Monoclonal Antibodies & Hybridoma Technology and its Application

1. MONOCLONAL ANTIBODIES
& HYBRIDOMA TECHNOLOGY
AND ITS APPLICATIONS
Shadab Khan
M. Pharm. I Sem.
Biotechnology & Bioinformatics

2. INTRODUCTION
 Antibodies or immunoglobulins are
protein or molecules produced by
B-lymphocytes. They are part of defense
system to protect the body against
invading foreign antigen. Each antigen
has specific epitopes located on it.
The antibody have complementary
determining regions (CDRs) which
are mainly responsible for the antibody specificity.
 Single antigen would usually induct more than once distinct clones of the
prevailing plasma cells and, therefore, it will give rise to the production of
antibodies bearing variant specificities. The serum of an animal
adequately immunized by a single antigen shall definitely comprise
antibodies with various specificities, but reacting particularly to the same
antigen. These specific variety of antibodies are invariably termed as
Polyclonal Antibodies because they are eventually produced by a good
number of different plasma cell-clones. Due to lack of specificity and
heterogenic nature, there are several limitation on the utility of polyclonal
antibodies for therapeutic and diagnostic purpose.

3.  Monoclonal antibody (MAb) is a single type of antibody that
is a single type of antibody that is directed against a specific
antigenic determinant (epitope).
 In the last 20 years or so, antibody-based therapeutics have
mainly focused upon the medical application of monoclonal
antibodies. Monoclonal antibody technology was first
developed in 1975, when George Kohler and Cesar Milstein
(Nobel Prize, 1984) successfully fused immortal myeloma
cells with antibody-producing B-lymphocytes. A proportion of
the resultant hybrids were found to be stable, cancerous,
antibody-producing cells. These ‘hybridoma’ cells
represented an inexhaustible
source of monospecific (monoclonal)
antibody. Hybridoma technology
facilitates the relatively
straightforward production of
monospecific antibodies against
virtually any desired antigen.

4. PRINCIPLE FOR CREATION OF HYBRIDOMA CELLS:
 The myeloma cells must not synthesize their own antibodies. After
fusion the hybrid cell must be separated
from intact parent cells. Selection is based
on inhibiting nucleotide synthesis. Synthesis
of nucleotides by two pathways:
 de novo synthesis
 Salvage pathway
 In de novo synthesis requires
tetrahydrofolate which is blocked by
aminopterin.
 In Salvage pathway, purine and pyrimidine directly convert into
nucleotide. Hypoxanthine guanine phosphoribosyl transeferase
(HGPRT) is key enzyme which converts hypoxanthine and guanine
into inosine monophosphate and guanine monophosphate.
Thymidine Kinase (TK) converts thymidine into thymidine
monophosphate. Mutation in one enzyme blocks salvage pathway.

5.  In HAT medium (hypoxanthine aminopterin and thymidine)
mutated cell die due to de novo synthesis inhibition. While
hybridoma cells survive because HGPRT functional gene obtained
from lymphocytes.

6. PRODUCTION OF MONOCLONAL ANTIBODIES
 Steps Involved:
 Immunization
 Cell fusion
 Selection of hybridoma
 Screening the products
 Cloning and propagation
 Characterization and storage
 IMMUNIZATION: Animal (usually mouse) immunize with
appropriate antigen. Freund’s complete or incomplete adjuvants
(adjuvants potentiate non specific immune response) are also
injected subcutaneously. Injections at multiple site at multiple
times increase stimulation of B lymphocytes. Immune stimulated
cells grown maximally by intravenous antigen administration three
days prior to killing. Serum antibody concentration assayed
frequently during immunization.
 When the serum concentration of the antibodies is optimal, the
animal is sacrificed.

