Monoclonal antibodies dr. asm

As per PCI Syllabus for M. Pharm (Phrmaceutics) students...

1. Monoclonal AntibodiesMonoclonal Antibodies
Preparation & application
Dr. Atish S. Mundada
Associate Professor,
SNJB’s SSDJ College of Pharmacy,
Chandwad, Dist. Nashik

2. Introduction:Introduction:
•Serum contains many different types of antibodies ( polyclonal)
that are specific for many different antigens.
•Even in hyperimmuneanimals, seldom are more than one-tenth of
the circulating antibodies specific for one antigen.
•The use of these mixed populations of antibodies creates a variety
of different problems in immunochemical techniques. Therefore,
the preparation of homogeneous, or monoclonal, antibodies with
a defined specificity was a long-standing goal of immunochemical
research.
•This goal was achieved with the development of the technology
for hybridomaproduction.
•The first isolation of a homogeneous population of antibodies
came from studies of B-cell tumors.
•Clonalpopulations of these cells can be propagated as tumors in
animals or grown in tissue culture.

3. •In the animal, antibodies are synthesized primarily by plasma cells,
a type of terminally differentiated B lymphocyte. Because plasma
cells cannot be grown in tissue culture, they cannot be used as an
in vitro source of antibodies.
•The advent of hybridomatechnology by Kohler and Milstein in
1975 for which they were awarded the Nobel prize in medicine in
1984 revolutionized many areas of biological and medical
research.
•In this technique, an antibody-secreting cell is isolated from an
immunized animal, then fused with a myeloma cell, a type of B-
cell tumor.
•These hybrid cells or hybridomascan be maintained in vitro and
will continue to secrete antibodies with a defined specificity.
•Antibodies that are produced by hybridomasare known as
monoclonal antibodies.

4. Characteristics of Monoclonal Antibodies:Characteristics of Monoclonal Antibodies:
•The usefulness of monoclonal antibodies stems from three
characteristics—their specificity of binding, their homogeneity, and
their ability to be produced in unlimited quantities.
•The production of monoclonal antibodies allows the isolation of
reagents with a unique, chosen specificity. Because all of the
antibodies produced by descendants of one hybridomacell are
identical, monoclonal antibodies are powerful reagents for testing
for the presence of a desired epitope.
•Hybridomacell lines also provide an unlimited supply of
antibodies.
•However, monoclonal antibodies are often more time-consuming
and costly to prepare than polyclonal antibodies.
•They are not necessarily the best choice for certain
immunochemical techniques.
•In practice, however, producing exactly the right set of monoclonal
antibodies is often a difficult and laborious job.

5. HybridomasHybridomas::
•In the early 1970s, several research groups worked on different
methods to extend the life span of antibody-secreting cells in
vitro.
•For murinecells, the practical aspects of this goal were solved by
applying techniques used in somatic cell genetics.
•By fusing two cells, each of which have properties necessary for a
successful hybrid cell line, Kohler and Milstein (1975) showed that
antibody secreting cell lines could be established routinely and
then maintained in vitro.
•The two cells that are commonly used as partners in these fusions
are antibody-secreting cells isolated from immunized animals and
myeloma cells.
•The myeloma cells provide the correct genes for continued cell
division in tissue culture, and the antibody-secreting cells provide
the functional immunoglobulin genes.

6. •In theory, the fusion between the myeloma cell and the antibody-
secreting cell can be effected by any fusogen.
•In practice, hybridomafusions became routine after the
introduction of the use of polyethylene glycol (PEG).
•The use of PEG as a fusing agent for mammalian cells was first
shown by Pontecorvo(1975) and was quickly adopted by somatic
cell geneticists.
•PEG is the method of choice for hybridomaproduction, allowing
the rapid and manageable fusion of mammalian cells.
•PEG fuses the plasma membranes of adjacent myeloma and/or
antibody-secreting cells, forming a single cell with two or more
nuclei.
•Even in the most efficient hybridomafusions, only 1% of the
starting cells are fused, and only about 1 in 105 form viable
hybrids. This leaves a large number of unfusedcells still in the
culture.
•The cells from the immunized animal do not continue to grow in
tissue culture and thus do not confuse further work.

