Abstract

Much strides have been made in the discovery of new, less conventional ways for the early
detection and treatment of different cancer. One such therapy is the use of therapeutic antibodies.
The main purpose of this paper is to how these antibodies can be used for these functions with
the focus being on the identification of cancers and use of the antibodies to inhibit the action of
various growth factors such as S100A4 and TAM as well as the effectiveness and challenges that
may occur.
Keywords: Tumor, Tumorgenesis, Angiogenesis, chimeric, humanized, monoclonal antibodies,
immune response, Complimentary Determining Regions.
Introduction
The use of therapeutic antibodies in the treatment of various types of diseases is a form of gene
therapy, which is still in the development stage in most aspects but is a quickly rising industry. In
is based on the principle that once the biology of the disease is known, i.e. once the disease
encoding genes are known, this knowledge can be used to identify the disease when present and
block expression of gene or reversion of pathogenicity. Therapeutic antibody treatment makes
use of monoclonal antibodies which are antibodies that are specific for only one antigen and can
occur naturally or be produced by genetic engineering. The high specificity of monoclonal
antibodies arises to the arrangement of both its light and heavy chains of the variable region.
These antibodies can be used to treat diseases such as autoimmune diseases, cancers and
infectious diseases. In this paper we would focus on the use of monoclonal antibodies in the
detection and treatment of cancers. The monoclonal antibodies that can be used to treat cancer
can be of three types;
1. Antibodies which can be used to trigger an immune response to destroy cancer cell
2. Antibodies which can bind and block growth factors for cancer
3. Antibodies that delivery drugs and treatment to cancer cells.

Figure 1: Schematic diagram of an antibody. (Khan, 2012)
The specificity of a monoclonal antibody can be increased with genetic modification and
engineering, this would increase the affinity and ability of the antibody to bind to its
corresponding antigen. In this therapy, various types of mouse monoclonal antibodies have been
isolated and the antigen it interacts with has been identified, and for a while the use of these
antibodies have been used in the identification and treatment of human diseases. The use of these
antibodies proved to be problematic as they can illicit and immune response from the host
organism, however the difficulties in the production, purification and use of fully humanized
antibodies does not make it a viable option at present. One way to avoid these problems was to
produce ad use chimeric and humanized antibodies (Figure 2) which contains components of
both human and mouse antibodies. In the chimeric antibodies the constant region of the antibody
is humanized, while the variable region consist of the variable region of a mouse antibody. In
these antibodies, while most of the antibody is human, the variable region is mouse which can, at
a lesser degree, illicit an immune response. For humanized antibodies (Figure 2) both the
constant and variable regions of the antibody are of a human nature with parts with mouse
complimentary determining region, CDRs being inserted within the variable region. These
regions in the antibody are known as hypervariable domains which allows for or would
determine which antigen would be to interacting within the binding region of the antibody. This
structure allows for a decrease chance of an immune response as the bulk of the antibody would
consist of human antibodies as well as allows for the wide range of the mouse CDRs to still be
used and incorporated into the new antibody.


Figure 2: Basic structure representations of human, mouse, chimeric and humanized antibodies.
(Lotipour and Hallaj Nezhadi, 2012)