7. The spleen is aseptically removed and
disrupted by mechanical or enzymatic
methods to release the cells. The
lymphocytes of the spleen are separated
from the rest of the cells by density
gradient centrifugation.
 CELL FUSION: The thoroughly washed
lymphocytes are mixed with HGPRT
defective myeloma cells. The mixture of
cells is exposed to polyethylene glycol
(PEG) for a short period (a few minutes),
since it is toxic. PEG is removed by
washing and the cells are kept in a
fresh medium. These cells are composed
of a mixture of hybridoma (fused cells), free myeloma cells and free
lymphocytes.
 SELECTION OF HYBRIDOMAS: When the cells are cultured in HAT
medium, only the hybridoma cells grow, while the rest will slowly
disappear. This happens in 7—10 days of culture.

8. The suspension of hybridoma cells is so diluted that the individual
aliquots contain on an average one cell each. These cell, when
grown in a regular culture medium, produce the desired antibody.
 SCREENING THE PRODUCTS: The hybridomas must be screened for
the secretion of the antibody of desired specificity. The culture
medium from each hybridoma culture is periodically tested for the
desired antibody specificity. The two techniques namely ELISA and
RIA are commonly used for this purpose. In both the assays, the
antibody hinds to the specific antigen (usually coated to plastic
plates) and the unbound antibody and other components of the
medium can he washed off. Thus, the hybridoma cells producing
the desired antibody can be identified by screening. The antibody
secreted by the hybrid cells is referred to as monoclonal antibody.
 CLONING AND PROPAGATION: The single hybrid cells producing the
desired antibody are isolated and cloned. Two techniques are
commonly employed for cloning hybrid cells.
 Limiting dilution method
 Soft agar method

9.  Limiting Dilution Method: In this procedure, the suspension of
hybridoma cells is serially diluted and the aliquots of each
dilution are put into microculture wells. The dilutions are so
made that each aliquot in a well contains only a single hybrid
cell. This ensures that the antibody produced is monoclonal.
 Soft Agar Method: In this technique, the hybridoma cells are
cultured in soft agar. It is possible to simultaneously grow many
cells in semisolid medium to form colonies. These colonies will
be monoclonal in nature.
 Both the above techniques are combined and used for maximal
production of MAbs.
 CHARACTERIZATION AND STORAGE: The monoclonal antibody
has to be subjected to biochemical and biophysical
characterization for the desired specificity. It is also important to
elucidate the MAb for the immunoglobulin class or sub-class,
the epitope for which it is specific and the number of binding
sites it possesses.

10.  The stability of the cell lines and the MAbs are important. The
cells (and MAbs) must be characterized for their ability to
withstand freezing and thawing. The desired cell lines are frozen
in liquid nitrogen at several stages of cloning and culture.

11.  LARGE SCALE PRODUCTION OF MAbs: The production MAbs in the
culture bottles is rather low (5-10 mg/ml).
The yield can be increased by growing the hybrid cells as Ascites in
the peritoneal cavity of mice. The ascitic fluid contains about 5—20
mg of MAb/ml. This is far superior than the in vitro cultivation
techniques. Drawbacks:-
 Costly
 Contamination
 Require immunodeficient mice
 Large number of mouse sacrificed
 In vitro fermentation produce low
concentration (0.1 to 0.5 mg/ml) have
certain advantages:-
 No contamination
 Cost effective
 Reliable
 Production can be done on various scales
 Encapsulation of hybridoma cells in alginate gels can increase MAb
production. Damon Biotech Company and Cell Tech use this
technology.

12. HUMAN MONOCLONAL ANTIBODIES
 Mice antibodies are stable but can only used in vitro but in vivo
it cause immunological complications. Human antibodies are
preferred but following are limitations:-
 Ethical reasons
 Hybrid cells are unstable
 Human myeloma cells are not suitable to replace mice
myeloma.
 Thus alternative arrangements are made
 Viral transformation of human
b-lymphocytes
 SCID mouse for producing human
MAbs
 Transgenic mouse for producing
human MAbs
 Bispecific MAbs have two different
epitopes produced by fusing two different
hybridoma cells or by genetic engineering,
useful for treatment of two different diseases.