7. Production of Monoclonal Antibodies:Production of Monoclonal Antibodies:
•Although hybridomascan be prepared from animals other than
mice. Similar techniques can be used for fusions of rat myelomas
and rat antibody secreting cells, as well as rabbit plasmacytomas
and rabbit antibody-secreting cells.
•Stages of HybridomaProduction:-
–1. Immunizing the mice
–2. Developing the screening procedure
–3. Producing Hybridomas
•Once a good humoralresponse (titer) has appeared in the
immunized animal and an appropriate screening procedure is
developed, a fusion can be considered.
•The sera from test bleeds are used to develop and validate the
screening procedure.
•After an appropriate screening assay has been established, the
actual production of the hybridomascan begin.

8. •Once a good immune response has developed in an animal and an
appropriate screening procedure has been developed, the construction of
hybridomasis ready to begin.
•For the actual fusion, antibody-secreting cells are isolated from the
appropriate lymphoid tissue (spleen and lymph nodes), mixed with
myeloma cells, centrifuged to generate good cell-to-cell contacts, and
fused with the aid of a fusing agent like PEG, Sendai virus, or electrofusion.

9. •The fused cells are then removed from the fusing agent, diluted into
selective medium and plated in multiwelltissue culture dishes.
•Beginning 1 wk later, samples of the tissue culture supernatants are
removed from wells that contain growing hybridomasand tested for the
presence of the appropriate antibodies.

10. •A strong immune response and the use of a good screening
method will make the production of the hybridomasan easier
task.
•Cells from positive wells are grown, single-cell-cloned, and
frozen. Finally, the monoclonal antibodies are collected and used.

11. List and types of monoclonal antibodies (FDA approved):List and types of monoclonal antibodies (FDA approved):
•abciximab(Reopro)
•adalimubab(Humira, Amjevita)
•alefacept(Amevive)
•alemtuzumab(Campath)
•basiliximab(Simulect)
•belimumab(Benlysta)
•bezlotoxumab(Zinplava)
•canakinumab(Ilaris)
•certolizumabpegol(Cimzia)
•cetuximab(Erbitux)
•daclizumab(Zenapax, Zinbryta)
•denosumab(Prolia, Xgeva)
•efalizumab(Raptiva)
•golimumab(Simponi, Simponi
Aria)
•inflectra(Remicade)
•ipilimumab(Yervoy)
•ixekizumab(Taltz)
•natalizumab(Tysabri)
•nivolumab(Opdivo)
•olaratumab(Lartruvo)
•omalizumab(Xolair)
•palivizumab(Synagis)
•panitumumab(Vectibix)
•pembrolizumab(Keytruda)
•rituximab(Rituxan)
•Tocilizumab(Actemra)
•Trastuzumab(Herceptin)
•secukinumab(Cosentyx)
•ustekinumab(Stelara)

12. Applications of Monoclonal Antibodies:Applications of Monoclonal Antibodies:
•The use of monoclonal antibodies to treat diseases is called
immunotherapy therapy because each type of monoclonal
antibody will target a specific targeted antigen in the body.
•There are currently several mAb-based products which have been
approved by the US Food and Drug Administration (FDA) and/or
the European Union Health Authorities for the treatment of a
wide range of diseases including autoimmunity, organ transplant
rejection, inflammation, infection as well as human cancers.
There are four types of applications of monoclonal antibodies.
(1) Diagnostic Applications
(2) Therapeutic Applications
(3) Protein Purification and
(4) Miscellaneous Applications.

13. 1. Diagnostic Applications:
•Monoclonal antibodies have revolutionized the laboratory
diagnosis of various diseases.
•For this purpose, MAbsmay be employed as diagnostic reagents
for biochemical analysis or as tools for diagnostic imaging of
diseases.
A) MAbsin Biochemical Analysis:
•Diagnostic tests based on the use of MAbsas reagents are
routinely used in radioimmunoassay (RIA) and enzyme-linked
immunosorbentassays (ELISA) in the laboratory.
•These assays measure the circulating concentrations of hormones
(insulin, human chorionic gonadotropin, growth hormone,
progesterone, thyroxine, triiodothyronine, thyroid stimulating
hormone, gastrin, renin) and several other tissue and cell products
(blood group antigens, blood clotting factors, interferon’s,
interleukins, histocompatibilityantigens, tumor markers).

14. B) MAbsin Diagnostic Imaging:
•Radiolabeled—MAbsare used in the diagnostic imaging of
diseases, and this technique is referred to as immunoscintigraphy.
•The radioisotopes commonly used for labeling MAbare iodine—
131 and technetium—99. The MAbtagged with radioisotope are
injected intravenously into the patients.
•These MAbslocalize at specific sites (say a tumor) which can be
detected by imaging the radioactivity.
•In recent years, single photon emission computed tomography
(SPECT) cameras are used to give a more sensitive three
dimensional appearance of the spots localized by radiolabeled—
MAbs.
•Immunoscintigraphyis a better diagnostic tool than the other
imaging techniques such as CT scan, ultrasound scan and magnetic
resonance.
•Mabsare successfully used in the diagnostic imaging of
cardiovascular diseases, cancers and sites of bacterial infections.

