mab

2. Dr. Satyabrata Sahoo
PDT Clinical & Experimental Pharmacology
School of Tropical Medicine, Kolkata

3. Introduction
 An antibody is a protein used by the immune system to
identify and neutralise foreign objects like bacteria and
viruses. Each antibody recognises a specific antigen unique
to its target.
 Polyclonal antibodies are antibodies that are derived from
different cell lines. They differ in aminoacid sequence
 Immunoglobulins are structurally related glycoproteins
that function as antibodies
 Monoclonal antibodies are antibodies that are identical
because they were produced by one type of immune cell,all
clones of a single parent cell

4. Cont..
Monoclonal antibodies:
 An antibody of unique specificity
 Derived from single B cell clone
 These can be made in large quantities in the labouratory
 Cornerstone of immunology
 These are also termed as magic bullets

5. Classification of antibodies
 Polyclonal antibodies
 Monoclonal antibodies
 Antibody fragments
 Chimeric antibodies
 Humanized antibodies
 Bispecific antibodies

6. Antibodies 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

7. Antibodies
Polyclonal Monoclonal
Produced by: Many B cell clones A single B cell clone
Bind to: Multiple epitopes of all
antigens used in the
immunisation
A single epitope of a
single antigen
Antibody class: A mixture of different
antibody classes(isotypes)
All of a single antibody
class
Ag-binding site: Different antigen binding
sites
All antibodies have the
same antigen binding site

8. History
 First monoclonal antibody was generated in 1975 by George
kohler and Cesar Milstein.
 Both of them and Niels Kaj Jerne shared the nobel prize in
physiology or medicine in 1984 for the discovery
 Fully licenced in 1986
 The first licenced monoclonal antibody was Orthoclone
OKT3(muromonab-CD3) approved in 1986 for use in preventing
kidney transplant rejection
 It is a monoclonal mouse IgG2a antibody whose cognate antigen
is CD3
 Its use was limited to acute cases due to reported side
effects(human anti-mouse antibody response)
 First fully human monoclonal antibody –Adalimumab(2003)
 Recently there are so many monoclonal antibodies generated
and used for diagnostic and therapeutic purpose

9. Principle of Mabs

11. Conventional production of Mabs
 The hybridoma technology:
Spleen cells from immunised mice are fused with the
murine myeloma cells
The several process had been developed at large scale.
According to the different cell culture methods, it can
classified into four fields
1. Robottle cell culture process
2. Membrane blinded cell culture process
3. Microcarrier cell culture process
4. Suspended cell culture process

12. Production in animals(In-vivo)
 Mouse ascites method:
 Hybridoma cells injected in mouse
 Produce ascites
 Fluid contains high concentration of antibodies
 No further concentration required
 Purification required
 Easy and inexensive
 Animal mortality

13. Production in cell culture(In-vitro)
 Batch tissue culture method:
 Grow hybridoma cells in batches
 Purify Mabs from the culture media
 Fetal bovine serum commonly used
 Low concentration
 Denature during concentration
 Semi permeable membrane based system:
 A barrier- hollow fibre or a membrane
 Larger compartment containing culture media
 Smaller chamber to isolate cells and Mabs
 High concentrations
 Method of choice for large scale production

14. Types of monoclonal antibody
 Naked monoclonal antibodies: those without any drug or
radioactive material attached to them
 Mark the cells for the immune system
 Attach to receptors- block binding of growth factors
E.g. 1. Transtuzumab- For advanced breast cancer(HER-2)
2.Cetuximab- For B cell non Hodgkin lymphoma(CD
20)
3.Bevacizumab- For metastatic colorectal
cancer(VEGF)
4.Alemtuzumab- For B cell chronic lymphocytic
leukemia (CD 52)

15. Types of Monoclonal antibodies
 Conjugated/loaded/labeled Mabs: Coupled with
drugs/toxins/radioactive atoms
 Chemo-labeled antibodies:
 Mabs conjugated with chemotherapeutic agents e.g.
Brentuximab vedotin and ado-transtuzumab emtasine
 Brentuximab vedotin,attached to a chemo drug(MMAE)
targets the CD 30 antigen(present on T and B- cells) in
treatment of Hodgkin lymphoma and non-responding
anaplastic large cell lymphoma
 Ado-transtuzumab emtansine, attached to DM1 chemo
drug, targets the HER2 protein antigen used for curing
advanced breast cancer patients

16. Types of Monoclonal antibodies
 Immune toxins: conjugated with toxins e.g.Denileukin diftitox
 Used to treat some cancers(cutaneous T- cell lymphoma and
many others)
 Consists of IL2 protein attached to a toxin( derived from the
germ causing diptheria)
 IL-2 normally attaches to cells that express the CD 25 antigen
and thus helps in delivering the toxin to these cells
 Radio-immune antibodies: e.g. Ibritumomab
 An Mab against the CD20 antigen on B cells (and lymphomas)
 Conjugated to either the radioactive isotope indium-111(111In) or
Yttrium-90(90Y) for treatment of lymphoma patients

17. Nomenclature

18. Mechanism of action

19. Pharmacokinetics:Mabs
 Routes of administration:
 Subcutaneously(Rituximab, Transtuzumab,Adalimumab)
 Intramuscularly(Palivizumab)
 Intravenously( Infliximab)
 Route for elimination of antibodies:
 Via uptake and catabolism by reticuloendothelial system
and target tissue

20. Pharmacokinetics:Mabs
 Half life
 Chimeric: 4-15 days
 Humanised: 3-24 days
 Recombinant human: 11-24 days
 Human antimouse antibody(HAMA) response develops 7-
10 days following exposure to murine antibody

