Monoclonal Antibodies

Introduction

Monoclonal antibodies are mono-

specific antibodies that are
produced in laboratory from
single clone of B cells that
recognizes only one antigen.
 Monoclonal Antibodies

• MAbs produced from a single clone of B cells

• Monoclonal antibodies all have identical antigen-binding

sites. Thus they all bind to the same epitope with the
same affinity.

• Mostly produced by fusing a B cell secreting the desired

antibody with a myeloma cell capable of growing
indefinitely in tissue culture
 Beginning of Monoclonal Era
• Georg Kohler and Cesar Milstein fused mouse lymphocytes
with neoplastic mouse plasma cells to yield hybridomas that
produced specific antibodies.
• This offers a limitless supply of monoclonal antibodies.
mAb Production

•There are currently two widely adopted

methods used to produce monoclonal Ab.
•In vivo & in vitro
•There are advantages and disadvantages
for both methods.
•The generation of mAb producing cells are
typically done in mice.
•This method involves following steps;
 Preparations of mAb
Step 1: Immunization of mice
• Mice are immunized with an antigen (attached to
adjuvant).
• The antigen can be whole cells, membrane fragment,
or complex molecules.
• Mice serum’s are screened using various techniques
such as ELISA. (Enzyme-linked immunosorbent assay is a plate-based
assay technique designed for detecting and quantifying substances such as
peptides, proteins, antibodies and hormones)
• When sufficient titer is reached the mice are
euthanized and spleen is removed as a source of cells
for cell fusion.
Step 2: Preparation of Myeloma Cells
• Myeloma cells are immortalized cells that are capable
of dividing indefinitely.
• These cells are treated with 8-azaguanine to ensure
sensitivity to HAT (hypoxanthine-aminopterin-
thymidine).
Step 3: Fusion of myeloma cells with Spleen cells
• Spleen cells harvested from mice are fused with myeloma
cells.
• Fission is done through co-centrifuging in polyethylene
glycol.
• Cells are plated in selection medium
• Hypoxanthine-aminopterin-thymidine (HAT) selection
medium-inhibitor of aminoterin which blocks nucleotide
synthesis.
• Only fused cells with grow on HAT.
• Cells are distributed on feeder cells (murine bone-
marrow) to promote growth of the hybridoma cells.
 Hybridoma Selection The “HAT Trick”

• Myeloma cells have been genetically engineered

(HGPRT-) such that they can not use Hypoxanthine,
Aminopterin, and Thymidine (HAT medium) as a source
for nucleic acid biosynthesis and will die in culture.
• Only B cells that have fused with the engineered
myeloma cells will survive in culture when grown in
HAT medium.
 Hybridoma Selection The “HAT Trick”

• This is possible because myeloma cells have lost the

ability to synthesize hypoxanthine-guanine-
phosphoribosyl transferase (HGPRT), an enzyme
necessary for the salvage synthesis of nucleic acids.
Unfused spleen cells cannot grow indefinitely because of
their limited life span.
• Only fused hybrid cells, referred to as hybridomas, are
able to grow indefinitely in the media because the spleen
cell partner supplies HGPRT and the myeloma partner has
traits that make it immortal (similar to a cancer cell).
Step 4: Cloning of Hybridoma cells
• A mouse is inoculated with the cell and thereby
becomes a factory for producing the mAB.
• Ascites are collected from the mouse.
• Ascites is the term that describes the fluid that fills
the peritoneal cavity due to inflammation. For in vivo
monoclonal antibody production, hybridoma cells are
injected into the peritoneal cavity to resulting in
ascites fluid containing the desired antibody.
Step 5: Ab are screened and Purified
•Ab are screened using specific Ag binding.
Advantage of in vivo process:
•Relatively inexpensive and easy
Disadvantage:
•Ethical concerns with using animals.

