Monoclonal antibody as targeting drug delivery system for immunosuppressant drug

1. Presented By-
Mr. Sagar. A. Kothawade
M. Pharmacy
Guided By-
Dr . C. R. Kokare
Asst. Professor
Sinhgad Technical Education Society’s
Sinhgad Institute Of Pharmacy,
Narhe, Pune-41
MONOCLONALANTIBODIES
: DRUG TARGETING
112/7/2019

2. Content
 Introduction
 Types of Antibodies
 Structure of Antibodies
 History Monoclonal Antibodies
 Production of Mab
 Advantages
 Limitation
 Characterization & Storage of Mab
 Targeted Drug Delivery
212/7/2019

3. Introduction
 Antibodies are group of glycoprotein molecules present
in blood serum and tissue fluid
 Antibodies are secreted by a class of blood cells known as
B-lymphocytes (plasma cells) in mammals
 Antibodies are produced when antigen are enters in body
312/7/2019

4. Cont....
 Antibodies are a part of defence system to protect the
body against the invading foreign substance namely
antigen.
 Antibodies are produced depends upon the antigen enters
412/7/2019

5. Types of antibodies
512/7/2019

6. Structure of antibodies
612/7/2019

7. History Of Monoclonal
Antibodies
 Mab technology is considered to be major revolutionary
landmarks in biotech
 1975 Kohler and Milstein provided the most outstanding
proof of the clonal selection theory by fusion of normal
and malignant cells. This resulted in the first monoclonal
antibodies, for which they received the Nobel Prize in
1984
712/7/2019

8. George Kohler & Cesar
Milstein in 1975 shared
the Nobel Prize in
Physiology or Medicine
in 1984 for discovery of
hybridoma technology.
812/7/2019

9. Production of Mab
 Immunization
 Generation of B-cell hybridomas (fusing B-cells &
myeloma cells )
 Screening
 Cloning by propagating the desired hybridomas
912/7/2019

10. Immunization
 Inject the microgram or milligram of immunogen mixed
with adjuvant (Aluminium salts , Freunds)
 Serum of animal assayed for relative conc. of antibodies
with various time interval
 When conc. of antibodies found to be nearly optimal.
 Animal sacrificed & spleens
 Plasma cells dissociated in to single splenocytes
1012/7/2019

11. Cell Fusion
 Splenocytes + plasmacytoma cell in medium
 High conc. (50%) PEG ( fusion proceed over a period of
time )
 The mixture is the washed and kept in a fresh medium.
 The mixture contains hybridomas, free myeloma cells,
and free lymphocytes
1112/7/2019

12. Screening , Cloning &
Propagation
 Screening is done for antibody specificity.
 Common tests like ELISA and RIA are used for this.
 The single hybrid cell producing the desired antibody are
isolated and cloned.
 Usually two techniques are commonly employed for this.
1212/7/2019

13. Cont....
A) Limiting dilution method:
Suspension of hybridoma cells is serially diluted so the
each dilution is having one hybrid cell . This ensures that
the antibodies produced is monoclonal.
B) Soft agar method:
In this method the hybridoma cells are grown in soft agar.
These form colonies and the colonies are monoclonal in
nature.
1312/7/2019

14. B cell
B cell has the enzyme HGPRT
But B cells die soon
They do not have the capacity to grow indefinitely
because of their limited life span
Scanning Electron Microscopic view of a B cell 1412/7/2019

15. Advantages Hybridoma
Technology
 Production of only pure molecular species
 Specificity for one antigenic determinants
 They can be easily tested for cross-reactivity
 High reproducibility with respect to specificity
 Mab are highly sensitive to small changes in both salt
concentration and pH
1512/7/2019

16. Disadvantages of Mab
 Minor changes in antigen epitope structure affect the
function of Mabs
 Mab production should be very specific to the antigen to
which it needs to bind
 Mab can prove expensive, laborious, and time consuming
12/7/2019 16

17. Characterization & Storage Of
Mab
 Biochemical biophysical characterization of
immunoglobulin.
 Immunochemically to defined its affinity for antigen
 Physical, chemical stability of antibody
 Mab must be characterized for their ability to withstand
freezing and thawing
1712/7/2019

