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Monoclonal antibodies
preparation and
applications
DEPARTMENT OF PHARMACEUTICS
• ANTIBODY:
Immunoglobulin synthesized by the
body’s immune system in response
to a foreign molecule(antigen).
Classification of
Antibodies
Antibodies are mainly classified into
following types
Polyclonal antibodies
Monoclonal antibodies
Chimeric antibodies
Humanized antibodies
Bispecific antibodies
Immunoconjugate.
These are the antibodies that are
obtained from different B cell
resources.
They are a combination of
immunoglobulin molecules secreted
against a specific antigen, each
identifying a different epitope.
Monoclonal Antibodies:
Monoclonal antibodies(MoAb’s) are specific
antibodies produced by fusing B-cells(Blymphocytes) derived from a single ancestral B-cell
with a tumour (myeloma) cell.
These cultures of B-cells are called
Monoclonal as they are derived
from a single ancestral B-cell.
The structure heavy and light
chains of all Mo Ab’s will be
identical.
These cells are used to harvest
single kind of antibodies called as
Monoclonal antibodies.
They are highly specific and
offer more consistent
efficacy.
Antibody Fragments:
Earliest MoAb’s were of non human origin
which are immunogenic and exhibit human
antimouse response(HAMA).To overcome
this problem, antibody was cleaved into Fc
and Fab fragments by papain digestion.
The Fab fragments are less immunogenic than
intact antibodies,penetrate more into tumour
cells, longer halflife.
But, antigen binding capacity is lost up to some
extent.
It contains Fc region of human
immunoglobulin(IgG)
and
Fab
regions are murine(mouse) origin.
 These are slightly immunogenic.
 These are produced by rDNA
technology.
 Majority of antibody framework is
human
in
origin,
but
the
CDR’s(responsible
for
antigen
binding) are murine.
Each of the two arms are
specific for two different antigens.
Target 1
Target 2
For MoAb’s targeted drug delivery, a drug is
bound covalently to an antibody
The resulting immunoconjugate
may contain a spacer between
drug and antibody (or) a
polymer to increase the number
of drug molecules that can be
bound to each antibody.
The drug can be incorporated
noncovalently into a liposome or
microsphere to which the
targeting antibody is bound to
surface is known as
Immunoliposome or
Immunomicrosphere.
Step 1: Immunization of Mice and Selection of
Mouse Donors for Generation of Hybridoma Cells
Mice are immunized
with an antigen that is
prepared for injection
either by emulsifying
the antigen with
Freund's adjuvant or
other adjuvants or by
homogenizing a gel
slice that contains the
antigen.
Intact cells, whole
membranes, and
microorganisms
are sometimes
used as
immunogens.
In almost all
laboratories, mice are
used to produce the
desired antibodies. In
general, mice are
immunized every 2-3
weeks but the
immunization
protocols vary among
investigators.
When a sufficient
antibody titer is
reached in serum,
immunized mice are
euthanized and the
spleen removed to
use as a source of
cells for fusion with
myeloma cells.
After several weeks of immunization, blood samples are obtained from
mice for measurement of serum antibodies. Several humane techniques
have been developed for collection of small volumes of blood from mice.
Serum antibody titer is determined with various techniques,
such as enzyme-linked immunosorbent assay (ELISA) and flow
cytometry.
If the antibody titer is high, cell fusion can be performed. If the titer is
too low, mice can be boosted until an adequate response is achieved, as
determined by repeated blood sampling.
When the antibody titer is high enough, mice are commonly boosted by
injecting antigen without adjuvant intraperitoneally or intravenously (via
the tail veins) 3 days before fusion but 2 weeks after the previous
immunization. Then the mice are euthanized and their spleens removed
for in vitro hybridoma cell production.
Step 3: Preparation of
Myeloma Cells
• Fusing antibody-producing spleen cells, which
have a limited life span, with cells derived from
an immortal tumor of lymphocytes (myeloma)
results in a hybridoma that is capable of
unlimited growth.
• Myeloma cells are immortalized cells that are
cultured with 8-azaguanine to ensure their
sensitivity to the hypoxanthine-aminopterinthymidine (HAT) selection medium used after cell
fusion.
• A week before cell fusion, myeloma cells are
grown in 8-azaguanine. Cells must have high
viability and rapid growth. The HAT medium
allows only the fused cells to survive in culture.
Step 4: Fusion of Myeloma Cells
with Immune Spleen Cells
• Transferred to 96 well plastic culture
plates.
• Supernatant assayed for particular
antigen specificity by
ELISA
RIA
 If a sample of supernatant contains
the desired antibody.
 It will bind to the antigen and will
remain associated with the well and
the unbound material is washed off.
