Monoclonal Antibodies

Monoclonal Antibodies are the antibodies that are made by identical immune cells that are all
clones of a unique parent cell. Monoclonal antibodies can have monovalent affinity, in that
they bind to the same epitope.
● The body naturally produces antibodies, which are elements of the immune system
produced by B-lymphocytes, that bind to foreign proteins in the body known as
antigens, which the aim of eliminating them.
● They naturally circulate in the body searching for foreign bodies (antigens) and once
they attach to the antigen, they destroy the antigen using various immune
mechanisms.
● Monoclonal antibodies (mAbs) have the ability to recognize unique binding sites
(epitopes) found on the specific antigens. This differentiates monoclonal antibodies
from polyclonal antibodies i.e monoclonal antibodies are derived from a single B-cell
clone to target single epitopes, unlike polyclonal antibodies that target multiple
epitopes.
● Monoclonal antibodies (mAbs) have been produced to target receptors or other
foreign proteins that are present on the surface of normal cells and cancer cells.
● Monoclonal antibodies are used in the treatment of many diseases including some
cancers

Types of monoclonal antibodies

A. Murine monoclonal antibodies
● They were named murine because of their origin from rodent hosts (mice and rats)
belonging to the Muridae family.
● Preclinical trials for its use in the treatment of Cryptococcus neoformans.
● The murine mAbs have played a crucial role in the development of modern antibody
production techniques and the potential applications of these artificial
immunoglobulins in therapeutics and analytical applications.
● In therapeutic applications, they are used as a framework for the development of
antibody and engineering techniques that include chimerization, humanization, and
development of bispecific antibodies from antibody fragments known as single-chain
antibody fragments (scFvs).
● Preclinical trials to use murine mAbs on the capsular polysaccharide and enhance the
therapeutic activity of amphotericin B administration.

B. Chimeric monoclonal antibodies

● These are structural chimeras that are made of a combination of mouse parts and
human parts, by fusion. This involves fusing variable regions of one species such as
mice and the constant regions of the other species such as the human species.
● Chimeric monoclonal antibodies are produced to reduce immunogenicity and to
increase the serum half-life when preparing them for therapeutic reasons.
● Chimeric antibodies retain the original antibody’s antigen specificity and affinity.
● The name of their treatments ends with -ximab.

C. Humanized monoclonal antibodies

● They are an extension of the chimeric monoclonal antibodies whereby, all regions of
the mouse antibody in chimeric mAbs are replaced with human ones except for the
complementarity-determining regions (CDRs) which are the amino acids that make
direct contact with the antigen. This means that humanized mAbs have small parts of
the mouse protein that are attached to the human protein.
D. Human monoclonal antibodies
● These are fully human proteins that have been manipulated by molecular biological
techniques so as to modify the amino acid sequences.
● This alters the specificity, affinity, or biological functions, acquiring sequences that
are not part of the human repertoire.

Production of monoclonal antibodies

● The production of monoclonal antibodies is an in vitro process by the use of tissue-
culture techniques.
● Producing monoclonal antibodies (mAbs) is initially done by identifying a specific
antigen, and immunizing an animal with the antigen multiple times. The most
commonly used animal models are laboratory mice.
● The B-cells of the immunized animals are removed from the spleen and then fused
with cancer B-cells known as the myeloma cells.
● The fusion of adjacent plasma membranes of the myeloma cells is done using
polyethylene glycol, however, it has a low success rate, and therefore the selective
medium must have the fusion activity as well to enhance cell growth.
● Myeloma cancer cells have an immortal characteristic of continuously proliferating,
unlike the normal B cells which proliferate for a period of 6-8 hours, and they
normally have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl
transferase (HGPRT), an enzyme necessary for the degradative synthesis of nucleic
acids.
● The myeloma cells are placed in a selective medium known as the Hypoxanthine
Aminopetrin Thymidine (HAT)-which is made up of hypoxanthine, aminopterin, and
thymidine, where it allows the growth and yield of fused hybridoma cells, and the
infused myeloma cells do not grow and infused B-cells die off.
● Hybridoma cells have the ability to grow continuously in culture as they produce
antibodies. They are then screened for the desired or specific monoclonal antibodies,
and those producing the desired mAbs are then transferred and grown in tissue
culture.
● Harvesting is done periodically and the monoclonal antibodies are then purified from
the medium.
● Growing and harvesting of these monoclonal antibodies are done for several weeks in
large media quantities in order to produce enough mAbs that can be used for
experimentation or to treat at least a single patient.
● The monoclonal antibodies produced are in millions of numbers and they are specific
for the antigen that was initially injected into the animal model.