18. Application & Uses
 Diagnostic reagents
 Therapeutic application
 Investigational & analytical application
 Drug targeting
 Miscellaneous.
1812/7/2019

19. Targeted Drug Delivery
System
Imag
1912/7/2019

20.  The very slow progress in the treatment of severe diseases
has led to the adoption of a multidisciplinary approach to
the targeted delivery and release of drugs.
 Minimize drug harmful side effects and enhance the
availability of the drug at the disease site.
2012/7/2019

21. The idea of a "magic
bullet" was first
proposed by Paul
Ehrlich at beginning of
20th century.
He postulated that if a
compound could be made
that selectively targeted a
disease-causing organism,
then a toxin for that
organism could be
delivered along with agent
of selectivity.
2112/7/2019

22. Objective
 To achieve a desired pharmacological response at a
selected sites without undesirable interaction at other sites,
there by the drug have a specific action with minimum
side effect & better therapeutic index.
 Ex-in cancer chemotherapy( Cytotoxic agent) and enzyme
replacement therapy.
2212/7/2019

23. Reasons For Drug Targeting
 Drug instability
 Low absorption
 Short half-life
 Large volume of distribution
 Low specificity
 Low therapeutic index
2312/7/2019

24. Principle Of Drug Targeting
 As several class of the drugs lack specificity for diseased
cells, they show their action on other site of action.
Ex- Cytotoxic action of chemotherapeutics agents is
directed against any rapidly proliferating cell population.
 Targeting is classified in to three categories
1.Passive targeting
2.Physical targeting
3.Active targeting
2412/7/2019

25. Strategies Of Drug Targeting
 Passive Targeting
 Inverse Targeting
 Active Targeting
 Dual Targeting
 Double Targeting
2512/7/2019

26. Passive Targeting
 Targeted to systemic circulation are characterized as
passive delivery systems.
 Ability some colloid to be taken up by the
Reticuloendothelial Systems (RES).
 Its determine by the inherent properties of the carrier like
hydrophobic & hydrophilic surface characteristics, particle
size , surface charge , particle number.
 Ex- Passive targeting of the lungs is made by modulating
the size of the particle to >7 micro m.
2612/7/2019

27. Inverse Targeting
 Avoid passive uptake of colloidal carrier by RES.
 To achieve inverse targeting, RES normal function is
suppressed by pre injecting large amount of blank
colloidal carriers or macromolecules like dextran sulphate.
 This leads to saturation of RES and suppression of
defence mechanism.
 Effective approach to target drug to non-RES organs
2712/7/2019

28. Active Targeting
 Active targeting is usually done by cell-specific ligands.
 Hence Mab targeting is adopted for active targeting. Mab
targeting is done by conjugating the drug antibody of the
specific targeting type.
 Hence antibody drug conjugates are used as active
targeting drug delivery systems.
2812/7/2019

29. Cont….
 In this approach carrier system bearing drug reaches to
specific site on the basis of modification made on its
surface rather than natural uptake by RES.
 Surface modification technique
1. Bio adhesive
2. Non-ionic surfactant (polysorbate 80)
3. Specific cell (MAb)
4. Albumin protein
2912/7/2019

30. Dual Targeting
 Carrier molecule itself have their own therapeutic activity
& thus increase the therapeutic effect of drug.
 Synergistic activity.
3012/7/2019

31. Double Targeting
 When temporal and spatial methodologies are combined
to target a carrier system ,then targeting may be called
double targeting.
 Spatial placement relates to targeting drug to specific
organs tissues , cells or even sub cellular compartment
whereas temporal delivery refers to controlling the rate of
drug delivery to target site.
3112/7/2019

32. Components Of Drug Targeting
Target:
Target means specific organ or a cell or group of cells,
which in chronic or acute condition need treatment.
Carrier or marker
They are engineered vector ,which retain drug inside them
via encapsulation an transport or deliver it into vicinity of
target cell .(Ex-MAb)
3212/7/2019

33. Mechanism of Mab as Target
Drug
 Immune system dependent drug - Mab interaction
 Target dependent drug - Mab interaction
3312/7/2019