Step 6: Desired Ab are cloned

• This is done in vitro on culture bottles
 Problems while using mouse mAb
• The therapeutic use of rodent monoclonal antibodies
in humans is limited by their immunogenic, short
circulating half-life, and inability to efficiently
trigger human effectors mechanisms.
• This is due to differences between the mouse and
humans.
• Also allergic response in human when mouse mAb
are introduced to a patients are anticipated.
• Also constant region of marine mAb are not
effective in interacting with human effectors
molecules.
 Chimeric mAb

• mAb are genetically engineered using a molecular

approach

• Chimeric Ab are obtained by genetically fusing the mouse

variable domains to human constant domains [Boulianne et
al.,1984; Morrison et al., 1984;Wright et al., 1992]

• Variable regions are Isolated using polymerase chain

reaction (PCR).
Chimeric mAb
Chimeric mAb
 Issues with Chimeric mAb

• There are problems that can arise from using mouse-

human Ab. sometimes the body may elicit an anti-chimeric
Ab in the present of these genetically engineered Ab.
Humanized mAb

• To address this problem, the complementary regions

(CDRs), which are the responsible for antigen binding
within the variable regions, have been transferred to human
frameworks creating ‘‘CDR-grafted’’ or ‘‘humanized’’
antibodies. This is, in essence a human Ab with small
segments containing mouse Ab genes.
 Polyclonal antibodies Monoclonal Antibodies

Produced by: Many B cell clones A single B cell clone

Bind to: Multiple epitopes of all A single epitope of a single

antigens used in the antigen
immunization

Antibody class: A mixture of different All of a single Ab class

Ab classes (isotypes)

Ag-binding sites: A mixture of Abs with All Abs have the same
antigen
different antigen-binding binding site
sites

Potential for cross-reactivity: High Low

Uses

• Measuring protein and drug levels in serum

• Typing tissue and blood
• Identifying infectious agents
• Identifying clusters of differentiation for the classification and
follow-up therapy of leukemias and lymphomas
• Identifying tumor metastasis
• Identifying and quantifying hormones
• Immunoaffinity Purification
Examples of FDA approved treatments of certain cancers

MAb Name Trade Name Used to Treat: Approved in:

Rituximab Rituxan Non-Hodglymphoma 1997

Trastuzumab Herceptin Breast cancer 1998

Gemtuzumab Acute myelogenous

Mylotarg 2000
ozogamicin* leukemia (AML)

Chronic lymphocytic
Alemtuzumab Campath 2001
leukemia (CLL)

Non-Hodgkin
Ibritumomab tiuxetan* Zevalin 2002
lymphoma
Non-Hodgkin
Tositumomab* Bexxar 2003
lymphoma

Colorectal cancer 2004

Cetuximab Erbitux
Head & neck cancers 2006

Bevacizumab Avastin Colorectal cancer 2004

APPLICATIONS

• Diagnostic use
• A breakthrough in Diagnostics
• Critical Diagnostic decisions
• Immunodiagnostic tests
• Finding solutions for Human use
• Conjugated monoclonal antibody therapy
• Cancer treatment
• herceptin
 Diagnostic use
• Although monoclonal antibodies were first produced in 1975 as research
tools, scientists quickly recognized their practical uses, especially in
diagnostic tests and in therapy. Several diagnostic procedures that use
monoclonal antibodies are now available
A breakthrough in Diagnostics

• A monoclonal antibody can be used to detect A breakthrough in

Diagnostics
• pregnancy only 14 days after conception. Other monoclonal
antibodies allow rapid diagnosis of hepatitis, influenza, herpes,
streptococcal, and Chlamydia infections .
Critical Diagnostic decisions

• Helps in Critical Diagnostic decisions. They can be used to detect for

the presence and quantity of this substance, for instance in a
Western blot test (to detect a substance in a solution) or an immune
fluorescence test.
Immunodiagnostic tests

• Monoclonal's helps In Immunodiagnostic tests . Monoclonal

antibodies can also be used to purify a substance with techniques
called immunoprecipitation and affinity chromatography.
Finding solutions for Human use

• In one approach, one takes the DNA that encodes the binding portion
of monoclonal mouse antibodies and merges it with human antibody
producing DNA, in order to make bacteria produce antibodies that
are half mouse and half human.
 Conjugated monoclonal antibody therapy
• Toxins or radioactive isotopes are bound to the constant region of the MAbs.
When the MAb binds to the surface cells of a tumor the toxin or radioactivity
will kill the cancer cells and all cells within a certain radius (a killing zone). In
this way cancer cells within the tumor will be killed.
 CANCER TREATMENT
• Possible treatment for cancer
involves monoclonal
antibodies that bind only to
cancer cells specific antigen
and induce immunological
response on the target cancer
cell (naked antibodies). mAb
can be modificated for
delivery of [toxin],
radioisotope, cytokine
Herceptin (trastuzumab and hyaluronidase
oysk)
• These monoclonal antibodies can be used against certain forms of
breast cancer and have passed clinical trials and approved for use by
the FDA.
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