Types, synthesis and uses of Monoclonal antibodies

1. MD. MUSTAFEED UDDIN
MONOCLONAL ANTIBODIES

2.  Introduction
 History of monoclonal antibodies
 Production
 Types
 Nomenclature
 Application and Side Effects
 Examples
 Conclusion

3. 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 are 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.

7.  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.
Given (almost) any substance, it is
possible to create monoclonal antibodies
that specifically bind to that substance;
they can then serve to detect or purify
that substance. This has become an
important tool in biochemistry, molecular
biology and medicine.

8.  Paul Ehrlich at the
beginning of the 20th
century theorized that a
cell under threat grew
additional side-chains
to bind the toxin, and
that these additional
side chains broke off to
become the antibodies
that are circulated
through the body.
 It was these antibodies
that Ehrlich first
described as "magic
bullets" in search of
toxins.

9.  1890 Von Behring and Kitasato discovered the
serum of vaccinated persons contained certain
substances, termed antibodies
 1900 Ehrlich proposed the “ side-chain theory”
 1955 Jerne postulated natural selection theory.
Frank Macfarlane Burnet expended.
 Almost the same time, Porter isolated fragment
of antigen binding (Fab) and fragment
crystalline (Fc) from rabbit y-globulin.

10.  1964 Littlefield developed a way to isolate
hybrid cells from 2 parent cell lines using the
hypoxanthine-aminopterin-thymidine (HAT)
selection media.
 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.

11. Prof.Georges J. F.
Köhler
Prof.César Milstein
Prof.Niels Kaj Jerne

12.  In the 1970’s the B-
cell cancer myeloma
was discovered, and it
was understood that
these cancerous B-
cells all produce a
single type of
antibody.
 This was used to study
the structure of
antibodies, but it was
not possible to
produce identical
antibodies specific to a
given antigen.

13.  lost the ability to synthesize hypoxanthine-
guanine-phosphoribosyl transferase (HGPRT).
◦ This enzyme enables cells to
synthesize purines using an extracellular source
of hypoxanthine as a precursor.
◦ Ordinarily, the absence of HGPRT is not a problem
for the cell because cells have an alternate (de
novo)pathway that they can use to synthesize
purines.
◦ However, when cells are exposed
to aminopterin (a folic acid analog), they are unable
to use de novo pathway and are now fully
dependent on HGPRT for survival.

14.  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

15.  CELLS FUSED:
◦ Spleen cells from a mouse that has been
immunized with the desired antigen
◦ Myeloma cells.
 FUSION AGENT:
◦ Polyethylene glycol
 MEDIUM:
◦ HAT Medium {Hypoxathine-Aminopterin-
Thymidine}

21. A. Murine source mAbs: rodent mAbs with excellent affinities
and specificities, generated using conventional hydrioma
technology. Clinical efficacy compromised by HAMA(human
anti murine antibody) response, which lead to allergic or
immune complex herpersensitivities.
B. Chimeric mAbs: chimers combine the human constant regions
with the intact rodent variable regions. Affinity and specificity
unchanged. Also cause human antichimeric antibody
response (30% murine resource)
C. Humanized mAbs: contained only the CDRs of the rodent
variable region grafted onto human variable region framework

24.  Whole of the antibody is of murine origin
 Major problems associated with murine antibodies
include
 reduced stimulation of cytotoxicity
 Formation of complexes after
repeated administration
 allergic reactions
 anaphylactic shock

25.  Chimeric antibodies are
composed of murine
variable regions fused onto
human constant regions.
 Antibodies are
approximately 65% human.
 This reduces
immunogenicity and thus
increases serum half-life.

26.  Humanised antibodies
are produced by grafting
murine hyper variable
amino acid domains into
human antibodies.
 This results in a
molecule of
approximately 95%
human origin

27.  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

28.  Every monoclonal antibody has the
following components in its name
 Variable-Target Substem-Source Substem-
Stem-Additional words(in special cases)
 Ex: Alacizumab pegol is
 Ala –ci-zu-mab-pegol

29. OLD NEW MEANING
-anibi- - Angiogenesis inhibitor
-ba(c)- -ba- Bacterium
-ci(r)- -ci- Circulatory system
-fung- -fu- Fungus
-ki(n)- -ki- Inlterleukin
-les- - Inflamattory lesions
-li(m)- -li- Immune system
-mul- - Musculoskeletal system
-ne(ur)- -n(e)-* Nervous system
-os- -s(o)- bone
-toxa- -tox(a)- toxin
- -tu- Tumour
-vi(r) -vi- virus

30. Letter Meaning
-a- Rat
-e- Hamster
-i- Primate
-o- Mouse
-u- Human
-xi- Chimeric
-zu- Humanized
-axo- Rat/mouse hybrid

