Monoclonal antibody

2. Monoclonal
Antibodies
Dr. Ashutosh Tiwari
M.D. Pharmacology 1st yr. stud.
SAIMS Indore
26/12/2013

3. Seminar layout
Antibody
Polyclonal Antibody
Monoclonal Antibody
Hybridoma technique
Production of Monoclonal Antibody
Evolution of Monoclonal Antibody
Nomenclature of Monoclonal Antibody
Types of Monoclonal Antibody
Pharmacokinetics & Adverse Effects
Therapeutic Potentials of Monoclonal Antibody

4. What are antibodies?
An antibody is a protein used by the immune
system to identify and neutralize foreign
objects like bacteria and viruses. Each
antibody recognizes a specific antigen unique
to its target.
Monoclonal antibodies (mAb) are antibodies
that are identical because they were produced
by one type of immune cell, all clones of a
single parent cell.
Polyclonal antibodies are antibodies that are
derived from different cell lines. They differ in
amino acid sequence.
Immunoglobulin (Ig) are structurally related
glycoproteins that function as antibodies

5. The Structure of an
Antibody
2 identical light chains (~220 amino
acids long)
Variable domain: VL
Constant domain: CL
2 identical heavy chains (~440 amino
acids long)
Variable domain: VH
3 Constant domains: CH1, CH2, CH3
Covalent, disulfide bonds between
cysteine residues
Flexible “hinge region”

6. Antibodies have two major functions:
• Recognize and bind antigen
• Induce immune responses after binding
The variable region mediates binding
• Affinity for a given antigen is
determined by the variable region
• The variable region confers absolute
specificity for an antigen
The constant region mediates immune
response after binding
• Different classes of constant regions
generate different isotypes
• Different isotypes of antibody have
differing properties
Antibody Function
Constant
region
Variable
region

7. Antibodies as Drugs
Antibodies are naturally occurring
Discovery of their innate properties hinted at
great therapeutic potential
High-specificity in binding
Already present in the body
Can activate and couple components of the immune
system
Modification to structure and refinement in
production methods have made antibodies a
viable modern drug

8. History
1975 :
Hybridoma Technology
George Kohler and Cesar
Milstein devised a method to
obtain large amounts of a mAb
1988 :
The Nobel Prize for Medicine
- In 1988, Greg Winter et al
pioneered the techniques to
humanize monoclonal
antibodies

9. History
1986 first monoclonal antibody reached
the market – Muromonab-CD3
2003 First fully human monoclonal
antibody – Adalimumab

10. Polyclonal antibodies are a mixture of antibodies with
different antigen binding sites that may bind to different
epitopes or antigens of the immunizing agent with
varying affinities.
Produced by immunizing an animal with the appropriate
antigen - wide array of B cells will be stimulated to
produce anti-protein antibodies.
Antibodies may be made to a number of different
epitopes of the protein.
Even antibodies that bind to the same epitope may have
different antigen-binding sites and bind the epitope with
different affinity.
Polyclonal Antibodies

11. The serum obtained from an immunized animal is
referred to as a polyclonal antiserum.
Contains antibody to different epitopes and different
antigens that were present in the immunizing inoculum
The immunized animal’s serum is collected.
Antibodies can then be purified from the serum.
Since one antigen induces the production of many
antibodies the result is a ‘polyclonal’ mixture of
antibodies.
ATG ( Anti thymocyte globulin)
ALG ( Anti lymphocyte globulin, Lymphocyte immune
globulin)

12. Monoclonal Antibodies
• A class of highly specific antibodies produced by the
clones of a single hybrid cell
• Produced by fusing a B cell secreting the desired
antibody with a tumour cell (myeloma cell) capable of
growing indefinitely
• Fused cell called hybridoma
• Monoclonal antibodies all have identical antigen-
binding sites
• bind to the same epitope with same affinity
• same antibody class (isotype)

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

14. 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

17. 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
Spleen removed
(source of cells)