• Single cells secreting the desired Ab are
isolated from positive cultures and propagated
into cell lines by two cloning techniques
• 1) Limiting dilution: Cells in the cultures are
diluted and aliquoted into new wells so that
only one cell found in any well.
Cells are allowed to re grow and the procedure
is repeated for several times to increase the
probability that all cells in be a given well are
monoclonal.
• 2)Soft agar: If the culture can be reliably
dispersed into single cells then the visible
colonies obtained which are picked out from
the agar said to be monoclonal.
Commercial production
of monoclonal
antibodies
Antibody based drug
delivery systems
Ideal Antibody directed delivery system
characterstics:
 Preparation by an efficient method which gives
high yield and capable of scale up.
 High stability of conjugate under shelf storage
conditions and in circulation after injection.
 Retention of antigen binding ability of antibody
while it is carrying the drug to target tissue.
 Upon reaching the target, the immunoconjugate it self should show the desired
pharmacological effect equivalent to free drug
(or) must release the free drug or a derivative
that is fully efficacious.
 The drug can be covalently bound to MoAb
directly or through a short spacer or through a
linker such as a water souble polymer.
 A carrier such as liposome or microsphere can
be used where in the drug is entrapped in (or)
bound to the carrier and the MoAb is bound to
the surface of carrier.
 A variety of coupling reagents are available that
aid in optimizing the properties of an
immunoconjugate.
 Amino,sulfhydryl and carboxyl groups are the
most common functional groups on the
antibody,carrier and drug molecules used for
coupling.
 If the drug lacks the desired group,
it may be possible to induce that
group.
 Eg: Alcohol / Amine Succinicanhydide Carboxyl
group.
Amine
2-iminothiolane
Sulfhydryl
 Classical protein cross linking
reagents are used for linkage of
drug to antibody.
Eg: Carbodiimide reagents like
Dicyclohexylcarbodiimide (DCC)
and its water soluble analogs link
amino groups with carboxyl group
via amide bonds.
Active ester method
Carboxyl groups of drug are linked to N-hydroxysuccimide in
the presence of a carbodiimide to form active ester derivative
of drug.It then reacts with Amino group of antibody.
 Dextran and similar carbohydrate linkers are used for
conjugation of drugs and antibody.
 Recently, a number of bifunctional reagents have been
developed that are more specific in forming linkages of antibody
to drug.
Eg: N-succimidyl-3-(pyridyldithio)propionate(SPDP) reagent is
used to derivatize drug with a pyridyl disulfide group.This
species react with the antibody containing free sulfhydryl
groups,yeilding immunoconjugate.
 Combinations of classical coupling methods with bifunctional
reagents have also been used for preparation of
immunoconjugates.
Eg: A 6 carbon spacer ending in a carboxyl group was
introduced into dextran and then mitomycin-c was coupled
to the spacer with a carbodiimide.
 The remaining carboxyl groups of spacer were
modified to amino groups and were then
coupled to MoAb-A7 by means of SPDP,with a
final MMC/ MoAb ratio of 40.
 The antibody activity of resulting conjugate
was almost equivalent to native MoAb A 7 and
release free MMC by chemical hydrolysis.
 Bifunctional reagents allow incorporation of a
metabolizable linker into the
immunoconjugate, releasing free drug or an
active derivative at a predictable rate.
 A number of studies have explored coupling
methods that allow control of the invivo rate of
release of active drug.
Eg: Free Doxorubicin was released from
conjugated antibody linked by hydrazone with
in 6 hours at 370 at pH 4.5.
 Daunomycin conjugates linked to MoAb-L6
via a polylysine and aconitate linkage, the
conjugate releases free drug at pH 6.0.
The MoAb’s interact with growth factor receptors such
as transferrin receptors show an anti tumour effect
because transferrin is essential for the growth of cells and
its receptors are predominantly present on proliferating
cells
Generic name
Type of MAb
application
Abciximab
Fab fragment of
chimeric anti7E3
Platelet
aggregation
inhibitor.
Rituximab
Chimeric antiCD20
Non-hodgkins
lymphome
Transuzumab
Humanized anti- Metastatic
HER2
breast cancer
In this approach, a MoAb is prepared against a given
tumour antigen.
 Rather than using it for immunotherapy directly, it is
used to inoculate mice, which produces a second
antibody against the idiotypic site of original
antibody(anti-idiotype).
 After cloning and administration to patients, this antiidiotypic Mo Ab would mobilize patient’s own
immune system to produce a 3rd antibody(anti-antiidiotype) that would have same idiotype of the 1st
antibody and thus bind to original antigen and lead to
cytotoxicity.