Example
Anti-cancer- Rituximab, Alemtuzumab ---- used in B cell leukemia
Anti-cancer and anti-viral- Bavituximab, Palivizumab---- used in cancer, hepatitis
C infection, RSV infections in children
Functions and Applications of Monoclonal Antibodies
1. Some monoclonal antibodies are designed to target specific tumor antigens. They
have been used to stimulate the production of anti-idiotypic antibodies stimulating a
strong antitumor immune response when they are inoculated in patients with B-cell
lymphoma. However, anti-idiotypic antibodies are developed in animal models, which
hiders production of monoclonal antibodies in humans. Though, humanized antiHer2
monoclonal antibodies Herceptin has proved effective in patients with chemotherapy-
resistant breast cancer.
2. Monoclonal antibodies are used to effectively bind the Tumor Necrotic Factor-alpha
(TNF-alpha), which is a cytokine that helps in the progression of Rheumatoid arthritis
(RA). Hence monoclonal antibodies are used as a therapeutic means for Rheumatoid
Arthritis.
3. Monoclonal antibodies have been generated against Tumor-specific Transplantation
Antigens (TSTAs). These are antigens that result from gene mutations that cause
altered proteins that are expressed by tumor cells. Practically, the patient tumor cells
are tagged with monoclonal antibodies that have toxins or radioactive materials. This
delivers a direct ‘magic-bullet’ therapeutic effect to the tumor and spares the healthy
cells.
4. They are used as an identification tool for several cancers and to also deliver drug
therapies to target cancer cells and initiate immune responses against the cancer cells.
5. Monoclonal antibodies are used in the diagnosis of several diseases by detecting
specific antigens circulating in the body tissues and detecting them by the use of
immunoassay techniques.
6. Currently, monoclonal antibodies are being studied by the COVID-19 Prevention
Network. for the treatment of COVID-19. Some trials have been rallied out in the US
to understand the role of monoclonal antibodies in providing short-term protection
against SARS-CoV-2 the causative agent of COVID-19.
Uses of Monoclonal Antibodies
Monoclonal antibodies have many practical applications in research, medical diagnosis,
and therapy. Some of their common applications include the following:
1. Identification of phenotypic markers unique to particular cell types. The basis for the
modern classification of lymphocytes and other leukocytes is the recognition of
individual cell populations by specific monoclonals These antibodies have been used
to define clusters of differentiation (CD) markers for various cell types.
2. The diagnosis of many infectious and systemic diseases relies on the detection of
particular antigens or antibodies in the blood, urine, or tissues by the use of
monoclonal antibodies in immunoassays.
3. Tumor identification. Labelled monoclonal antibodies specific for various cell
proteins are used to determine the tissue source of tumours by staining histological
tumor sections.
4. Advances in medical research have led to the identification of cells and molecules that
are involved in the pathogenesis of many diseases. Monoclonal antibodies, because of
their exquisite specificity, provide a means of targeting these cells and molecules. A
number of monoclonal antibodies are used therapeutically.
5. Functional analysis of cell surface and secreted molecules.

Limitations of Monoclonal Antibodies

1. Monoclonal antibodies are most easily produced by immunizing mice, but patients
treated with mouse antibodies may make antibodies against the mouse Ig, called
human anti-mouse antibody (HAMA). These anti- Ig antibodies block the function or
enhance clearance of the injected monoclonal antibody and can also cause a disorder
called serum sickness.
2. Genetic engineering techniques have been used to expand the usefulness of
monoclonal antibodies. The complementary DNAs (cDNAs) that encode the
polypeptide chains of a monoclonal antibody can be isolated from a hybridoma and
these genes can be manipulated in vitro.
3. Fully human monoclonal antibodies are also in clinical use. These are derived using
phage display methods or in mice with B cells expressing human Ig transgenes.
Humanized antibodies are far less likely than mouse monoclonals to appear foreign in
humans and to induce anti-antibody responses.