18. PRODUCTION OF MONOCLONAL ANTIBODY
Step 2: - Screening Of Mice For Antibody Production
HYBRIDOMA TECHNOLOGY
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

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

20. PRODUCTION OF MONOCLONAL ANTIBODY
Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells
&
Selection of Hybridoma Cells
HYBRIDOMA TECHNOLOGY
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

21. PRODUCTION OF MONOCLONAL ANTIBODY
Step 4: - Cloning of Hybridoma Cell Lines by “ Limiting
Dilution” or Expansion
HYBRIDOMA TECHNOLOGY
A. Clone Each +ve Culture
B. Test Each Supernatant for Antibodies
C. Expand +ve Clones
Mouse
Ascites
Method
Tissue
Culture
Method

22. Myeloma cells have been genetically
engineered such that they can not use
hypoxanthine, aminopterin, and thymidine
(HAT medium) as a source for nucleic acid
biosynthesis and will die in culture (lack
HGPRT enzyme)
Spleen cells (B cells) have limited life span
Only B cells that have fused with the
engineered myeloma cells will survive in
culture when grown in HAT medium.
Hybridoma Selection The “HAT Trick”

24. Conventional production of mAbs
The hybridoma technology:
spleen cells from immunized 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 calisifed into four fields
1. Robottle cell culture process.
2. Membrane binded cell culture process
3. Microcarrier cell culture process
4. Suspended cell culture process

25. Production in animals ( In-vivo )
Mouse ascites method
•Hybridoma cells injected in mouse
•Produce ascites
•Fluid contains high concentration of
Ab’s
•No further concentration required
•Purification required
•Easy and inexpensive
•Animal mortality

26. Ethical issues:
 Freund’s 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.
 Shock - rapid fluid loss
 FCA and pristane should not be used where it is possible to
use no adjuvant or less irritant adjuvants.
 FCA should not be used more than once in individual mice.
 The volume of FCA and pristane used should not exceed
0.1ml and 0.2ml respectively.
 Individual mice should not be inoculated with adjuvant more
than 3 times.
 A priming agent should not be used in individual mice more
than once.
 Ascites fluid should only be harvested once at the time of
euthanasia.

27. 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
• denaturation 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

28. Origin
First generation
 Murine, rabbit or rat proteins purified after
immunisation with antigen
 Abs to these proteins (Ag) generated in
patients: human antimurine antibody (HAMA)
 Block effectiveness of therapy
 Adverse events serum sickness or anaphylaxis

29. Origin
Second generation
 DNA technology or genetic engineering used
to construct hybrids composed of human Abs
regions with murine
Chimeric Abs
Humanized
Human

30. EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC
2. LIBRARIES
a.BACTERIOPHAGE
b. mRNA
c. Cell Surface
Ist
generation
mab
2nd generation mab
Daclizumab

31. Types of mAbs
Purple denotes human component orange murine component

33. Murine
 Derived from mice
 Patients treated with murine mAbs develop a
human antimouse antibody (HAMA) response
Rapid clearance of the mAb
Poor tumour penetration
Hypersensitivity reactions
 90Y-ibritumomab
 131I -Tositumomab

34. Chimeric Abs
Antigen binding parts (variable region) of mouse
with effector parts (constant region) of human
Infliximab
Abciximab
Rituximab

35. Humanized
Human Ab with complimentary determining region
(CDR) or hypervariable region from non human
source
Daclizumab
Trastuzumab

36. Human Abs
Recombinant DNA technology:
 Genes for variable Fab portion of human Abs
is inserted in genome of bacteriophages &
replicated
 Mixed with Ag & complementary Ab
producing phages selected
e.g. Adalimumab

37. Human mAbs
Transgenic Mice
Phage Display

38. Human Abs
Even human Ab can provoke anti-idiotype
responses
HACA (human antichimera antibodies)
HAHA (human antihuman antibodies)
Plant Derived Mabs
- Plant Genetic Engineering
- Transgenic Plants
- Transgenic tobacco, soyabeen, alfalfa etc.
Lack of animal pathogenic contaminants
Low production cost
Ease of agricultural scale up