13. GENETIC ENGINEERING STRATEGIES FOR THE PRODUCTION OF HUMAN- MOUSE MABS:
 With the advances in genetic engineering, it is now possible to add certain human
segments to a mouse antibody. This is truly a hybridized antibody and is referred to
as humanized antibody or chimeric antibody.
 Substitution of Fv region of human Ig by mouse Fv: The DNA coding sequences for
Fv regions of both L and H chains of human immunoglobulin are replaced by Fv DNA
sequence (for L and H chains) from a mouse monoclonal antibody. The newly
developed humanized MAb has Fc region of Ig being human. This stimulates proper
immunological response.
 Substitution of Human Ig by Mouse CDRs: Genetic engineers have been successful
in developing human MAbs containing mouse complementary determining regions
(CDRs). This is made possible by replacing CDRs genes (CDR1, CDR2, and CDR3) of
humans by that of
mouse. These chimeric
antibodies possess
the antigen binding
affinities of the mouse
and they can serve as
effective therapeutic
agents.

15.  High quality, one molecular species, homogenous.
 Specificity for one antigen.
 Antibodies with high avidity.
 Immure immunogen can be used.
 Both in vitro and in vivo production is possible.
 Maintenance of farm/animals is not required.
 Immortal cell lines.
 Antiserum having specific antibody with constant property can
be obtained worldwide.
 High reproducibility.
 Radiolabelling and fluorescent conjugation or enzyme marking
of MAbs are easy.
ADVANTAGES OF MABS
LIMITATIONS OF MABS
 Laborious and time consuming.
 Mice carry several viruses (adenovirus, hepatic virus, retrovirus,
reovirus, thymic virus) whose presence has been detected in
hybridomas.
 Virus free antibody is not guaranteed.

17. APPLICATIONS OF MONOCLONAL ANTIBODIES
 Diagnostic applications
 Therapeutic uses
 Protein purification
 Miscellaneous applications
 DIAGNOSTIC APPLICATIONS: MAbs have revolutionized the
laboratory diagnosis of various diseases.
 MAbs IN BIOCHEMICAL ANALYSIS: Used in Radio Immuno Assay
(RIA) and Enzyme Linked Immuno Sorbent Assays (ELISA). These
assays measures circulating concentration of hormones (insulin,
HCG, Growth Hormone, progesterone, thyroxine,
triiodothyronine, TSH, gastrin, renin) and several other tissue
and cell products (blood group antigens, blood clotting factors,
interferons, interleukins, histocompatibilty antigens, tumor
markers).
 Commercial diagnostic kits available for diagnosis:
 Pregnancy Kits: The hCG Card Pregnancy Test is a rapid
chromatographic immunoassay for the qualitative detection of
human chorionic gonadotropin in urine to aid in the early
detection of pregnancy.

18.  Cancer: MABs work by recognizing and
finding specific proteins on cancer cells.
Each MAB recognizes one particular
protein. So different MABs have to be
made to target different types of cancer.
Depending on the protein they are
targeting, they work in different ways
to kill the cancer cell or stop it from growing.
Colorectal cancer, prostate cancer, etc. estimated through MAbs.
 Hormonal Disorders
 Infectious Diseases: Detection of antigens of Neisseria gonorrhea
and herpes simplex virus for diagnosis STD.
 MAbs IN DIAGNOSTIC IMAGING: Radiolabelled MAbs are used in the
diagnostic imaging of diseases, and this technique is referred to as
immunoscintigraphy. The radioisotopes commonly used for labeling
MAb are I131 and Tc99. The MAb tagged with radioisotope are injected
intravenously into the patients. These MAbs localize at specific sites
(say a tumor) which can be detected by imaging the radioactivity.
Immunoscintigraphy is a better diagnostic tool than the other imaging
techniques because it can differentiate between cancerous and non-
cancerous growth, since radiolabelled MAbs are tumor specific.