 The advantage of this approach is the multiplicative
effect of the vaccine and more specific and safer than
using the antigen itself as vaccine.
These are the MoAb’s to which radio nuclides have been
conjugated to provide cytotoxic radiation after the
MoAb binds to its target antigen.
 Iodine-131 and Yttrium-90 are the isotopes
commonly used.
 They have more ability to kill tumour cells that are
poorly accessible and/or antigen negative.
 Unlike conventional radiation therapy. Radio
immuno conjugates provide continuous radiation
from the decay of radionuclide which allows less
opportunity for the tumour cells to repair the damage.
 Depending on the type of MoAb, the antibody itself
may trigger CDC and ADCC mechanisms that
supplement the effect of the radionuclide.
Problems associated with MoAb’s
 Cancer cells are heterogenous, so those cells that are
not recognised by MoAb can escape and proliferate.
 Some tumours contain semidead cores with poor
circulation and thus cannot be reached by
monoclonals.
 MoAb’s can interact with circulating target antigens
before reaching this target.
 Patients can experience possible immunogenic
reactions.
 A MoAb is not as specific invivo as would be
predicted from invitro studies.i.e, tumour antibodies
may bind to normal cells as well as target cells.
 MoAb’s have poor penetration incase of solid tumors
due to lack of vasculature.
For these reasons, it is proven more effective to combine
MoAb with standard chemotherapeutic agents.
Applications of Monoclonal antibodies in
Drug delivery
Principle of targeting:
MoAb’s are used for active targeting of immunotoxins and
drugs(i.e,drug immunoconjugates).
 A number of anti tumour agents including chlorambucil,
methotrexate, Daunomycin and Doxorubicin conjugated to
tumour specific antibodies.
Eg: Doxorubicin-BR96 immunoconjugate. It is a chimeric
MoAb specific for lewis antigen found on the surface of
tumour cells.
 CMA-676 which is a conjugate of an anti-CD33 MoAb and
calicheamicin, an anticancer drug is 1000 times more potent
than Doxorubicin.
 Bispecific MoAb’s composed of anti-CD3 or anti-CD2
MoAb, chemically conjugated to antitumour antibody and
coated on lymphokine activated killer(LAK) cells have
been clinically investigated for the treatment of malignant
glioma,lymphoma and ovarian cancer.
APPLICATIONS
A. Diagnostic tests
1. Diagnostic imaging
(a)Cardiovascular diseases
(b)Sites of bacterial infection
(c)Cell surface markers
(d)Detection of circulating antigens
(e)Cancer
(f)Hormones
(g)Immunometric assay procedures
B.
Therapeutic applications
1.
General
(a)MAB itself
(b) Radio isotope immuno conjugates
(c) Toxin drug conjugates
Transplantation
(a)Organ
(b)Bone marrow
Infectious diseases
(a) micro organisms
(b) parasites
Enzyme and proteins
Cardiovascular disease
Auto immune disease
Cancer
Antidotes
2.
3.
4.
5.
6.
7.
8.
C. Investigational and analytical
applications
1.Lymphocyte phenotying
2.Purification of proteins
3.Radioimmuno assay
D. Drug targeting
1.Immunotoxins
2.Supresser deletion
3. Site specific modification
4. Antibody enzyme conjugates
E. Miscellaneous
1.Abzymes
2.Auto antibody finger printing
• Pure one molecular species only
• Specificity for one antigenic determinant.
• Antibodies titer values obtained are very
high
• Antibodies with avidity can be produced
• Invitro or invivo production is possible
with high production rate
• Maintainance of farm/animals is not
required for immunization and bleeding
• Antiserum having identical antibody
with an identical specificity and
constant property can be obtained
world wide
• High reproducibility with respect to
specificity and avidity
• Producion of cell lines to individual
components of a mixture is possible
• Radiolabelling and flourescent
conjugation or enzyme marking of
MAb’s are easy
CONCLUSION
Monoclonal antibodies (mAb) are
important reagents used in biomedical
research, in diagnosis of diseases, and
in treatment of such diseases as
infections and cancer.
As antibody-based drugs come to the
forefront of therapeutics derived from
biotechnology, they provide a range of
new and exciting opportunities to
treat a number of currently unmet
medical needs
REFERENCES
•
A REPORT OF THE COMMITTEE ON
METHODS OF PRODUCING MONOCLONAL
ANTIBODIES NATIONAL RESEARCH
COUNCIL.
• ENCYCLOPEDIA OF PHARMACEUTICAL
TECHNOLOGY
• PHARMACEUTICAL BIOTECHNOLOGY
S.P VYAS AND V.K DIXIT