39. Nomenclature of Monoclonal Antibodies
Prefix Target Source Suffix
variable
-o(s)- bone -u- human
-mab
-vi(r)- viral -o- mouse
-ba(c)- bacterial -a- rat
-li(m)- immune -e- hamster
-le(s)- infectious lesions -i- primate
-ci(r)- cardiovascular -xi- chimeric
-mu(l)- musculoskeletal -zu- humanized
-ki(n)- interleukin -axo- rat/murine hybrid
-co(l)- colonic tumor
-me(l)- melanoma
-ma(r)- mammary tumor
-go(t)- testicular tumor
-go(v)- ovarian tumor
-pr(o)- prostate tumor
-tu(m)- miscellaneous tumor
-neu(r)- nervous system
-tox(a)- toxin as target

40. Nomenclature
Suffix
Human: -umab
Humanized: -zumab
Murine: -momab
Chimeric: -ximab

41. Examples
ab- + -ci- + -xi- + -mab: chimeric
monoclonal antibody used on the
cardiovascular system.
tras- + -tu- + -zu- + -mab:
humanized monoclonal antibody
used against a tumor.
Pali- + -vi- + -zu- + -mab
humanized mab used against a virus
(RSV)

42. Types of Monoclonal Antibody
Naked Monoclonal Antibody: 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. Trastuzumab - For advanced breast cancer (HER-2)
2. Rituximab - For B cell non Hodgkin lymphoma (CD 20)
3. Cetuximab - For advanced colorectal cancer ( HER-1)
4. Bevacizumab - For metastatic colorectal cancer (VEGF)
5. Alemtuzumab - For B cell chronic lymphocytic
leukemia. (CD 52)

43. Types of Monoclonal antibodies
Conjugated/loaded/labeled Mabs : Coupled
with drugs / toxins / radioatiactive atoms
Chemo-labeled antibodies:
○ MAbs conjugated with chemotherapeutic agents
e.g. brentuximab vedotin and ado-trastuzumab
emtansine.
○ Brentuximab vedotin, attached to a chemo drug
(MMAE) targets the CD30 antigen (present on T
and B-cells) in treatment of Hodgkin lymphoma
and non-responding anaplastic large cell
lymphoma.
○ Ado-trastuzumab emtansine, attached to DM1
chemo drug, targets the HER2 protein antigen
used for curing advanced breast cancer patients.

44. 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 IL-2 protein attached to a toxin (derived
from the germ causing diphtheria).
○ IL-2 normally attaches to cells that express the
CD25 antigen and thus helps in delivering the
toxin to these cells.
Radio-immune Abs: 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

45. Pharmacokinetics: mAbs
Routes of administration:
 Subcutaneously (Rituximab, Trastuzumab, Adalimumab)
 Intramuscularly (Palivizumab)
 Intravenously
IV route: preferred because of 100% bioavailability
Route for elimination of antibodies
 Via uptake & catabolism by reticuloendothelial system &
target tissue.

46. P/K: mAbs
Half-life
Chimeric : 4 –15 days
Humanized: 3 - 24 days
Recombinant human: 11– 24 days
Human antimouse antibody (HAMA) response
develops 7–10 days following exposure to murine
antibody

47. Adverse Effect of Mabs
Related to three mechanism:
Xenogenetic nature of Mab used
Suppression of physiological function
Activation of inflammatory cells or mediators after
binding of Mab to its target

48. Adverse Effect of Mabs
Naked Mabs :
Mild ; often allergic reaction on 1st infusion
Cytokine release syndrome
infusion toxicity, cytopenias
Conjugated Mabs:
More A/E’s ; depend on substances attached
Antilymphocytes Mabs
Immunosupression
increase risk of infection
Cancer
Anti TNF-α Mab
reactivation of tuberculosis
lymphomas

49. Mechanisms of Action
1. Blocking action of molecular targets
Can work antagonistically by binding a receptor to
prevent activation
Can also bind the antigen and prevent activation
2. “Magic Bullet”
Compound with target specificity is coupled with
various effector groups
○ Toxins, radionuclei, enzymes, DNA
3. Signal molecules
Coupled to mediators of apoptosis, cell division,
etc.