19.  Cardiovascular Disease:
 Myocardial infarction: The cardiac protein myosin gets exposed
wherever myocardial necrosis (death of cardiac cells) occurs.
Antimyosin MAb labeled with radioisotope indium chloride (111 In)
is used for detecting myosin and thus the site of myocardial
infarction. It is possible to detect the location and the degree of
damage to the heart by using radiolabelled antimyosin MAb.
 Deep vein thrombosis (DVT): DVT refers to the formation of
blood clots (thrombus) within the blood veins, primarily in the
lower extremities. For the detection of DVT, radioisotope labeled
MAb directed against fibrin or platelets can be used. Fibrin
specific MAbs are successfully used for the detection of clots in
thigh, pelvis, calf and knee regions.
 Atherosclerosis: Thickening and loss of elasticity of arterial walls
is referred to as atherosclerosis. Atherosclerosis has been
implicated in the development of heart diseases. MAb tagged
with a radiolabel directed against activated platelets can be used
to localize the atherosclerotic lesions by imaging technique.

20.  Cancers: Tumors can be located in patients using radioisotope
labeled MAbs specific to the protein(s), particularly of membrane
origin.
 It has been possible to detect certain cancers at early stages (lung
cancer, breast cancer, ovarian cancer, melanoma, colorectal cancer)
by employing MAbs. About 80 per cent specificity has been achieved
for detecting cancers by this approach.
 An iodine (131l) labeled monoclonal antibody specific to breast cancer
cells when administered to the patients detects (by imaging) the
spread of cancer (metastasis) to other regions of the body. This is not
possible by scanning techniques. The imaging technique by using
MAb can also be used to monitor therapeutic responses of a cancer.
 There are certain limitations in using MAb in cancer diagnosis and
prognosis. These include the difficulty in the selection of a specific
MAb and the access of MAb to the target site of the tumor which
may be less vascularized.
 MAbs in immunohistopathology of cancers: The pathological
changes of the cancerous tissue can be detected by
immunohistochemical techniques. This can be done by using MAb
against a specific antigen.

21.  THERAPEUTIC APPLICATIONS: Monoclonal antibodies have a wide
range of therapeutic applications. MAbs are used in the treatment of
cancer, transplantation of bone marrow and organs, autoimmune
diseases, cardiovascular diseases and infectious diseases. The
therapeutic applications of MAbs are broadly grouped into 2 types:
 Direct use of MAbs as therapeutic agents
 MAbs as targeting agents
 MAbs AS DIRECT THERAPEUTIC AGENTS: Monoclonal antibodies
can be directly used for enhancing the immune function of the
host.
 In destroying disease-causing organisms: MAbs promote
efficient opsonization of
pathogenic organisms (by coating
with antibody) and enhance
phagocytosis.

22.  In the treatment of cancer: MAbs, against the antigens on the
surface of cancer cells, are useful for the treatment of cancer.
The antibodies bind to the cancer cells and destroy them. This
is brought out by antibody—dependent cell-mediated
cytotoxicity, complement-mediated cytotoxicity and
phagocytosis of cancer cells (coated with MAbs) by
reticuloendothelial system.
 Limitations for direct use of MAbs in cancer:
 The MAbs produced in mice and directly used for
therapeutic purposes may lead to the development of anti-
mouse antibodies and hypersensitivity reactions.
 All the cancer cells may not carry the same antigen for
which MAb has been produced. Thus, MAbs may not be
attached to some cancer cells at all.
 The free antigens (of target cells) present in the circulation
may bind to MAbs and prevent them from their action on
the target cells.

23.  In the immunosuppression of organ transplantation: In
recent years, MAbs specific to T-lymphocyte surface
antigens are being used for this purpose. The monoclonal
antibody namely OKT3, was the first MAb to be licensed by
U.S. OKT3 specifically directed against CD3 antigen of T-
lymphocytes is successfully used in renal and bone
marrow transplantations. CD3 antigen activates T-
lymphocytes and plays a key role in organ transplant
rejection.
 MAbs AS TARGETING AGENTS IN THERAPY: Toxins, drugs,
radioisotopes etc., can be attached or conjugated to the
tissue-specific monoclonal antibodies and carried to target
tissues for efficient action. This allows higher concentration
of drugs to reach the desired site with minimal toxicity. In
this way, MAbs are used for the appropriate delivery of drugs
or isotopes.
 MAbs in use as immunotoxins: The toxins can be coupled
with MAbs to form immunotoxins and used in therapy
e.g., diphtheria toxin, Pseudomonas exotoxin, toxins used
for cancer treatment.