32.  Rituximab
◦ Ri- Variable
◦ tu- Tumour
◦ xi- Chimeric
◦ mab- Monoclonal Antibody
 So Rituximab is a Chimeric Monoclonal
Antibody targetting a Tumour

33.  Bevacizumab
◦ Beva- Variable
◦ ci- Circulatory System
◦ zu- Humanised
◦ mab- Monoclonal Antibody
 So Bevacizumab is a Humanised Monoclonal
Antibody targetting a protein in Circulatory
System

34. A second word following the name of the antibody indicates that another
substance is attached.
1)An antibody can be PEGylated (attached to molecules of polyethylene
glycol)
◦ to slow down its degradation by enzymes and to decrease
its immunogenicity
 Ex:alacizumab pegol
2)A cytotoxic agent can be linked to an anti-tumor antibody for drug
targeting purposes.
◦ vedotin,(monomethyl auristatin E) which is toxic by itself but
predominantly affects cancer cells if used in conjugates
 Ex:glembatumumab vedotin
3)A chelator for binding a radioisotope can be attached.
◦ Pendetide, a derivative of pentetic acid, in capromab pendetide to
chelate indium-111.If the drug contains a radioisotope, the name of
the isotope precedes the name of the antibody.
 Ex: indium (111In) capromab pendetide

35. 1)Homogeneity:
Monoclonal antibody represents a single antibody molecule that
binds to antigens with the same affinity and promote the same
effectors functions.
2) Specificity:
The product of a single hybridoma reacts with the same epitope on
antigens.
3) Immunizing Antigen:
Need not be characterized and is ultimately not needed in large
quantities to produce large quantities of antibody.
4) Selection:
It is possible to select for specific epitope specificities and generate
antibodies against a wider range of antigenic determinants.
5) Antibody Production:
Unlimited quantities of a single well-defined monospecific reagent

36.  Average affinity of monoclonal antibodies are generally
lower than polyclonal antibodies
 Monoclonals against conformational epitopes on native
proteins may lose reactivity with antigens.
 Antibodies sometimes display unexpected crossreactions
with unrelated antigens.
 Immune rejections
 Time and effort commitment: VERY LARGE.

38. They are used in
 Western blot test
 ELISA TEST
 Antigen capture assays
 Immuno dot blot tests to detect the specific protein on a
membrane.
 Naked eye dipstick tests
 Immuno-histochemistry , which detect antigen in fixed tissue
sections and
 Immuno-fluorescence test, which detect the substance in a
frozen tissue section or in live cells.
 Radio immuno assays
 Tissue typing
 Serotyping of Microorganisms
 They are also used in the diagnosis of lymphoid and myeloid
malignancies

39. Number of Monoclonal Antibodies have
been developed in detection and diagnosis
of
 Parasites like
◦ Trichomonas vaginalis
◦ Leishmania donovani
◦ Trypanosoma congolense
◦ Babesia bovis
 Human Viruses like
◦ Influenza virus
◦ Rotavirus
◦ Rabies Virus etc
 Animal Viruses like
◦ Bovine herpes virus,
◦ Cervine herpes virus type I

40. MAbs can be used to detect pregnancy with Antibody to
Beta HCG.

41.  Capture of parasite antigen from
peripheral blood using monoclonal
antibodies prepared against a
malaria antigen target and
conjugated to either a liposome
containing selenium dye or gold
particles in a mobile phase.
 A second or third capture
monoclonal antibody applied to a
strip of nitrocellulose acts as the
immobile phase.
 The migration of the antigen-
antibody complex in the mobile
phase along the strip enables the
labeled antigen to be captured by
the monoclonal antibody of the
immobile phase, thus producing a
visible colored line

42.  Immunoprecipitation
(Micral test): It is based
on the colour shift of
monoclonal antibody
to human albumin
labelled with gold.

43.  Monoclonal antibodies
can also be used to
purify a substance with
techniques called
immunoprecipitation
and affinity
chromatography.