21. Adverse effects of Mabs
 Naked Mabs:
 Mild, often allergic reaction on 1st infusion
 Cytokine release syndrome
 Infusion toxicities, cytopenias
 Conjugated Mabs:
 More Adverse effects, depend on substances attached
 Antilymphocytes Mabs:
 Immunosuppression
 Increase risk of infection
 Cancer
 Anti TNF-α Mabs:
 Reactivation of tuberculosis
 lymphomas

23. Applications of Mabs
 Diagnostic applications
 Mab is used to detect pregnancy as early as weak or two after
conception by reacting with human chorionic gonadotrophin
 HIV diagnostic kits
 Rapid diagnosis of hepatitis, inflenza, herpes,
streptococcal,chlamydia and corona virus
 Identification and characterisation of tumor specific antigen:
classification of cancer
 Imaging: localisation of cancer
 Diagnostic tools in research and labouratory
 Different technologies in which Mabs are used include: western
blot, immunodot blot, ELISA, RIA,flow cytometry,
immunohistochemistry, fluorescence microscopy, electron
microscopy, confocal microscopy

24. Therapeutic uses
 Immunosuppression
 Autoimmune diseases
 Malignancies
 Antiplatelet therapy
 Infectious diseases
 Asthma
 Osteoporosis

25. Cancer
 Strategies for the Mabs in cancer:
 Immune reaction directed destruction of cancer cells
 Interference with the growth& differentiation of malignant
cells
 Antigen epitope directed transport of anticancer agents to
malignant cells
 Variety of agents conjugated to Mabs for selective delivery
to cancer cells

26. Future applications
 Fight against bioterrorism
 Inhalational anthrax(potent biological terrorism) is caused
by breathing the bacterial spores of Bacillus anthracis
 Raxibacumab injection is used to treat infectious
inhalational anthrax when alternative therapies are failed

29. FDA approved Mabs

37. Ethical issues
 Freunds complete adjuvant(FCA)(to enhance the immune
response): painful lesions at the injection site
 Pristane as a priming agent- granulomatous reactions
 Respiratory distress due to ascites
 The volume of FCA and pristane used should not exceed 0.1
ml and 0.2 ml respectively
 Individual mice should not be inoculated adjuvant more
than 3 times
 Ascites fluid should only be harvested once at the time of
euthanasia

38. Limitations of Mabs
 The typical doses of Mab drugs needed for treatment are
significantly higher than those required for other drugs( or
products)
 The huge demand to increase production of these drugs and the
drive to lower the cost of these expensive medicines is a
continuous challenge to the present industry
 Hybridoma technology is labourious and time consuming
 The presence of retroviruses as a part of the mammalian
chromosomes is a common occurence
 This possess a great danger, since there is no guarentee that Mab
production is totally virus-free, despite the purification. For this
reason USFDA insists that Mab for human use should be totally
free from all pathogenic organisms including viruses

39. Summary
 The monoclonal antibody production technology has
revolutionized the world of biotechnology
 Advances in genetic engineering over the years have provided
numerous ways to design Mabs that are more robust and
efficacious compared with their original murine version
 Animals are utilise to produce Mabs but these antibodies are
associated with immunogenicity and ethical problems
 Recombinant DNA technology, genetic engineering and
transgenic animals are used to produce humanized Mabs or
pure human Mabs with fewer ADRs
 Used for treatment of cancer, autoimmune disorders, graft
rejections , infections, asthma, osteoporosis etc
 Somany newer Mabs are under trial phase now for future benefit

41. References
1.Goodman & Gilman’s the pharmacological basis of therapeutics 13th
edition
2. Leavy O. Therapeutic antibodies: past, present and future. Nat Rev
Immunol 2010;10(5):297.
3. Kohler G, Milstein C. Continuous cultures of fused cells secreting
antibody at predefined specificity. Nature 1975;256: 495-7.
4. Robert A. Burger, M.D., Mark F. Brady, Ph.D., Michael A. Bookman,
M.D.,Gini F. Fleming, M.D., Bradley J. Monk, M.D., Helen Huang, M.S.,
n engl j med 365;26 nejm.org december 29, 2011
5. CLINICAL MICROBIOLOGY REVIEWS, JUly 1988, p. 313-329 Vol. 1, No.
30893-8512/88/030313-17$02.00/0Copyright © 1988, American Society
for Microbiology
6. L. Gandhi, D. Rodriguez-Abreu, S. Gadgeel, E. Esteban, E. Felip, F. De
Angelis,M. Domine, P. Clingan, M.J. Hochmair, S.F. Powell ,N Engl J
Med 2018;378:2078-92.DOI: 10.1056/NEJMoa1801005
7. Patrick Mayrhofer and Renate Kunert,Department of Biotechnology,
University of Natural Resources and Life Sciences (BOKU), 1190
Vienna, Austria, Human Antibodies 27 (2019) 37–51 37DOI
10.3233/HAB-180347IOS Press

42. References
8. Masami Suzuki1, Chie Kato1, and Atsuhiko Kato1, J Toxicol
Pathol 2015; 28: 133–139
9. Patrick Chames1, Marc Van Regenmortel2, Etienne Weiss2
and Daniel Baty1, British Journal of Pharmacology (2009),
157, 220–233 Journal compilation © 2009 The British
Pharmacological Society No claim to original French
government works All rights reserved 0007-1188/09
10. Monoclonal Antibody Production
http://www.nap.edu/catalog/9450.html
11. The CAMMS223 Trial Investigators*, n engl j med 359;17
www.nejm.org october 23, 2008
12. Maria Alice Vieira Willrich1*, 454 JALM | 454–457 | 02:03 |
November 2017

43. Thank you