Monoclonal antibodies have two basic function, the first of which is to identify its antigen and
the second is elimination of the disease cell. In the first basic function, mABs can be used for
identification as each antibody is highly specific for one antigen. Once the mABs come into
close proximity of its corresponding antigen it interacts to for an antibody antigen complex and
simultaneously promotes the production of more like antibodies. Therefore the presence of an
antibody at high concentrations can be used as an indicator that the equivalent pathogen or
disease is also present in the body. Once mABs have identified and interacted with its antigen it
can facilitate the elimination of the disease cell by either activating an immune response cascade
or by inhibiting the activity of growth and stimulating factors.
While the use of antibodies can be very effective in the use for disease treatment, like every other
approach there are always difficulties in the application one of which is the aggregation of
antibodies. Antibodies are protein in nature and like proteins they are not very stable when
compared to other small molecules and can aggregate when they come in close contact. Knowing
this with the fact that for therapeutic antibody treatment relatively high concentrations of the
antibody is required per dosage it increase the chances of self-aggregation which makes it
problematic in handling and administering the treatment. Another challenge that may arise with
the use of mABs is the loss of activity after humanizing.
As stated earlier, the focus of this paper is the use of monoclonal antibodies in the identification
and treatment of cancer but for this therapy to be applicable first the biology of cancer must be
known. Cancer can be thought of a loss of a cells ability to control its growth and division as
well as the ability to avoid apoptosis which lead to the generation of mass of unwanted cell
known as a tumor, can either be benign or malignant. This formation of tumor occurs in a multi-
step process known as tumorigensis. In tumorigensis a series of mutations must occur for tumor
growth to occur, but these mutations must occur at specific parts along a genome which would in
turn stimulate tumor growth. As the tumor grows, the centre of the mass is being moved away
from a blood supply and as such lacks the nutrients required for continued growth, therefore for a
tumor to grow beyond 2mm a process known as angiogenesis must occur. Angiogensis is also
required for metastasis. This is by which new blood vessels, specifically capillaries, are formed
connect the original blood supply to the tumor which would now supply the oxygen and nutrients
required for further growth. Other factors can also aide in tumor growth such as growth factors
example S100A4 and environmental conditions such as exposure to radiation, availability of
oxygen and nutrient as the mutations in the tumor cells give it an advantage for survival in
harsher conditions that normal healthy cells.
As stated earlier there are three main ways in which mABs can be used to identify and treat
cancers, in this paper the main focus would be on one of the three above mentioned types. The
use of antibodies to the inhibition of tumor growth factors.
Thesis statement: Monoclonal antibodies can be used to both identify and treat different cancers
either directly by targeting the disease cells as with PAM4 or indirectly by the inhibition of the
tumor growth factors as with S100A4 and CSF -1.

Evidence
Use of mABs in the detection of cancers
The use of mABs in the identification of specific cancers can be appreciated by examining the
antibody PAM4 also known as the humanized form of clivatuzumab which can be used to
identify the presense of pancreatic ductal adenocarcinoma, PDAC. In PDAC there is an
abnormal expression of mucin glycoproteins which are heavily glycosylated proteins with high
molecular weights. PAM4 was created by indroduction of a polyclonal antiserum to pancreatic
ductal mucin which was shown to have a particular epitope that was used for the development of
the antibody. PAM4 works by identifying a biomarker present in cells of PDAC, and if it is
present there is a high possibility that is present in the patient. It has a high specificity for the
cells of PDAC with very minute reactivity to normal and benign tissue and also has little
reactivity to other adenocarcinimoas that occur in organs other than the pancreas example lungs.
PAM4 is thought to react with the MUC5AC mucin glycoprotein (Figure 3 and 4), specifically at
the C-terminus region and that the PAM4 epitope is either blocked or changed during antibody
interaction with the MUC5AC antigen. While the interaction between the mABs and antigen has
been confirmed the location of the PAM4 epitope has not. Studies to confirm the location using
EIAs were negative which is not an accurate confirmation. A negative result may be due to
changes in epitope conformation from that of it in native mucin or can be altered during reaction
of the mABs with the C-terminus even if it resides in a non C terminus region. The
biomarker recognised by PAM4 is also present in the precursors for pancreatic intraepithelial
neoplasia and intraductal papillary mucinous neoplasia and so PAM4 can be used for the earlier
identification of cancers.