44.  Mab helps to identify a
specific
molecule/substance in
a mixture of
substances

45.  MAbs have tremendous application not only
in the field of diagnostics but also in
◦ therapeutics and targeted drug delivery systems for
infectious diseases caused by bacteria, viruses,
protozoa and for cancer, metabolic and hormonal
disorders.
◦ They are also used in the immunological
intervention with passive antibody, magic bullet
therapy with cytotoxic agents coupled with anti
mouse specific antibody

46.  Auto immune Conditions
◦ Rheumatoid arthritis,
◦ Crohn's disease
◦ Ulcerative Colitis
by their ability to bind and inhibit TNF Alpha
 Acute rejection of organ transplants by
inhibiting Interleukin-2 on activated T cells
 Allergic Asthma by inhibiting IgE Antibodies
 Malignancies of solid organs

47.  Leukemias
 Lymphomas
 Osteoporosis
 Platelet Aggregation Inhibitors

48. Anti-cancer monoclonal antibodies can be targeted
against malignant cells by several mechanisms:
1)Radioimmunotherapy (RIT) involves the use
of radioactively conjugated murine antibodies
against cellular antigens to limit radiation
exposure.
 Murine antibodies were especially chosen, as their
high immunogenicity promotes rapid clearance
from the body.
◦ Ex:Tositumomab in non-Hodgkins lymphoma.

49. 2)Antibody-directed enzyme prodrug therapy
(ADEPT)
 It involves the application of cancer
associated monoclonal antibodies which are
linked to a drug-activating enzyme.
 Subsequent systemic administration of a
non-toxic agent results in its conversion to
a toxic drug, and resulting in a cytotoxic
effect which can be targeted at malignant
cells

50. 3)Immunoliposomes are antibody-
conjugated liposomes.
 Liposomes - carry drugs or therapeutic nucleotides
- conjugated with monoclonal antibodies -
directed against malignant cells.
 Tissue-specific gene delivery using
immunoliposomes has also been achieved in brain,
and breast cancer tissue

51. mAbs treatment for cancer cells
ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody
dependent cell-mediated cytotoxicity; CDC, complement dependent
cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.

52. DRUGS

53.  Target:
◦ VEGF-A(Vascular Endothelial Growth Factor) that
stimulates neovascularisation
 Indications:
◦ Metastatic Breast,Colon Renal and Brain Cancers
◦ Diabetic Retinopathy,Retinopathy of Prematurity
and Choroidal neovascularisation membrane
 Adv Eff:
◦ Poor wound Healing, Impaired Collateral Formation
around Atherosclerotic Vessels, Hypertension and
Bleeding

54.  Target:
◦ TNF-Alpha
 Indications:
◦ Rheumatoid Arthritis, Ulcerative Colitis, Crohn`s
disease,Psoriatic Arthritis, Ankylosing Spondylitis
 Adverse Effects:
◦ Infections,Lymphomas,Reactivation of Hep
B,Tuberculosis,Drug induced Lupus,Vitiligo

55. ANTIBODY TARGET USES
Abciximab CD41(integrin alpha
receptor)
Platelet aggregation
inhibitor
Adalimumab TNF ALPHA RA, Crohn's,
Psoriasis, Psoriatic
Arthritis, Ankylosing
Spondylitis, Juvenile
Idiopathic
Arthritis, Hemolytic
disease of the
newborn
Alemtuzumab CD 52 CLL

56. ANTIBODY TARGET USES
Basiliximab CD 25(alpha chain of
interleukin 2)
Prevention of Organ
Transplant Rejection
Belimumab BAFF NHL
Bevacizumab VEGF -A Metastatic Cancer,
Diabetic Retinopathy
Brentuximab Vedotin CD 30 Hematologic Cancers
Canakinumab IL-1 RA
Cetuximab EGFR Metastatic Colorectal
Cancer and H&N
Cancer
Certolizumab pegol TNF alpha Crohns disease
Daclizumab CD 25(alpha chain of
interleukin 2)
Prevention of Organ
Transplant Rejection

57. ANTIBODY TARGET USES
Denosumab RANKL Osteoporosis & Bone
Cancer
Eculizumab C5 PNH
Efalizumab LFA-1 (CD11a) Psoriasis
Gemtuzumab CD33 AML
Infliximab TNF Alpha rheumatoid arthritis,
ankylosing spondylitis,
psoriatic arthritis,
psoriasis, Crohn's
disease, ulcerative
colitis
Muromonab CD33 Prevention of Organ
Transplant Rejection

58. ANTIBODY TARGET USES
Natalizumab Integrin alpha 4 Multiple
Sclerosis,Crohn`s
Omalizumab Ig E Fc region Allergic Asthma
Panitumumab EGFR Colorectal Cancer
Ranibizumab VEGF-A Macular Degeneration
Rituximab CD20 Lymphomas,Leukemia
Tocilizumab IL-6 Receptor RA
Tositumomab CD20 Follicular Lymphoma
Trastuzumab HER2neu Breast Cancer

59.  MAbs are used in research purpose also in
analysing
◦ Human Lymphocytes
◦ MHC Antigens
◦ HLA system
 Analysis of antigenic differences between
virus and viral related proteins
 Antigenic Characterization of viruses, bacteria
and parasites

61.  Fever
 Chills
 Weakness
 Headache
 Nausea
 Vomiting
 Diarrhea
 Rashes
 Hypo/Hypertension
DRUG/
TARGET
RELATED SIDE
EFFECTS

62.  Immune response against monoclonal
Antibodies, producing HAMA ("human anti-
mouse antibodies").
 Quickly eliminated
 Immune complexes - Renal Damage
 Monoclonal antibodies raised in humans
would lessen the problem, but few people
would want to be immunized in an attempt to
make them, and most of the attempts that
have been made have been unsuccessful.

