Presentation by Andreas Schleicher Tackling the School Absenteeism Crisis 30 ...
monoclonal antibodies and engineered antibodies
1. Seminar on
MONOCLONAL ANTIBODIES
AND
ENGINEERED ANTIBODIES
PRESENTED BY
MOHAMMED MUNAWAR ALI
(M.PHARM. 2ND SEMESTER) DEPT. OF PHARMACEUTICS
ST.PETER’S INSTITUTE OF PHARAMACEUTICAL SCIENCES,
VIDYANAGAR, HANAMKONDA. 506001.
AFFILIATED TO KAKATIYA UNIVERSITY
2. Contents
Introduction
Advantages and disadvantages
Production methods
Problems associated
Applications
Engineering antibodies
Conclusion
References 2
3. Introduction
Antibody(Ab) is a protein used by the immune system to identify and
neutralize foreign objects like bacteria and viruses.
An antibody is called monoclonal (mAb)
when each immunoglobulin is produced
by a single clone of cells and hence is
identical to every other molecule in the
preparation, in terms of heavy as well as
light chain structure.
Polyclonal antibodies (pAb) are
produced by B-lymphocyte which
respond to many epitopes of antigen.
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4. Polyclonal antibodies
(Polyclonal antiserum)
Harvest Ab
Monoclonal antibodies
B B B B B B B B
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5. Advantages
Specificity.
Affinity.
Potential to generate large quantities of Abs under precisely controlled
conditions.
In vivo and in vitro production is possible with high production rate.
Immortal cell lines.
Disadvantages
mAb cant differentiate between two antigens if the body is directed to an
epitope common to antigens.
mAb to viral strains are so specific that they don’t react to other minor strains of
same virus.
pAb have potential for co-operatively binding to respective antigens, so
stabilising the overall affinity and binding forming precipitating complexes
while mAbs form network with antigens.
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8. In vivo production
Hybridoma cells are injected into pristane primed rodents, the cell line
proliferates and are stored in ascitic fluid.
10-50 mL of fluid is collected containing several mg/mL of antibody.
mAbs produced this way are considered unsuitable because of viral
contamination.
Widely used in research applications.
The inherent variability in animals can result
in lack of consistency, while some lines
produce solid rather than diffuse tumors,
and some produce none or
kill the host.
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9. In vitro production
Fermentation is most widely employed for production of monoclonal
antibodies because of the problems associated with above listed
methods.
No contamination with normal mouse immunoglobulin is seen with
fermentation.
Apart from this, bacterial cell cultures, transgenic animals and
transgenic plants are also used for production of monoclonal antibodies
but to a very limited extent.
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10. Problems associated with mAb therapy
The main problem is the mouse antibodies are recognised by human
immune system as foreign material and the patients develop a immune
response against them, producing HAMA (Human Anti-Mouse
Antibodies).
The doses of monoclonal antibodies to treat chronic diseases are
typically large.
Stability issues concerned with oxidation, deamidation, aggregation,
fragmentation and other forms of chemical modification with alter or
probably nullify the antibody function.
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11. Stabilisation
Freeze dried and again recovered Spray dried Ab added to
for moisture uptake. polylactide-co-glycolide (PLGA)
Carbohydrates are added to produce microspheres.
Ex- Trehalose, sucrose etc., Characterization.
Monitoring 11
12. Applications- Diagnosis and therapy
Identification of tumors as they express specific membrane proteins.
Measurement of circulation steroid harmones and in differentiating viral
strains.
Bacterial infections and STD.
Drug immunoconjugates.
Radioisotopes anchored mAbs.
Analytical applications
RIA and ELISA.
Purification of proteins.
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15. Engineering antibodies
Antibodies exhibit four main effector functions: antibody-dependent
cellular cytotoxicity (ADCC), phagocytosis, complement-dependent
cytotoxicity (CDC), and half-life/clearance rate.
Each of these effector functions is mediated through interaction with a
specific set of receptors and cell types: ADCC and phagocytosis through
interaction of cell-bound mAbs with Fc gamma receptors (Fc γ R), CDC
through interaction of cell-bound mAbs with the series of soluble blood
proteins that constitute the complement system (e.g. C1q, C3, C4, etc.),
and half-life/clearance rate through binding of antibodies to the neonatal
Fc receptor (FcRn).
C1q binding and complement activation.
FcγR binding and ADCC.
FcRn and half-life/clearance rate.
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16. Genetically engineered antibodies
Chimeric antibody- As HAMA response of patients is due to Fc
portion of murine Abs, murine Fv region was fused with human Fc to
produce Chimeric genes.
Ex- Infliximab, rituximab, and abciximab
Humanised antibody- This consists of fusion of hyper variable region,
aminao acids responsible for antigen binding with human antibody thus
replacing its own hyper variable region.
Ex- Mylotarg®, Herceptin, Xolair®
Chimeric antiody
Humanised antiody
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17. Myelotarg®
mAb + Calcichemicin
Antibody portion of molecule targets CD33 (a cell surface molecule), abundant
on the surface of acute myeloid leukemia cells (AML) & absent from normal
blood stem cells.
AML cells accumulate in the bone marrow and prevent normal bone marrow
from growing an functioning properly.
Calcichemicin is a potent anti-cancer drug which intercalates into DNA and
breaks it, because of which cells undergo apoptosis.
Antibody targets calcichemicin to AML cell specifically through CD33 cell
surface molecule
Calcichemicin kills AML cells.
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21. Conclusion – Challenges and Opportunities
Successful clinical application of these novel agents requires the development
of stable formulations that can be used for specific delivery methods.
Antibodies, because of their endogenous nature, have built-in features that may
pose problems for stability as bio-therapeutics.
Lyophilization and pH dependent modification.
Several modes of targeting is achieved successfully.
Linking up the technology to gene therapy will ensure highly specific treatment
Examples of such an approach include studies performed using an antibody
directed against the Her2/neu antigen to delivery liposomes containing a
chemotherapeutic have been described, antibodies used to deliver cationic
liposomes for the administration of nucleic acid material for gene therapy.
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22. References
L.G. Presta, Engineering antibodies for therapy, Curr. Pharm. Biotechnol. 3
(2002) 237– 256.
J.W. Park, K. Hong, P. Carter, H. Asgari, L.Y. Guo, G.A. Keller, C. Wirth, R.
Shalaby, C. Kotts, W.I. Wood, et al., Development of anti-p185HER2
immunoliposomes for cancer therapy, Proc. Natl. Acad. Sci. U. S. A. 92 (1995)
1327– 1331.
A.L. Daugherty, R.J. Mrsny, Formulation and delivery issues for monoclonal
antibody therapeutics, Advanced Drug Delivery Reviews. 58 (2006) 686–706.
Leonard G. Presta, Engineering therapeutic antibodies to minimize
immunogenicity and optimize function, Advanced Drug Delivery Reviews 58
(2006) 640–656.
Lee K. Tan and Sherie L. Morrison, Antibody structure and antibody
engineering, Advanced Drug Delivery Reviews, 2 (1998) 129-142.
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