Figure 3: Schematic diagram aa sequence for MUC5AC ( Moniaux, et al. 2001)


Figure 4: Schematic representation of MUC5AC peptide structure. (Atlas of Genetics and
Cytogenetics in Oncology and Haematology, 2014)

Use of mABs for the treatment of cancers
As discussed earlier, for tumor growth and proliferation it would need access to a blood supply
in which it can get oxygen and other nutrients and this is accomplished by the formation of new
blood vessels connecting the to the growing tumor, therefore, one way of treating cancers if the
inhibition of the process. This is observed with the inhibition of the protein S100A4, also known
as metastasin, which is a member of the S100 family of calcium binding proteins and take part in
various cell process, example cell growth and differentiation. The S100A4 protein is secreted by
both tumor and stromal cell but is overexpressed in tumor cell. It takes part in the motility and
metastatic capacity of cancer cells due to its angiogenic characteristics, cell proliferation due to
inhibition of the p53 protein and cell adhesion and detachment. S100A4 promotes angiogenesis
by interacting synergetically with vascular endotheial growth factor (VEGF) via the receptor for
advanced glycation endproducts (RAGE). The mechanism of promotion can be explained by the
expression of kinase insert domain receptor (KDR) which is also known as vascular endothelial
growth factor receptor 2 (VEGFR-2). In the presence of both VEFG and S100A4, KDR
expression increases by 12 fold each with a 2 fold increase of KDR phosphoralyation,
S100A4 also increases the expression and activity of matrix metallopoteinases (MMPs) which
then secretes a more active form of MMPs 9 when compared to that in the absence of S100A4
which would result in cell movement and invasion. Therefore inhibition of S1004A leads to
lower levels of KDR and MMPs 9 to be present in the cell as well as decreases the impact of
VEGF would have on the organism. The mAB 5C3 inhibits the activity of S1004A and as such
destroys the synergy between the VEGF and S1004A, so only migration that occur is that
stimulate by the VEGF only which in comparison is much less. 5C3 also has total inhibition for
the increased production of MMP 9.
Another case that can be studied for the use of mABs in the treatment of cancer is the use of the
antibody AFS98 and its role for the inhibition of CD115. The differentiation and
functionalization of myeloid cell types, example macrophages and osteoclasts, are dependant on
the CD115/CSF-1 pathway. It regulated by the CD115 which belong to the receptor tyrosine
kinase family, class 3. It is the only known receptor for colony stimulating factor 1 (CSF-1),
a cytokine that is involved with the regulating differentiation, proliferation and migration. CSF
1 is believed to have a major role in cancer growth progression and metastasis, due to its ability
to recruit macrophages, with an overexpression of both this and the CD115 to occur in many
types of epithelial tumors, example ovarian and breast. During the metastasis, the metastastic
cells require CD115 positive macrophages as well as tumor associated macrophages (TAMs)
which regulate angiogenesis. The mAB AFS98 blocks the binding of the CSF -1 to its CD115
receptor and inhibits the CD115/CSF-1 pathways and hence preventing the creation of the
macrophages. This would decrease the chances or probability of the tumor to metastasis. This
type of inhibition is thought to be competitive as it would compete with the CSF-1 ligand, so
high concentrations of the AFS98 is required per dosage to achieve desired results. Also
combining this antibody treatment with other types of treatment like chemotherapy can increase
the effects of the chemotherapy. One study done was the use of the chemotherapy drug Paclitaxel
in combination with the AFS98 antibody showed significant reduction in the tumor size of mice ,
much greater than the reduction that occurred used only one of the treatments.



Conclusion
From the research done and the information gather it is shown that monoclonal antibodies can be
used to successfully detect and treat various types of cancers once the biology the cancer is
known. There are three main ways in which these antibodies can be used to treat these disease
once identified and these are; to activate an immune response, inhibit its growth factors as with
S100A4 and AFS98 and the use of the antibody to amplify the impact of drugs as with AFS98.
However like all treatment there would be some difficulties that would arise in the use of these
antibodies, some of which is the ability of the antibodies to aggregate as well as the risk of
activating an immune response by using chimeric antibodies.