63.  Genetic engineering  mouse-human hybrid
antibodies to reduce the problem of HAMA.
 Chimeric antibodies.
◦ Antigen-binding parts (variable regions) of the mouse
antibody with the
◦ Effector parts (constant regions) of a human antibody.
◦ Ex: Infliximab, rituximab, and abciximab
 Humanized antibodies.
◦ Only the amino acids responsible for making the antigen
binding site (the hypervariable regions) of a mouse (or
rat) antibody with
◦ the rest of a human antibody molecule.
◦ Ex:Daclizumab, Gemtuzumab, Transtuzumab

64. Fully Human
Monoclonal
Antibody
Parts of Human Antibody
Parts of Mouse Antibody

65.  Transgenic mice.
 Have human antibody gene loci inserted into their
bodies (using the embryonic stem cell method).
 have had their own genes for making antibodies
"knocked out".
 The result is a mouse that
◦ can be immunized with the desired antigen
◦ produces human, not mouse, antibodies against the
antigen
◦ can yield cells to be fused with myeloma cells to
manufacture all-human monoclonal antibodies.
 Phage display is another technique for making all-
human monoclonal antibodies

66. THE LATEST UPDATE

67.  The production of recombinant monoclonal antibodies
involves technologies, referred to as cloning or phage
display/yeast display.
 Involves the use of viruses or yeast to create antibodies,
rather than mice.
 Rely on rapid cloning of immunoglobulin gene segments to
create libraries of antibodies from which antibodies with
desired specificities can be selected.
 These techniques can be used to enhance
◦ The specificity with which antibodies recognize antigens
◦ The stability in various environmental conditions
◦ Their therapeutic efficacy and
◦ Their detectability in diagnostic applications

68.  mab: whole monoclonal antibody
 Fab: fragment, antigen-binding (one arm)
 F(ab')2: fragment, antigen-binding, including
hinge region (both arms)
 Fab': fragment, antigen-binding, including
hinge region (one arm)
 Variable fragments:
 scFv: single-chain variable fragment
 di-scFv: dimeric single-chain variable fragment
 sdAb: single-domain antibody
 Bispecific monoclonal antibodies:
 3funct: trifunctional antibody
 BiTE: bi-specific T-cell engager

69.  Antibodies can bind to epitopes expressed at the
surface of target cells (as well as to soluble
molecules) but are not effective against the
peptide fragments that antigen-presenting cells
contain tucked within their histocompatibility
molecules.
 T-cell receptors are the ligands needed for that
job
 So monoclonal antibodies are not effective
against intracellular antigens, e.g. virus-encoded
proteins and tumor-specific antigens.
 But now progress is being made toward the
development of monoclonal T-cell receptors (αβ
TCRs).

70.  Preparing a fusion protein of
◦ the engineered TCR conjugated to
◦ an effector molecule
◦ Cytotoxic Agent
to destroy cells expressing the target MHC-peptide
complex.
 These could then be introduced into a cancer patient to
target the tumor-specific antigens or into an AIDS patient
to target HIV-infected cells.

71.  Since 2000, the therapeutic market for monoclonal antibodies
has grown exponentially.
 The current “big 5” therapeutic antibodies on the market are
◦ BEVACIZUMAB
◦ TRASTUZUMAB
◦ ADALIMUMAB
◦ INFLIXIMAB
◦ RITUXIMAB
accounted for 80% of revenues in 2006.
 In 2007, eight of the 20 best-selling biotechnology drugs in the
U.S. are therapeutic monoclonal antibodies.
 This rapid growth in demand for monoclonal antibody
production has been well accommodated by the industrialization
of mAb manufacturing

72.  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.
 MAbs have not only been used as diagnostics,
therapeutics, research reagents, drug targettor for
various infectious diseases but also cancerous,
metabolic and hormonal disorders.
 MAb technology in conjunction with recombinant
DNA technology has successfully led to the
reconstruction of chimeric, humanized and fully
human antibodies and has enormous potentials for
therapeutic uses.

74.  Katzung's - Basic and Clinical Pharmacology
12th edition
 Roitt’s Essential Immunology 11th edition
 medscape
 www.whatisbiotechnology.org/exhibitions/mi
lstein/monoclonals