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Monoclonal antibodies
1.
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
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
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
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
22.
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
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
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