1. MONOCLONAL ANTIBODIES
MICBIO: applications of monoclonal antibodies

2. OUTLINE
CONCEPT OF MONOCLONAL ANTIBODIES
Structure of immunoglobulin
Polyclonal Vs monoclonal antibodies
TYPES
 1. Murine 2.Chimeric 3.Humanised 4.Human
PRODUCTION AND SELECTION TECHNICHES
Hybridoma technology
APPLICATIONS

3. MONOCLONAL
ANTIBODIES (mabs)
• DEFINITION: - They are called Monoclonal because they originate,
following appropriate:
 SCREENING
 SELECTION, and
 from ONLY ONE CELL CLONE
 SPECICIFIC for SINGLE EPITOPE

4. Epitopes: Antigen Regions that Interact
with Antibodies

5. ANTIBODIES
Derived from different B
Lymphocytes cell lines
POLYCLONAL. MONOCLONAL.
Derived from a single B cell
clone
Batch to Batch variation
affecting Ab reactivity &
titre
mAb offer Reproducible,
Predictable & Potentially
supply of Ab with exquisite
specificity
Enable the development of
secure immunoassay systems.
NOT Powerful tools for
clinical diagnostic tests

6. Georges Köhler César Milstein, and Niels
Kaj Jerne in 1975 who shared the Nobel
Prize in Physiology or Medicine in 1984 for
the discovery hybridoma technology

7. In 1988 Greg Winter and his team pioneered the techniques to
humanize monoclonal antibodies, removing the reactions that
many monoclonal antibodies caused in some patients.

8. Types of monoclonal antibodies
Types of
MOOCLONAL
ANTIBODIES
Murine antibody Chimeric
antibodies
Humanized
antibody
Human
Monoclonal
antibody

9. Murine antibody
Whole of the antibody is of murine origin
Major problems associated with murine antibodies include
1. Formation of complexes after repeated administration
2. Allergic reactions
3. Anaphylactic shock
4. human anti-mouse antibodies (HAMA)
5. Formation of immune complexes that can damage to the kidneys

10. Chimeric antibodies
Chimeric antibodies are composed of
murine variable regions fused onto human
constant regions.
has human segments
retains its original antigen-binding specificity

11. HUMANIZED ANTIBODY
Substitution into human antibodies
only the CDRs of the rodent monoclonal antibodies
95% human and 5% mouse DNA sequences
antigen-binding affinities similar to original rodent mAb
 more effective therapeutic agents.

12. Human Monoclonal antibody
Human monoclonal antibodies are produced by transferring human
immunoglobulin genes into the murine genome
after which the transgenic mouse is vaccinated against the desired
antigen, leading to the production of monoclonal antibodies

13. PRODUCTION
FUNDAMENTAL OBJECTIVE for the applied use of antibodies, was
• to discover how to create a cell line that could be grown in culture medium
• that would produce a single type of antibody molecule (mAb)
• Have a high affinity for a specific target antigen
Such a cell line would provide
• a consistent
• continuous source of identical antibody molecules
Unfortunately, the B cells do not reproduce in culture
However,
• hybrid cell type could be created : leading to Hybridoma Technology

14. Hybridoma technology

15. Step 1: Immunization of Mice and Selection of Mouse Donors for
Generation of Hybridoma Cells
Immunization:
Emulsifying the
antigen
Homogenizing a
gel slice that
contains the
antigen
Intact cells, whole
membranes
Selection of
Mouse Donors
mice are
immunized every
2-3 weeks
On having a
sufficient antibody
titer
spleen removed to
use as a source of
cells for fusion
with myeloma cells.