50. Pharmaceutical Antibodies
The fastest growing segment of the biopharmaceutical market
$65 billion in sales for 2012
there are 5 antibody drugs listed in the top 10 best selling drugs of
2012
The antibody drugs featured in the top 10 in the year 2012:
#8 = Roche’s Avastin (bevacizumab), with sales of US$6.260 billion
#7 = Johnson & Johnson’s Remicade (infliximab), with sales of
US$6.139 billion
#5 = Roche’s Hereceptin (trastuzumab), with sales of US$6.397 billion
#3 = Roche’s Rituxan (rituximab), with sales of US$7.285 billion
#1 = AbbVie’s Humira (adalimumab), with sales of US$9.265 billion.
therapeutic antibody drugs are in high demand and are playing a
significant role in the generation of revenue for drug companies
Many of the leading pharmaceutical companies have entered the mAb
sector, attracted by quicker and less costly development, higher success
rates, & premium pricing

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

54. Autoimmune disease
mAB Target immune cells:
remove them,
block their function
decrease cytokine levels

55. 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
Anti idiotype vaccines
Variety of agents conjugated to Mabs for
selective delivery to cancer cells

56. Applications of mABs
Diagnostic tools in research and
laboratory
Different technologies in which MAbs are
used include Western blot, immunodot blot,
ELISA, radioimmuno assay (RIA), flow
cytometry, immunohistochemistry,
fluorescence microscopy, electron
microscopy, confocal microscopy

57. Applications of mABs
Diagnostic applications
MAb is used to detect pregnancy as early as a
week or two after conception by reacting with
human chorionic gonadotrophin
HIV diagnostic kits
Rapid diagnosis of hepatitis, influenza, herpes,
streptococcal & chlamydia infections
Identification & characterization of tumor specific
antigen: classification of cancer
Imaging: Localization of cancer
○ Arcitumomab : colorectal carcinoma
○ Nofetumomab : small cell lung cancer

58. 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 have failed

59. Future aspects
Zanolimumab : Cutaneous T cell lymphoma
Tremelimumab : Advanced melanoma
Ruplizumab : Lupus nephritis
Efungumab : Invasive Candida Infection
Panobacumab : Pseudomonas Infection
Solanezumab : Alzheimer’s Disease
60

60. 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.

61. Interesting Variations
Small antibody fragments (Fv or Fab) are also effective in
blocking cytokines
Benefit: More readily penetrate tissue
Coupling of antibody fragments to form dimers and
tetramers
Increases avidity and cross-linking
Engineered Diabodies (Bispecific mABs)
Two different antigen specificities
○ One against the target
○ The other against effectors
Can cross-link effector cells

62. Nanobodies
A Nanobody/single-domain antibody is a
peptide chain of about 110 amino acids
long, comprising one variable domain
(VH) of a heavy-chain antibody, or of a
common IgG
1989 - Raymond Hamers
Discovered in camels
Completely lack the light chain!
Same antigen affinity as their four-
chain counterparts
Structure makes them more
resistant to heat and pH
May lead to development of oral
nanobody pills
Orally available single-domain
antibodies against E. coli-induced
diarrhoea in piglets have been
developed and successfully tested
Other diseases of the gastrointestinal
tract, such as inflammatory bowel
disease and colon cancer, are also
possible targets for orally available
sdAbs

63. TO SUMMARISE :
MAbs are highly specific Abs produced by a clone of
single hybrid cells formed by fusion of B cell with the
tumor cell.
The hybridoma formed yields higher amount of
MAbs.
MAbs can be produced in vitro and in vivo .
Animals are utilized 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 etc.

64. Finally, the dreams of Paul Ehrlich
who considered antibodies
a magic bullets have become
reality.
Monoclonal antibodies have
established themselves as the most
important and rapidly expanding
class of drugs in oncology.
Paul Ehrlich
(1854-1915)