The document discusses monoclonal antibodies (mAbs), including their production using hybridoma technology. It describes how mouse B cells are fused with myeloma cells to generate immortal hybridoma cell lines that secrete a single antibody clone. The monoclonal antibodies produced through this process have various therapeutic applications for treating cancer, transplant rejection, and inflammatory/autoimmune diseases.

1. HAFIZ M WASEEM
UE LAHORE PAKISTAN

2.  Antibodies are glycoproteins produced by B lymphocytes
(cells of immune system) which neutralize the
pathogens/antigens
 B lymphocytes are cells of acquired immune system
 Immune system is derived from Latin term “IMMUNIS” which
refers to immunity or protection against the pathogens
 It is integrated system of organs, tissues, cells & cell
products (antibodies, cytokines, etc.) that differentiates self
from non – self & neutralizes potentially pathogenic
organisms/antigens

3.  Innate immune system
• primitive
• non specific
• Various types of cells
 Acquired immune
system
• advanced
• specific
• B and T lymphocytes
(give humoral & cell
mediated immunity
respectively)
RESPONSE
Foreign invaders - viruses, bacteria, allergens, toxins and
arasites- constantly bombard our body.

4.  Antibodies are found in extracellular
fluids (blood plasma, lymph, mucus,
etc.) and on the surface of B cells
 There are about 10 million different
types of B lymphocytes
 B cells on stimulation will actively
secrete antibodies and are called
plasma cells
 Provide defense against bacteria,
bacterial toxins and viruses which
circulate freely in body fluids

6. ANTIBODIES
Derived from different B
lymphocytes cell lines
POLYCLONAL. MONOCLONAL.
Derived from a single B cell
clone
Batch to batch variation
affecting Ab reactivity &
titre
Offer reproducible &
inexhaustible supply with
exquisite specificity
Enable the development of
secure immunoassay systems
Not powerful tools for
clinical diagnostic tests

7.  Each antibody has at least
• Two heavy chains
• Two light chains
 Variable Regions: Two regions at the
end of Y’s arms. Identical on the same
antibody, but vary in others
 Constant Regions: Stem of monomer
and lower parts of Y arms which bind to
complement or cells
Structure of antibody and antigen binding cleft

8. IMMUNOTHERAPY
Treatment of the disease by Inducing, Enhancing or
Suppressing the Immune System
Active Immunotherapy
It stimulates body’s own
immune system to fight
disease
Passive Immunotherapy
It rely on the immune system
components (antibodies)
created outside the body

9. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
 Hypoxanthine guanine phosphoribosyl
transferase negative (HGPRT)¯
myeloma cells are selected
 HGPRT enzyme enables cells to
synthesize purines by salvage pathway
 Ordinarily absence of HGPRT is not a
problem because of de novo pathway
 But cells exposed to Aminopterin are
unable to use de novo pathway and are
dependent on salvage pathway only
 HGPRT+ B cells are fused with the
myeloma cells to produce hybridoma
and are grown in HAT medium

10. PRODUCTION OF MONOCLONAL ANTIBODY
Step 1: - Immunization of mice
ANTIGEN (purified
proteins, etc.) +
ADJUVANT
(emulsification)
Serum antibody titre is determined
Titre too low
BOOST
(Pure antigen)
Titre High
BOOST
(Pure antigen)
2 weeks

11. PRODUCTION OF MONOCLONAL ANTIBODY
Step 2: - Removal of spleen for antibody producing B cells
After several
weeks of
immunization
High Ab titre in Serum
Spleen is removed
(source of B cells)

12. PRODUCTION OF MONOCLONAL ANTIBODY
Step 3: - Formation of hybridoma cells
FUSION
PEG
MYELOMA CELLS
SPLEEN CELLS
HYBRIDOMA CELLS
ELISA PLATE
Feeder cells
Growth medium
HAT Medium
1. Plating of cells in
HAT selective
medium
2. Scanning of viable
hybridomas

13. PRODUCTION OF MONOCLONAL ANTIBODY
Step 4: - Cloning of hybridoma cell lines
A. Clone each +ve Culture
B. Test each supernatant for antibodies
C. Expand +ve clones
Mouse
ascites
method
Tissue
culture
method

14.  Cells, cell debris, lipids, and clotted material from supernatant
or ascitic fluid are removed by filtration with a 0.45 µm filter
 Antibodies are commonly separated by protein A/G affinity
chromatography
 mAbs selectively bind to Protein A/G and can be afterwards
eluted
Step 5: - Separation & purification of monoclonal antibodies

15.  Therapeutic applications
Transplant rejection (Muronomab-CD3)
Cardiovascular disease (Abciximab)
Cancer (Rituximab)
Infectious Diseases (Palivizumab)
Inflammatory disease (Infliximab)
 Clinical applications
Purification of drugs, Imaging the target
 Future applications
Fight against Bioterrorism

16.  Hematologic malignancies
 CD20 is highly expressed in B
cell malignancies (95% in
follicular lymphomas)
 Low grade non Hodgkin
lymphoma is being successfully
treated with rituximab
 Rituximab is combined with
chemotherapy for intermediate
grade or diffuse large cell non
Hodgkin lymphoma

17.  Solid tumors
 Solid tumors do not have many specific
targets
 Edrecolomab targets 17-1A antigen (Ep-
CAM) in colon and rectal cancer by
ADCC & CDC methods
 Trastuzumab is being used to target
HER-2 (Human Epidermal growth factor
Receptor 2, CD340) antigen in breast
cancers alone or in combination with
chemotherapy

18.  Muromonab-CD3 (OKT3) binds to CD3 molecule on
surface of T cells and prevents rejection of organs
 Humanized versions of OKT3 show promise in inhibiting
autoimmune destruction of beta cells in Type 1 diabetes
 Infliximab binds to TNF-α and is used in inflammatory
diseases such as rheumatoid arthritis
 Omalizumab binds to IgE thus preventing IgE from
binding to mast cells (allergic asthma)
 Abciximab inhibits clumping of platelets by binding
receptors on their surface and helpful in preventing
clogging of coronary arteries