It includes general introduction to antibodies; Monoclonal antibodies; comparison between Polyclonal & Monoclonal antibodies; Hybridoma Technology & Hyridoma Selection; advantages & disadvantages of mABs; Applications of mABs; Recombinant Monoclonal antibodies production through Antibody Engineering.

1. PRESENTED BY :-
ARUNDHATI MEHTA
B.Sc. BIOTECH VI SEMESTER
© Arundhati Mehta 2016

2. An antibody is a protein ( Antigen binding ) used by the
immune system to identify and neutralize foreign objects like
bacteria and viruses. Each antibody recognizes a specific
antigen unique to its target. These are present on the B-cell
membrane and secreted by
plasma cells.

3. 2 Identical Light chains (~ 220 amino
acid long)
Variable domain : VL
Constant domain: CL
2 Identical Heavy chains (~440 amino
acid long)
Variable domain : VH
3 Constant domain : CH1, CH2, CH3
Covalent Disulphde bonds between
Cysteine residues.
Flexible “ Hinge region ”

4.  In 1890, Von Behring & Kitasato discovered antibodies.
 In1900, Ehrlich proposed the “ side-chain theory.”
 In 1955, Jerne postulated “Natural selection theory.” which
F.M. Burnet expended.
 In the same same time (1955) , Porter isolated Fragment of
antigen binding (Fab) & Fragment crystalline (Fc) frm rabbit y-
globulin.

5. In 1975,Kohler and Milstein provided the most
outstanding proof of the clonal selection theory by fusion of
normal and malignant cells i.e., Hybridoma Technology.
In 1964, Littlefield developed a way to isolate hybrid cells
from 2 parent cell lines using the hypoxanthine-aminopterin-
thymidine (HAT) selection media.

6.  In 1986-1990, the first monoclonal antibodies reached the
market - Muromonab- CD3 ( produced by Milstein).
 In 1988, Greg Winter et al pioneered the techniques to
humanise monoclonal antibodies.
 Paul Ehrlich at the beginning of the 20th century theorized
that a cell under threat grew additional side-chains to bind the
toxin, and that these additional side chains broke off to become
the antibodies that are circulated through the body. It was these
antibodies that Ehrlich first described as the
"magic bullets" in search of toxins.
 In 2003, First Fully Human monoclonal
antibody – Adalimumab

7. Prof. Niels K. Jerne Prof. Georges J.F. Köhler Prof. César Milstein
"for theories concerning the specificity in development and control of the immune
system and the discovery of the principle for production of monoclonal
antibodies".

8.  Polyclonal antibodies are a mixture of antibodies with different antigen binding
sites that may bind to different epitopes or antigens of the immunizing agents with
varying affinity.
 It is produced by immunizing an animal with the appropriate antigen- wide array of
B cells will be stimulated to produce anti- protein antibodies.
 The serum obtained from immunized animal referred to as “Polyclonal Serum”

9.  Monoclonal antibodies (mAb) are antibodies that are identical because
they were produced by one type of immune cell, all clones of a single parent
cell.
 These are a class of highly specific antibodies produced by the clones of a
single hybrid cell.
They all have identical antigen- binding sites.
 Bind to the same epitope with same affinity.
 same antibody class ( isotope)

10. CHARACTERISTICS POLYCLONAL MONOCLONAL
PRODUCED BY : Many B cell clones A single B cell clone
BIND TO : Multiple epitopes of all
antigens used in the
immunization
A single epitope of a
single antigen
ANTIBODY CLASS
:
A mixture of different Ab
classes (isotopes)
All of a single Ab class
ANTIGEN -
BINDING SITES :
Different antigen-binding
sites
All antibodies have the
same antigen binding
sites

11. CHARACTERISTICS POLYCLONAL MONOCLONAL
COST Less expensive More expensive
YIELD Limited supply Infinite supply
EASE
Easily
Rapidly produced
Time consuming
More technical skill
POTENTIAL FOR
CROSS-REACTIVITY High Low

12.  Monoclonal antibodies
(mAb) are directed
against a specific epitope
(antigen, antigenic
determinant) .
 Typically made by
fusing myeloma cells with
the spleen B cells from a
mouse that has been
immunized with the
desired antigen or a single
Hybridoma cell line.

14. STEP 1 :- Immunization of Mice & Selection of Mouse
Donor for generation of Hybridoma Cells.
ANTIGEN
(intact cell/ whole cell
membrane/microorganisms)
+
ADJUVANT
(emulsification)
Antibody titre
reached in Serum
Spleen removed
( source of cells )

15. STEP 2 :- Screening of Mice for Antibody Production
After several weeks
of immunization
Serum Antibody Titre Determined
( Technique :- ELISA / Flow cytometry )
Titre too low
BOOST
(Pure Antigen)
Titre high
BOOST
(Pure Antigen)

16. STEP 3 :- Preparation of Myeloma Cells
8 - Azaguanine
Myeloma cells
Immortal Tumour of Lymphocytes
Myeloma cells
HGPRT ¯
High Viability & Rapid Growth

17. STEP 4 :- Fusion of Myeloma cells with Immune
Spleen Cells & Selection of Hybridoma
Cells
Spleen Cells Myeloma Cells
HYBRIDOMA CELLS
ELISA PLATE
HAT Medium
Feeder cells
Growth Medium

18. STEP 4 :- Cloning of Hybridoma Cell Lines by
“Limiting Dilution” or Expansion
A. Clone each positive Culture .
B. Test each Supernatent for
Antibodies
C. Expand positive clones

19. Myeloma cells have been genetically engineered
such that they can not use hypoxanthine,
aminopterin & thymidine (HAT Medium ) as a
source of nucleic acid biosynthesis and will de in
culture ( lack of HGPRT enzyme).
Spleen cells ( B cells) have limited life span
Only B cells that have fused with the engineered
myeloma cells will survive in culture when grown
in Hat medium.