16. PRODUCTION OF MONOCLONAL
ANTIBODY
Step 1: - Immunization Of Mice & Selection Of Mouse
Donor For Generation Of Hybridoma cells
HYBRIDOMA TECHNOLOGY
ANTIGEN ( Intact
cell/ Whole cell
membrane/ micro-
organisms ) +
ADJUVANT
(emulsification) Ab titre reached in Serum

17. Step 2: Screening of Mice for Antibody Production
Blood samples are
obtained from mice for
measurement of serum
antibodies
Serum antibody titer is
determined:
Enzyme-linked
immunosorbent assay
(ELISA)
Flow cytometry
(passage of labelled cells
in front of a laser so
they can be detected)

18. Step 2: - Screening Of Mice For Antibody Production
After several
weeks of
immunization
Serum Antibody Titre Determined
(Technique: - ELISA / Flow cytometery)
Titre too low
BOOST
(Pure antigen)
Titre High
BOOST
(Pure antigen)
2 weeks
PRODUCTION OF MONOCLONAL ANTIBODY

19. Step 3: Preparation of Myeloma Cells
Myeloma cells
Immortalized cells, cultured with 8-azaguanine
ensure their sensitivity to the hypoxanthine-aminopterin-
thymidine (HAT) selection medium* used after cell fusion.
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

20. Step 3: - Preparation of Myeloma Cells
Immortal Tumor Of Lymphocytes
+ 8 - Azaguanine
Myeloma Cells
High Viability & Rapid Growth
HGPRT-
Myeloma Cells
PRODUCTION OF MONOCLONAL ANTIBODY

21. Step 4: Fusion of Myeloma Cells with Immune Spleen Cells
Fusion is
accomplished by:
• Co-centrifuging freshly harvested
• Spleen cells and
• Myeloma cells in polyethylene glycol (PEG),
• a substance that causes cell membranes to fuse
• Fused cells are transferred to a growth medium
containing
• hypoxanthine, aminopterin, and thymidine (HAT
medium).
Fusion can be of
various type:
• Nonfused spleen cells (S)
• Nonfused myeloma cells (M)
• Fused spleen-spleen cells (S-S)
• Fused myeloma-myeloma cells (M-M)
• Fused hybrid spleen-myeloma cells (S-M)

22. Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells
&
Selection of Hybridoma Cells
FUSION
PEG
MYELOMA CELLSSPLEEN CELLS
HYBRIDOMA CELLS
ELISA PLATE
Feeder Cells
Growth Medium
HAT Medium
1. Plating of Cells in
HAT selective
Medium
2. Scanning of Viable
Hybridomas
PRODUCTION OF MONOCLONAL ANTIBODY

23. The HAT procedure for selecting hybrid spleen–myeloma
(hybridoma)
cells.

24. Step 5: - Cloning of Hybridoma Cell Lines by “ Limiting
Dilution” or Expansion
A. Test Each Supernatant for Antibodies
C. Expand +ve Clones
Mouse
Ascites
Method
Tissue
Culture
Method
PRODUCTION OF MONOCLONAL ANTIBODY

26. PRODUCTION OF MONOCLONAL ANTIBODY

27. Cont’d

28. In vitro methods

29. In vivo methods

30. Applications of Monoclonal Antibodies
Diagnostic Applications
To detect the presence of this substance/antigen
Gene cloning
mAb that recognizes that the gene product is available, it can be used as a probe for
detecting those cells that make the product
Protein purification
MAb affinity columns eg, Sepharose
Therapeutic Applications
Transplant rejection
Cardiovascular disease
Cancer
Infectious Diseases
Inflammatory disease
Future Applications
Fight against Bioterrorism

31. MAbs in Biochemical Analysis
Number of diagnostic kits using mAbs have become commercially
available
 Detecting the urinary levels of human chorionic gonadotropin for pregnancy
 Estimation of plasma carcinoembryonic antigen in colorectal cancer
 Prostate specific antigen for prostate cancer
 Infectious diseases by detecting the circulatory levels of antigens specific to
the infectious agent

32. MAbs in Diagnostic Imaging
• Radiolabeled—MAbs are used in the diagnostic imaging of diseases,
• MAbs localize at specific sites can be detected by imaging the
radioactivity
• Differentiate between cancerous and non-cancerous growth, since
radiolabeled—MAbs are tumor specific
• Antimyosin MAb labeled with radioisotope indium chloride (111 In) is
used for detecting myosin and thus the site of myocardial infarction

33. • Deep vein thrombosis (DVT) refers to the formation of blood clots
(thrombus) within the blood veins
• Radioisotope labeled MAb directed against fibrin or platelets can be used for DVT
• MAb tagged with a radiolabel directed against activated platelets can be
used to localize the atherosclerotic lesions by imaging technique