24.  MAbs in drug delivery: The drugs can be
coupled with MAb (directed against a
cell surface antigen of the cells, say
a tumor) and specifically targeted
to reach the site of action.
In the treatment of certain
diseases, a pro-drug (an inactive
form of the drug) can be used.
This can be enzymatically
converted to active drug in the target tissues.
For this purpose, the enzyme (that converts pro-drug to drug) is
coupled with MAb that is directed against a specific cell surface
antigen. This approach, referred to as antibody-directed enzyme
pro-drug therapy (ADEPT), allows an effective delivery of the drug
to the cells where it is required.
 MAbs in the dissolution of blood clots: A monoclonal antibody
directed against fibrin can be coupled to tPA and used for
degradation of blood clots. MAb-tPA complex due to a high affinity
gets attached to fibrin. . Due to the concentration of tPA at the
target spots, there is more efficient conversion of plasminogen to
plasmin which in turn dissolves blood clot (fibrin).

25.  PROTEIN PURIFICATION: Monoclonal antibodies can be produced
for any protein. And the so produced MAb can be conveniently
used for the purification of the protein against which it was raised.
MAbs columns can be prepared by coupling them to cyanogen
bromide activated Sepharose (chromatographic matrix). The
immobilized MAbs in this manner are very useful for the
purification of proteins by immunoaffinity method.
 Advantage:
 Specificity
 Very efficient and high degree of purification.
 More than 5,000 fold purification of interferon-α2 is possible
to achieve.
 Disadvantages:
 not possible to achieve 100% purity
 cannot distinguish between the intact target protein and a
fragment of it with the antigenic site.

26.  MISCELLANEOUS APPLICATIONS:
 Catalytic MAbs (Abzymes): Catalysis is the domain of enzymes. The
most important common character between enzymes and
antibodies is that both are proteins. The binding of both is specific
with high affinity and involves weak and non-covalent interactions.
The striking difference is that the enzyme alters the substrate while
the antigen bound to antibody remains unaltered.
 Certain similarities between enzyme-substrate interaction and
antibody-antigen interaction have tempted researchers to explore
the possibility of using antibodies in catalysis. The antibody
enzymes, appropriately regarded as abzymes, are the catalytic
antibodies. There is a difference in the antibody recognition of an
antigen and enzyme recognition of a substrate. While the
antibodies recognize in ground state, the enzymes recognize in a
transition state.
 Advantages: The number of naturally occurring enzymes and their
catalytic functions are limited. Antibodies, on the other hand, are
unlimited, and may be developed to possess recognized site
structures, appropriate for the catalytic functions.
 Limitations: It is doubtful whether they will ever match the natural
enzymes in their catalytic function.

27.  Autoantibody Fingerprinting: A new category of individual
specific (IS) autoantibodies have been discovered in recent
years. These IS-autoantibodies are produced after birth and
reach maximum in number by 2 years, and then remain
constant for the later part of life. Monoclonal antibodies
produced against IS-autoantibodies can be used for their
detection, and identification of individuals. The autoantibodies
collected from samples such as blood, saliva, semen and tears
can be used.
 Adverse Effects: They are biologic products and can elicit a
number of immune-mediated and other reactions and adverse
effects. In general, mAb treatment carries fewer side effects
than traditional chemotherapy treatments. However, mAb
treatment for cancer may cause rare side effects that can be
very serious. The standard infusion reactions or the most
common side effects caused include: allergic reactions, such as
hives or itching; flu-like signs and symptoms, including chills,
fatigue, fever, and muscle aches and pains; nausea; vomiting;
diarrhea; skin rashes; and low blood pressure.