Side effects of Monoclonal Antibodies

● Needle site reaction associated with Pain, swelling, soreness, redness, itchiness, rash
● Flu-like symptoms associated with chills, fatigue, nausea, vomit, fever, diarrhea,
muscle ache, pain
● Effects of monoclonal antibodies associated:
● Mouth and skin sores that can lead to serious infections,
● High blood pressure,
● Congestive heart failure,
● Heart attacks, Inflammatory lung disease
Monoclonal antibody treatment for cancer
Mechanisms of Action of Monoclonal Antibodies in Cancer Therapy Monoclonal
antibodies (mAbs) have revolutionized cancer therapy by leveraging highly specific
targeting mechanisms to combat malignant cells. These antibodies function through
various pathways that either directly inhibit tumor growth or engage the immune system
to enhance tumor elimination. The primary mechanisms of action include direct tumor
antigen targeting, immune-mediated cytotoxicity, complement activation, and immune
checkpoint inhibition.
Mechanisms of Action
1. Direct Tumor Targeting and Growth Inhibition
Monoclonal antibodies can bind to specific tumor-associated antigens, blocking
key pathways necessary for cancer cell proliferation and survival. Trastuzumab,
for instance, targets the HER2 receptor in HER2- positive breast cancer,
preventing dimerization and signaling, thereby reducing uncontrolled growth.
Similarly, Cetuximab, an anti-EGFR antibody, inhibits epidermal growth factor
receptor activation, leading to reduced cell division in colorectal and head-and-
neck cancers. Another major category of direct tumor-targeting mAbs includes
antibody-drug conjugates (ADCs). These mAbs are chemically linked to cytotoxic
agents and deliver the therapeutic payload directly to the cancer cells.
Trastuzumab emtansine (T-DM1) exemplifies this approach, where trastuzumab is
conjugated to a microtubule inhibitor, selectively killing HER2-expressing tumor
cells while sparing normal cells.
2. Antibody-Dependent Cellular Cytotoxicity (ADCC)
One of the primary immune-mediated mechanisms of monoclonal antibodies is
antibody-dependent cellular cytotoxicity (ADCC). In this process:
• The Fab region of the monoclonal antibody binds to the target antigen on the
cancer cell surface.
• The Fc region of the antibody interacts with Fc gamma receptors (FcγRs) on
immune effector cells such as natural killer (NK) cells, macrophages, and
dendritic cells.
• The activated effector cells release perforins and granzymes, leading to
apoptosis and tumor cell death. Examples of monoclonal antibodies that induce
ADCC include Rituximab (anti-CD20 for B-cell malignancies), Daratumumab
(anti-CD38 for multiple myeloma), and Atezolizumab (anti-PD-L1 in lung and
urothelial cancers).
 3. Complement-Dependent Cytotoxicity (CDC)
Complement-dependent cytotoxicity (CDC) is another immune-mediated
mechanism that involves the activation of the complement cascade upon antibody
binding to the tumor antigen. This leads to the formation of the membrane attack
complex (MAC), which creates pores in the tumor cell membrane, leading to lysis
and cell death. Monoclonal antibodies such as Ofatumumab and Rituximab, which
target CD20 in B-cell malignancies, have demonstrated CDC activity in clinical
settings, enhancing tumor clearance through complement activation.
4. Immune Checkpoint Inhibition
Some cancers evade immune destruction by exploiting immune checkpoints,
which are regulatory pathways that suppress immune system activation.
Monoclonal antibodies targeting these checkpoints help restore immune function
and enable T-cell-mediated tumor destruction. Two key classes of immune
checkpoint inhibitors are:
• PD-1/PD-L1 Inhibitors: Programmed death-1 (PD-1) is an inhibitory receptor
on T-cells, and its ligand, PD-L1, is often overexpressed in tumors. Nivolumab
