suppressive mechanisms by.Treg cells
suppressed the expansion of co-transferred CD4+ Thelper cells, when suppressing T-cell
help of tumor-reactive T cells; this will prevent CD8+ T cells from being properly
activated. Knowing that CD8+ T cell recognizing 5T4 is important as anti-tumor effectors
in the immune defense.

IX. Conclusion:

As we see 5T4 play a double role in biological and pathological progression .its a major candidate
for immunotherapy for cancer. However information about the protein 5T4 activities in
impanation, metastasis, interaction with immune effecter to decrease immunogenic is difficult to

11
gather it by studying the human tissue but many research found that mouse and human 5T4 protein
sequences show 81 % homology and with the possibility of cloning the coding region of the
human and mouse 5T4 genes. This opens a wide view on study of 5T4 oncofetal antigen giving
more opportunity to develop therapeutic strategy targeting this protein in cancer and other
diseases.

Glossary:

Antibody-dependent cell-mediated cytotoxicity: it is a mechanism of cell mediated immunity

whereby an effecter cell of the immune system actively lyses a target cell that has been bound by
specific antibodies. It is mediated by natural killer cell & monocytes

Carcinoma: is any malignant cancer that arises from epithelial cells.

MVA: (Modified Virus Ankara) It is a viral vector, derived from the replication-
competent strain Ankara, is a highly attenuated, replicative-defective vaccinia strain
incapable of virion assembly.

Lentiviral vectors: are types of retrovirus that can infect both dividing and non dividing
cells because their preintegration complex (virus “shell”) can get through the intact
membrane of the nucleus of the target cell. .
ABR-214936: anatumomab mafenatox tumor targeted super antigen.

Adenovirus: any virus belonging to the family adenoviridae a family of DNA viruses
with a double-stranded genome, generally with a narrow host ranges, and transferred by
direct or indirect transmission.

Melanoma: is a malignant tumor of melanocytes which are found predominantly in skin

but also in the bowel and the eye .It is one of the rarer types of skin cancer but causes the
majority of skin cancer related deaths.

scFvHIgG1: single chain human immunoglobin (Ig) antibodies

TGF-β: Transforming growth factor beta controls proliferation, differentiation, and other
functions in most cell types.

12
CD4: (cluster of differentiation 4) is a glycoprotein expressed on the surface of T helper
cells, regulatory T cells. On T cells, CD4 is the co-receptor for the T cell receptor (TCR).
It amplifies the signal generated by the TCR. It forms the T helper with ctyotoxic cell Tc.

CD8 :( cluster of differentiation 8) is a transmembrane glycoprotein which serves as a co-

receptor for the T cell receptor (TCR). Cytotoxic T cells with CD8 surface protein are
called CD8+ T cells.

LRR motif: leucine rich region motif, involved in cell signaling.

References:

1. Carsberg et al., (1996) Int J Cancer 1996 Sep. 27; 68(1):84-92. 5T4 proposed for tumor progression
and metastasis potential
Online: http://www.freshpatents.comPolypeptide-dt20070118ptan20070014808.phptype=description

2. R W Griffiths1, D E Gilham1, A Dangoor2, V Ramani3, N W Clarke3, P L Stern2 and R E Hawkins1 Expression of

the 5T4 oncofoetal antigen in renal cell carcinoma: a potential target for T-cell-based immunotherapy.
British Journal of Cancer (2005) 93, 670677.
doi:10.1038/sj.bjc.6602776 published online 13 September 2005

3. Peter L stem1 , Robert Ehawkins2 ,David M Shaw1 , Noel B Connolly2, Poulam M Patel4, David Hastings5 , Peter
Julyan5 , Jamal Zweit3, Suzanne Kilany6 , Orjan Nordle6, Gunnar Hedlund6 , Goran Forserg6 . A phase 2 Study of a
Tumor-Targeted Superantigen (ABR-214936) Recognizing the 5T4 Oncofetal Antigen in Patient with Advanced
Renal Cell Carcinoma.

4. Andrew M Woods, Who W Wang, David M Shaw, Christopher M Ward, Miles W Carroll, Buddug R
Rees, and Peter L Stern. CRUK Immunology Group, Paterson Institute for Cancer Research, Christie
Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, U.K. Characterization of the murine 5T4
oncofoetal antigen: a target for immunotherapy in cancer.

5. CJ Carsberg, KA Myers, GS Evans, TD Allen and PL Stern CRC Department of Immunology, Paterson Institute
for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. Metastasis-associated 5T4 oncofoetal
antigen is concentrated at microvillus projections of the plasma membrane

6. Kevin A Myers1, Matthew G Ryan1, Peter L Stern2, David M Shaw2, M Jim Embleton3, Susan M Kingsman1 and
Miles W Carroll1. Targeting immune effectors molecules to human tumor cells through genetic delivery of 5T4-
specific scFv fusion proteins

7. S D Patel, M Moskalenko, D Smithb, B Maske, M H Finer and J G McArthura. Cell Genesys Inc., Department of
Preclinical Biology and Immunology, 344 Lakeside Drive, Foster City, CA 94404, USA. Impact of chimeric
immune receptor extracellular protein domains on T cell function

8. Sumia Ali1, Kate Mulryan1, Taher Taher1 and Peter L. Stern1. Importance of vaccine vector to achieve specific
cellular immune response.