28. MONOCLONAL/ENGINEERED ANTIBODIES APPROVED FOR MEDICAL USE
Product Company Indication
CEA-Scan (Arcitumomab, murine Mab fragment
(Fab), directed against human (CEA) Immunomedics
Detection of
recurrent/metastatic
colorectal cancer
MyoScint (Imiciromab-Pentetate, murine Mab
fragment directed against human cardiac myosin) Centocor Myocardial infarction imaging
agent
Orthoclone OKT3 (Muromomab CD3, murine
Mab directed against the T-lymphocyte surface
antigen CD3)
Ortho Biotech Reversal of acute kidney
transplant rejection
ReoPro (Abciximab, Fab fragments derived from
a chimaeric Mab, directed against the platelet
surface receptor GPIIb/IIIa)
Centocor Prevention of blood clots
Verluma (Nofetumomab murine Mab fragments
(Fab) directed against carcinomaassociated
antigen)
Boehringer
Ingelheim/ NeoRx
Detection of small cell lung
cancer
Simulect (Basiliximab, chimaeric Mab directed
against the α chain of the IL-2 receptor) Novartis
Prophylaxis of acute organ
rejection in allogeneic renal
transplantation
LeukoScan (Sulesomab, murine Mab fragment
(Fab) directed against NCA 90, a surface
granulocyte non-specific cross-reacting antigen)
Immunomedics
Diagnostic imaging for
infection/ inflammation in
bone of patients with
osteomyelitis
Humira (EU & USA; also sold as Trudexa in EU)
(adalimumab; r (anti-TNF) human monoclonal
antibody created using phage display technology
Cambridge Antibody
Technologies &
Abbott (USA) Abbott
(EU)
Rheumatoid arthritis
Xolair (omalizumab; humanized monoclonal
which binds immunoglobulin E at the site of high
affinity IgE receptor binding)
Genentech/Novartis/
Tanox/Sankyo
Treatment of
adults/adolescents with
moderate to severe persistent
asthma
Avastin (humanized monoclonal raised against
vascular endothelial growth factor).
Genentech (USA)
Roche (EU)
Carcinoma of the colon or
rectum

29. CONCLUSION
 The commercial development of therapeutic monoclonal
antibodies commenced in the early 1980s, and by 1986 the first
therapeutic monoclonal antibody, Orthoclone OKT3, was
approved for prevention of kidney transplant rejection. Since the
approval of OKT3, therapeutic monoclonal antibodies and
antibody-related products such as Fc-fusion proteins, antibody
fragments, and antibody-drug conjugates (collectively referred
to hereafter as monoclonal antibody products) have grown to
become the dominant product class within the
biopharmaceutical market.
 Monoclonal antibody products today are approved for the
treatment of a variety of diseases, ranging from those that treat
patient populations of a few thousand or less for such orphan
indications as paroxysmal nocturnal hemoglobinuria or the
cryopyrin-associated periodic syndromes to those treating
hundreds of thousands of patients for some cancers and
multiple sclerosis or even millions of patients for diseases such
as asthma and rheumatoid arthritis.

30.  So far, about 80 MAbs have been approved by the FDA to detect,
diagnose, and treat many different diseases.
 There are over 500 monoclonal antibodies includes approved and
investigational drugs as well as drugs that have been withdrawn from
market.
 The continued interest in antibody product development is partially
driven by the rapid advancement of our understanding of disease at a
molecular level.
 Monoclonal antibody products often provide the most rapid route to a
clinical proof-of-concept for activating, inhibiting, or blocking these new
targets. Since the production of most monoclonal antibody products is
easily amenable to efficient platform-based approaches and antibodies
are generally well-tolerated and highly specific, the risk of unexpected
safety issues in human clinical trials of monoclonal antibody products is
lower than with many other types of therapeutic products.
 Since 2000, the therapeutic market for monoclonal antibodies has grown
exponentially. In 2007, eight of the 20 best-selling biotechnology drugs
in the U.S. are therapeutic monoclonal antibodies. This rapid growth in
demand for monoclonal antibody production has been well
accommodated by the industrialization of MAb manufacturing.

31. THANK YOU....