and Pembrolizumab (anti-PD-1) and Atezolizumab and Durvalumab (anti-PD-L1)
block this interaction, allowing T-cells to recognize and destroy cancer cells.
• CTLA-4 Inhibitors: Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)
downregulates immune activation by inhibiting co-stimulatory signals required for
T-cell activation. Ipilimumab (anti-CTLA4) enhances immune response by
blocking this suppressive mechanism, particularly in melanoma treatment
Clinical Applications
Monoclonal Antibodies in Hematologic Malignancies
Hematologic cancers, including leukemias, lymphomas, and multiple myelomas, have been
extensively treated using monoclonal antibodies. Several antibodies have been approved for
targeting specific markers expressed on blood cancer cells.
• Rituximab (Anti-CD20):
Used in Non-Hodgkin’s Lymphoma (NHL) and Chronic Lymphocytic Leukemia (CLL).
Binds to CD20 on B cells, leading to immune-mediated B cell destruction via antibody-
dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and
apoptosis induction.
Often combined with chemotherapy regimens like R-CHOP (Rituximab, Cyclophosphamide,
Doxorubicin, Vincristine, and Prednisone).
• Daratumumab (Anti-CD38):
Approved for Multiple Myeloma.
Induces tumor destruction through ADCC, CDC, and direct apoptosis.
Enhances the effects of standard chemotherapy regimens such as Daratumumab +
Lenalidomide + Dexamethasone.
• Blinatumomab (CD19/CD3 Bispecific Antibody):
Used for Acute Lymphoblastic Leukemia (ALL).
Bridges CD19-expressing B cells and CD3-expressing T cells, leading to T cell-mediated B
cell destruction
2. Monoclonal Antibodies in Solid Tumors
Monoclonal antibodies have revolutionized the treatment of solid tumors by targeting tumor-
specific antigens and disrupting key pathways that promote cancer progression.
• Trastuzumab (Anti-HER2):
HER2-positive Breast and Gastric Cancer.
Inhibits HER2 receptor signaling, preventing cancer cell proliferation.
Used in Trastuzumab + Chemotherapy (Paclitaxel, Carboplatin) combination therapy.
• Bevacizumab (Anti-VEGF):
Colorectal, Lung, Renal, and Ovarian Cancer.
Blocks vascular endothelial growth factor (VEGF), preventing tumor angiogenesis and
starving tumors of blood supply.
Often combined with chemotherapy (Bevacizumab + FOLFIRI in colorectal cancer).
• Cetuximab (Anti-EGFR):
Colorectal Cancer and Head & Neck Squamous Cell Carcinoma.
Inhibits epidermal growth factor receptor (EGFR), reducing tumor cell growth.
Effective in KRAS wild-type colorectal cancer when combined with chemotherapy
(FOLFIRI + Cetuximab).
3. Immune Checkpoint Inhibitors:
Expanding Monoclonal Antibody Therapy Checkpoint inhibitors are a unique class of
monoclonal antibodies that reinvigorate the immune system to attack cancer cells.
• Nivolumab and Pembrolizumab (Anti-PD-1):
Non-Small Cell Lung Cancer (NSCLC), Melanoma, Hodgkin’s Lymphoma. o Block
PD-1, restoring T cell-mediated immune response.
Combined with chemotherapy or Ipilimumab (CTLA-4 inhibitor) for synergistic
effects.
• Atezolizumab and Durvalumab (Anti-PD-L1):
Bladder, Lung, and Breast Cancer.
 Target PD-L1, preventing tumor immune evasion.
Approved in combination with chemotherapy for NSCLC and triple-negative breast
cancer.
4. Antibody-Drug Conjugates (ADCs):
Enhancing Tumor-Specific Killing Antibody-drug conjugates (ADCs) combine the
specificity of monoclonal antibodies with the cytotoxic power of chemotherapy drugs,
allowing for precise targeting of tumor cells.
• Trastuzumab emtansine (T-DM1): HER2-positive breast cancer.
Challenges and Future Directions
● Resistance: Some patients develop resistance to mAb therapies, necessitating ongoing
research to overcome this hurdle.
● Side Effects: While generally fewer than traditional therapies, mAbs can still cause
adverse effects like cytokine release syndrome.