34. Therapeutic Applications
• In destroying disease-causing organisms: mAbs promote
efficient opsonization
• In the treatment of cancer
• In the immunosuppression of organ transplantation eg: OKT3
 MAbs in drug delivery
• In the treatment of autoimmune diseases
• mAbs in use as immunotoxins
• Ricin is a cytotoxic protein is conjugated to mAb that is specific to
cancer cells, it blocks ribosomal function, leading to the death of
cancer cells

35. • MAbs in the dissolution of blood clots
• mAbs directed against fibrin can be coupled to Tissue plasminogen
activator (tPA), used for degradation of blood clots
• More the concentration of tPA at the target spots, more efficient
conversion of plasminogen to plasmin which in turn dissolves blood
clot

37. Therapeutic applications
OKT3 (Orthoclone) monoclonal antibody
First FDA approved monoclonal antibody in 1986.
OKT3 binds to a cell surface receptor called CD3*, which is present
on all T cells
Resulting, a blocked immunological response, and the transplanted
organ is not rejected
*CD3 receptor, a membrane protein on the surface of T cells

38. Rituximab and Ibritumomab (Rituxan/Mabthera)
Works against CD20* found on the surface of B cells
Destroys B cells and treat diseases characterized by overproduction or
dysfunctional B cells
Non-Hodgkin Lymphoma, transplant rejections, autoimmune diseases
*CD20 = B-lymphocyte antigen CD20 = protein on the surface of B lymphocytes
(component of immune system)

39. Alemtuzumab (Campath/MabCampath)
Used to treat some patients with chronic lymphocytic leukemia (CLL)
 Alemtuzumab binds to the CD52* antigen, which is found on cells called
lymphocytes (which include the leukemia cells)
Once attached, the antibody attracts immune cells to destroy these cells
*CD52 = CAMPATH-1 antigen – antigen on surface of some white bloodcells
(lymphocutes

40. Trastuzumab (Herceptin)
Antibody against the human epidermal growth factor 2 (HER2)* protein
Breast and stomach cancer cells sometimes have large amounts of HER2 on
their surface
When activated, it helps these cells grow
 Trastuzumab binds to these proteins and stops them from becoming active
*HER2 = human epidermal growth factor receptor 2. Often mutated in breast cancers.

41. Ibritumomab tiuxetan (Zevalin)
• Radiolabeled mAbs: uses high-energy radiation to destroy cancer cells and
shrink tumors
• Antibody against the CD20 antigen, found on lymphocytes called B cells
• The antibody delivers radioactivity directly to cancerous B cells
• Used to treat some types of non-Hodgkin’s lymphoma

42. Brentuximab vedotin (Adcetris)
• Chemolabeled mAbs: have powerful chemotherapy drugs attached to them
• Also known as antibody-drug conjugates
• A CD30*-specific, antibody-drug conjugate, was approved by the FDA in
2011
• Used for the treatment of Hodgkin’s lymphoma and systemic anaplastic
large-cell lymphoma.
*CD30: Cell membrane protein of the tumor necrosis factor receptor family and tumor
marker

43. Ado-trastuzumab emtansine (Kadcyla)
Chemolabeled mAbs: An antibody that targets the HER2 protein, attached
to a chemotherapy drug called DM1
Used to treat some breast cancer patients whose cancer cells have too much
HER2

44. OMALIZUMAB: ANTI
IgE
• Acts by binding to free IgE in the
circulation and prevent them from
binding to mast cells and from
further degranulation

46. refrences
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Rodney J. Y. Ho, Milo Gibaldi., 2013. Biotechnology and
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Helmout Modjtahedi, Sumaira Ali, Sharadah Essapen. 2012.
Therapeutic application of monoclonal antibodies in cancer:
advances and challenges. British Medical Bulletin. 104: 41–59.
Brenard R. Glick, Jack J. Pasternak, Cheryl L. Pattern. 2010.
Molecular Biotechnology. 4th Edition. ASM press

47. Feng Li, Natarajan Vijayasankaran, Amy Shen, Robert K., Ashraf
Amanullah. 2010. Cell culture processes for monoclonal antibody
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Michael J. Waites, Neil L. Morgan, John S. Rockey, Gary Higton.
2001. Industrial Microbiology: An Introduction. Blackwell science.