15. 2. Therapeutic Applications:
•Monoclonal antibodies have a wide range of therapeutic
applications.
•MAbsare used in the treatment of cancer, transplantation of bone
marrow and organs, autoimmune diseases, cardiovascular diseases
and infectious diseases.
•The therapeutic applications of MAbsare broadly grouped into
2 types:
–(A) Direct use of MAbsas therapeutic agents
–(B) MAbsas targeting agents.
(A) MAbsas Direct Therapeutic Agents:
•Monoclonal antibodies can be directly used for enhancing the
immune function of the host.
•Direct use of MAbscauses minimal toxicity to the target tissues or
the host.

16. •MAbspromote efficient opsonizationof pathogenic organisms (by
coating with antibody) and enhance phagocytosis.
•Thus help In destroying disease-causing organisms.
•MAbs, against the antigens on the surface of cancer cells, are
useful for the treatment of cancer.
•Limitations for direct use of MAbsin cancer:
1. The MAbsproduced in mice and directly used for therapeutic
purposes may lead to the development of anti-mouse antibodies
and hypersensitivity reactions.
2. All the cancer cells may not carry the same antigen for which MAb
has been produced. Thus, MAbsmay not be attached to some
cancer cells at all.
3. The free antigens (of target cells) present in the circulation may
bind to MAbsand prevent them from their action on the target
cells.

17. B) MAbsas Targeting Agents in Therapy:
•Toxins, drugs, radioisotopes etc., can be attached or conjugated to
the tissue-specific monoclonal antibodies and carried to target
tissues for efficient action.
•This allows higher concentration of drugs to reach the desired site
with minimal toxicity. In this way, MAbsare used for the
appropriate delivery of drugs or isotopes.
•The toxins can be coupled with MAbsto form immunotoxinsand
used in therapy e.g., diphtheria toxin, Pseudomonas exotoxin,
toxins used for cancer treatment.
•The drugs can be coupled with MAb(directed against a cell
surface antigen of the cells, say a tumor) and specifically targeted
to reach the site of action.
•Antibody-directed enzyme pro-drug therapy (ADEPT), allows an
effective delivery of the drug to the cells where it is required.

18. 3. Protein Purification:
•Monoclonal antibodies can be produced for any protein. And the
so produced MAbcan be conveniently used for the purification of
the protein against which it was raised.
•MAbscolumns can be prepared by coupling them to cyanogen
bromide activated Sepharose(chromatographic matrix).
•The immobilized MAbsin this manner are very useful for the
purification of proteins by immunoaffinitymethod.
•There are certain advantages of using MAbsfor protein
purification. These include the specificity of the MAbto bind to
the desired protein, very efficient elution from the
chromatographic column and high degree of purification.
•It is not possible to achieve 100% purity of the target protein by
immunoaffinity. This is due to the fact that a small quantity of
MAbleaks into the elution.

19. 4. Miscellaneous Applications:
A) Catalytic MAbs(ABZYMES):
•Catalysis is the domain of enzymes. The most important common
character between enzymes and antibodies is that both are
proteins.
•Further, the binding of an antibody to its antigen is comparable to
the binding of an enzyme to its substrate.
•In both instances, the binding is specific with high affinity and
involves weak and non-covalent interactions (electrostatic,
hydrogen and van der Waals forces).
•The striking difference is that the enzyme alters the substrate (to a
product) while the antigen bound to antibody remains unaltered.
•Certain similarities between enzyme-substrate interaction and
antibody-antigen interaction have tempted researchers to explore
the possibility of using antibodies in catalysis.
•The antibody enzymes, appropriately regarded as abzymes, are
the catalytic antibodies.

20. B) Autoantibody Fingerprinting:
•The occurrence of autoantibodiesand their involvement in
certain diseases is well known (e.g. rheumatic arthritis).
•A new category of individual specific (IS) autoantibodieshave
been discovered in recent years. These IS-autoantibodiesare
produced after birth and reach maximum in number by 2 years,
and then remain constant for the later part of life.
•Monoclonal antibodies produced against IS-autoantibodiescan
be used for their detection, and identification of individuals.
•This technique referred to as autoantibody finger­printing, is
particularly useful for the detection of criminals, rapists etc.