22. MURINE
These are derived from
Mice. Patients treated
with murine mAbs
develop Human Anti-
Mouse Antibody (HAMA)
response
Eg:
90 Y-ibritumomab
CHIMERIC
They combine the
Antigen binding parts
(variable region) of
mouse with effector
parts (constant region)
of human.
Eg:
Infliximab
HUMANISED
These are human
antibody with
complementary
determining region
(CDR) or hypervariable
region from non human
source (rodent) grafted
onto human variable
region.
Eg:
Daclizumab

23. HOMOGENEITY
mABs represent a single Ab
molecule that binds to
antigen with the same
affinity & promote the same
effectors functions.
SPECIFITY
The product of a single
hybridoma reacts with the
same epitope on antigen
IMMUNIZING ANTIGEN
Need not to be pure or
characterized and is
ultimately not needed to
produce large quantities.
SELECTION
It is possible to select for
specific epitope
specificities and generate
antibodies against a wider
range of antigenic
determinants.
ANTIBODY PRODUCTION
Unlimited quantities of a
single well-defined
monospecific reagent.

24. AFFINITY
Average affinity of Mabs are
generally lower than polyclonal
antibodies
EFFECTOR FUNCTIONS
It may not produce the
desired biological response
SPECIFICITY
Monoclonals against
conformational epitopes on
native proteins may lose
reactivity wth antigens.
CROSS REACTIONS
Antibodies sometimes
display unexpected
crossreactions with
unrelated antigens.
TIME & EFFORT
COMMITMENT
Very large

25. THERAPEUTIC USES-
Immunosuppression, Malgnancies,
Asthama, auto immune
disease,Cancer, etc
RESEARCH TOOL –
In western blot, ELISA, RIA, flow
cytometry, Immunodot blot ,etc.
DIAGNOSTIC APPLICATONS –
In HIV, hepatitis, influenza, herpes,
pregnancy detection ec.

26. ADEPT- antibody
directed enzyme
prodrug therapy;
ADCC- antibody
dependent cell-
mediated
cytotoxicity;
CDC- complement
dependent
cytotoxicity; MAb-
monoclonal
antibody;
ScFv- single-chain Fv
fragment

28.  The growing knowledge of antibody gene structure and regulation has
made possible what Cesar Milstein, one of the inventors of monoclonal
antibody technology, has called “man-made antibodies.” It is now possible
to design and construct genes that encode immunoglobulin molecules in
which the variable regions come from one species and the constant regions
come from another.
 New genes have been created that link nucleotide sequences coding
nonantibody proteins with sequences that encode antibody variable regions
specific for particular antigens.
 Finally, by replacement of the immunoglobulin loci of one species with
that of another, animals of one species have been endowed with the capacity
to respond to immunization by producing antibodies encoded by the donor’s
genetically transplanted Ig genes.

29. Engineering an antibody to clone recombinant DNA containing the
promoter, leader, and variable region sequences from a mouse antibody
gene and the constant-region exons from a human antibody gene.
Production of chimeric mouse-
human monoclonal
antibodies. Chimeric mouse-
human heavy- and light-chain
expression
vectors are produced. These
vectors are transfected into Ab
myeloma cells. Culture in
ampicillin medium selects for
transfected
myeloma cells that secrete the
chimeric antibody.

30. These are hybrids of two different antibody molecules which can be
constructed by chemically cross linking two different antibodies or by
synthesizing them in hybridomas consisting of two different monoclonal-
antibody-producing cell lines that have been fused.
A CHIMERIC IMMUNOTOXIN
is chimeric monoclonal
antibody in which the
terminal Fc domain is
replaced by toxin chains
(white).
A HETEROCONJUGATE in
which onehalf of the mouse
antibody molecule is specific
for a tumor antigen and the
other half is specific for the
CD3/T-cell receptor
complex.

31. Generating monoclonal antibodies employing the polymerase chain reaction
(PCR) to amplify the DNA that encodes antibody heavy-chain and light-chain
Fab fragments from hybridoma cells or plasma cells.

32. The capacity of mice to
rearrange Ig heavy- and
lightchain
gene segments was
disabled by knocking out
the C and C loci. The
antibody-producing
capacity of these mice was
reconstituted by
introducing long stretches
of DNA incorporating a
large
part of the human germ-
line and heavy-chain loci
(miniloci). Chimeric mice
were then bred to
establish a line of
transgenic mice bearing
both heavy- and light-
chain human miniloci.
Immunization of these
mice results in the
production of human
antibody specific for the
target antigen.

33. Monoclonal antibodies are given intravenously (injected into a vein). These
are often more like an allergic reaction & are more common while the drug
is first being given. Possible side effects can include :
mAbs
Anaphylaxis
Malignancy
Fever &
chills
Nausea &
vomiting
Low blood
pressure
Rashes &
hypersensiti
vity

35.  The First approved mAbs was OKT-3 [1986] which is a murine IgGa2
protein to deplete T cells in patients with acute rejection of renal
allotransplant.
 Until Feb 24, 2013, 312 mAbs were approved by FDA, which were
applied in the treatment of organ transplant, Cancer, Asthma, Hematopoietic
malignancies and psoriasis.