13
9. Recombinant modified vaccinia Ankara-5T4-vaccine.
Online: http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=449683

10. B16h5t4 melanoma

Online: http://www.jimmunol.org/cgi/content/full/177/7/4288

11. Kate Mulryan, Matthew G. Ryan, Kevin A. Myers, David Shaw, Who Wang, Susan M. Kingsman,
Peter L. Stern,1 and Miles W. Carroll1. Attenuated Recombinant Vaccinia Virus Expressing Oncofetal
Antigen (Tumor-associated Antigen) 5T4 Induces Active Therapy of Established Tumors.

12. What is active immunotherapy

Online: http://www.pharmexa.com/cms/site.aspx?p=242

13. By Kenneth Hung,* Robert Hayashi,§ Anne Lafond-Walker, Charles Lowenstein, Drew Pardoll,* and
Hyam Levitsky* The Central Role of CD4+ T Cells in the Antitumor Immune Response

14. Christopher J. Kirk2, Dennis Hartigan-O’Connor and James J. Mulé3 The Dynamics of the T-Cell
Antitumor Response .Chemokine-secreting Dendritic Cells Can Prime Tumor-reactive T Cells
Extranodally1

15. Paul Andrew Antony and Nicholas P Restifo .From the Surgery Branch, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Mark O. Hatfield Clinical Research Center,
Bethesda, Maryland. CD4+CD25+ T Regulatory Cells, Immunotherapy of Cancer, and Interleukin-2
Online:http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1242172#R52#R52

16. Drew M Pardolla and Suzanne L Topalianb,the role of CD4+ in antitumor immunity.

17. M. Eastham, H. Spencer, F. Soncin, S. Ritson, C. L.R. Merry, P. L. Stern, and C. M. Ward. Epithelial-
Mesenchymal Transition Events during Human Embryonic Stem Cell Differentiation. Cancer Res.,
December 1, 2007; 67(23): 11254 - 11262.
Online: http:// www.MBConline.com

18. Zhao Y, Wang YX. 5T4 Oncotrophoblast Glycoprotein: Janus Molecule in Life and a Novel Potential
Target against Tumors
Online: http:// www.unboundmedline.com

19. A. M. Eastham, H. Spencer, F. Soncin, S. Ritson, C. L.R. Merry, P. L. Stern, and C. M.

Ward.Epithelial-Mesenchymal Transition Events during Human Embryonic Stem Cell Differentiation
Cancer Res., December 1, 2007; 67(23): 11254 – 11262.

20. Hoffmann, R., Valencia, A. A gene network for navigating the literature. Nature Genetics 36, 664
(2004) and as "iHOP
Online: http://www.ihop-net.org/

21. Wikipedia online, the free encyclopedia.

22. Figure 1 and 2 Molecular Biology of the Cell.H. L. Spencer, A. M. Eastham, C. L.R. Merry, T. D. Southgate, F.
Perez-Campo, F. Soncin, S. Ritson, R. Kemler, P. L. Stern, and C. M. Ward. E-Cadherin Inhibits Cell Surface
Localization of the Pro-Migratory 5T4 Oncofetal Antigen in Mouse Embryonic Stem Cells
Mol. Biol. Cell, August 1, 2007; 18(8): 2838 - 2851.
Online: http:// www.journalofcellscience.com

23. fig: 3 online : http://www.answers.com/topic/monoclonalab-jpg-1

14
24. Fig:4 Peter L stem1 , Robert Ehawkins2 ,David M Shaw1 , Noel B Connolly2, Poulam M Patel4, David Hastings5 ,
Peter Julyan5 , Jamal Zweit3, Suzanne Kilany6 , Orjan Nordle6, Gunnar Hedlund6 , Goran Forserg6

25. Fig: 5 http://www.bmj.com/cgi/content/full/323/7324/1289#F4

26. Fig: 6 online: http://mct.aacrjournals.org/content/vol1/issue12/images/large/gd1220111001.jpeg

27. fig:7 online: http://www.csa.com/discoveryguides/cancer/review.php

Cancer Vaccine Fact Sheet, (National Cancer Institute. National Institutes of Health)
Online:http://www.cancer.gov/newscenter/pressreleases/cancervaccines

28. Fig : 8 Trevor K.T., et.al. "Generation of dendritic cell-tumor cell hybrids by electro fusion for clinical vaccine
application" Cancer Immunology Immunotherapy (2004) 53:705-714
Online http://www.csa.com/discoveryguides/cancer/review.php

29. Fig: 9 http://www.pubmedcentral.nih.gov-PubMed Central, FIGURE 2: J Immunotherapy (1997)

15