34. Formation of
Ig(A,D,E,G,M)
Immuno
suppressnt
drugs
Administration of
Mab
Degradation
of Mab by
immune
system
Reduced
Clearance of
Mab
Fig. Immune System Dependent Drug –Mab
Interaction
3412/7/2019

35. Administration
of MAb
Small
drugs
molecule
target
expression
Deceased
or
increased
clearance
of MAb
Formulation of
complex
between MAB &
target
Degradation of Mab
target dependent
cellular uptake
Target Dependent Drug Mab Interaction
3512/7/2019

36. Drug Conjugates
 Toxin conjugates (immunotoxins)
Ex- Diphtheria toxin, ricin have been conjugated to the
tumour specific antibodies Ricin has tow chains. Among
these A-chain is cytotoxic and B-chain is non-specific.
Hence B-chain is removed and the toxin is conjugates to
tumour specific antibody. Thus we increase the toxins by
using Mab as active drug targeting systems.
3612/7/2019

37.  Drug immunoconjugates :
Agents like chlorambucil, methotrexate and doxorubicin are
conjugated with tumour specific antibodies.
Ex- Dixirubicin-BR96 immunoconjugates for Lewis antigen
found on the surface of tumour cells.
3712/7/2019

38.  They are homogenous in nature.
 They are specific to a particular antigen with a particular
epitope.
 Ex-Rituximab (Rituxan,anti-CD20) is a good example -
this antibody is used for the treatment of lymphoma.
3812/7/2019

39. FDAApproval
The first approved Mab was OKT-3 (1986), which is a
murine IgGa2 protein to deplete T cells in patients with
acute rejection of renal allotransplant.
3912/7/2019

40. @ Prevents acute
rejection of
kidney
transplants
Prevents
autoimmune
destruction of islet
cells in type I
diabetes mellitus
OKT3
4012/7/2019

41. Mab For Tumour Therapy
 Cell depletion
Ex- Rituxan, Campath (naked), Myelotarg (drug), Zevalin,
Bexxar (radioisotope)
 Blocking receptor
Ex- Herceptin
 Attacking Vasculature
Ex- Avastin , Erbitux
 Vaccination against idiotype
Ex- Panoex
4112/7/2019

42. Mab Against HIV Receptor
Well Characterized
Mab directed against
HIV receptor complex
for treatment of
HARRT resistant
AIDS patients
through entry
inhibition.
4212/7/2019

43. Highlights Of Current Research
4312/7/2019

44. Limitation
 As they are specific to a particular antigen, they cannot
distinguish molecule as a whole.
 Some times they cannot distinguish group of different
molecules is not distinguished.
 Mice used in Mab production carry Adenovirus, Hepatic
virus, Retrovirus, Cytomegalovirus, Thymic virus.
4412/7/2019

45. Side Effects
 Rare but more serious side effects of Mab therapy may
include:
Infusion reaction, Severe allergy-like reaction can occur
and in very few cases lead to death.
Dangerously very low blood cell counts. Low levels of
RBC’s, WBC’s & platelets may lead to serious
complications.
4512/7/2019

46. Key References
 U. Satyanarayan, 2005 Pharmaceutical Biotechnology,
Books and Allied Pvt. Ltd. 213-226.
 R. Panchuhnula and C. Dey, 1997, Monoclonal Antibodies
in Targeted Drug, Journal of clinical pharmacy and
Therapeutics, vol-22, 7-19.
 D. C. Balkey. 2009. Drug Targeting with monoclonal
Antibodies, Journal of Acta Oncologic, 91-97
4612/7/2019

47. Key References
 Jagadevappa Patil. 2016, Functional Application of
Monoclonal Antibodies in Cancer Therapy Via drug
Targeting Approach, Journal of Pharmacovigilance . 4-5
 E.Bhargav, et al: 2013, Targeted Drug Delivery. World
Journal of Pharmacy and Pharmaceutical Sciences. vol-3,
150-169.
 Rollett A,et al: 2013. HSA nanocapsules functionalized
with monoclonal antibodies for targeted drug delivery.
International Journal of Pharmaceutics. 458 . 1-8
4712/7/2019

48. 4812/7/2019