Monoclonal Antibodies

1. PRESENTER
MOHAMMED AMEER AHMED
M .PHARMACY
ROLL.NO 170215886003
(DEPARTMENT OF PHARMACEUTICS)
Under the guidance of
Mrs. Pavani
M PHARM, Ph. D
&
Mrs Mounika Nijahwan
M PHARM, Ph. D
GOKARAJU RANGARAJU COLLEGE OF
PHARMACY

3. DEFINITION
TYPES OF ANTIBODIES
STRUCTURE OF ANTIBODY
PRODUCTION OF MONOCLONAL ANTIBODIES
THERAPEUTIC APPLICATIONS
LIMITATIONS OF MCA

4. 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.

5. Isotypes
According to differences in their heavy chain constant
domains, immunoglobulins are grouped into five classes, or
isotypes: IgG, IgA, IgM, IgD, and IgE.
IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood
and tissue liquid.
IgA:IgA1 (90%) and IgA2 (10%), stomach and intestines
IgM: normally pentamer, ocassionally hexamer, multiple
immunoglobins linked with disulfide bonds
IgD:1% of proteins in the plasma membranes of B-
lymphocytes, function unknown
IgE: on the surface of plasma membrane of mast cells, play
a role in immediate hypersensitive and denfensive for
parasite

6. The structure of antibodies
• http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html

8. Immunization of Mice and
Selection of Mouse Donors
*Mice are immunized with an antigen that is prepared for injection
either by emulsifying the antigen with Freund's adjuvant or other
adjuvants or by homogenizing a gel slice that contains the antigen.
*Intact cells, whole membranes, and microorganisms are sometimes
used as immunogens. In almost all laboratories, mice are used to
produce the desired antibodies.

9. *In general, mice are immunized every 2-3 weeks but the
immunization protocols vary among investigators.
*When a sufficient antibody titer is reached in serum, immunized mice
are euthanized and the spleen removed to use as a source of cells for
fusion with myeloma cells.
Immunization of Mice and
Selection of Mouse Donors

10. Screening of Mice for Antibody
Production
*After several weeks of immunization, blood samples are obtained
from mice for measurement of serum antibodies.
*Several humane techniques have been developed for collection of
small volumes of blood from mice .
*Serum antibody titer is determined with various techniques, such as
Enzyme-Linked Immunosorbent Assay (ELISA).
*If the antibody titer is high, cell fusion can be performed. If the titer
is too low, mice can be boosted until an adequate response is
achieved, as determined by repeated blood sampling.

11. *When the antibody titer is high enough, mice are commonly boosted
by injecting antigen without adjuvant intraperitoneally or
intravenously (via the tail veins) 3 days before fusion but 2 weeks
after the previous immunization.
*The mice are then euthanized and their spleens removed for in vitro
hybridoma cell production.
Screening of Mice for Antibody
Production

12. Preparation of Myeloma Cells
*Fusing antibody-producing spleen cells, which have a limited life
span, with cells derived from an immortal tumor of lymphocytes
(myeloma) results in a hybridoma that is capable of unlimited
growth.
*Myeloma cells are immortalized cells that are cultured with 8-
azaguanine to ensure their sensitivity to the hypoxanthine-
aminopterin-thymidine (HAT) selection medium used after cell
fusion.

13. *A 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.
Preparation of Myeloma Cells

14. Fusion of Myeloma Cells with
Immune Spleen Cells
*Single spleen cells from the immunized mouse are fused with the previously
prepared myeloma cells.
*Fusion is accomplished by co-centrifuging freshly harvested spleen cells and
myeloma cells in polyethylene glycol, a substance that causes cell
membranes to fuse.
*Only fused cells will grow in the special selection medium. The cells are
then distributed to 96 well plates containing feeder cells derived from saline
peritoneal washes of mice.
*Feeder cells are believed to supply growth factors that promote growth of
the hybridoma cells.

15. *Commercial preparations that result from the collection of media
supporting the growth of cultured cells and contain growth factors are
available that can be used in lieu of mouse-derived feeder cells.
*It is also possible to use murine bone marrow-derived macrophages
as feeder cells.
Fusion of Myeloma Cells with
Immune Spleen Cells

16. Cloning of Hybridoma Cell
Lines by “Limiting Dilution”
*At this step new, small clusters of hybridoma cells from the 96 well
plates can be grown in tissue culture followed followed by selection
for antigen binding or grown by the mouse ascites method with
cloning at a later time.
*Cloning by “limiting dilution” at this time ensures that a majority of
wells each contain at most a single clone.
*Considerable judgment is necessary at this stage to select hybridomas
capable of expansion versus total loss of the cell fusion product due
to underpopulation or inadequate in vitro growth at high dilution.

17. *In some instances, the secreted antibodies are toxic to fragile cells
maintained in vitro.
*Also, it is the experience of many that a brief period of growth by the
mouse ascites method produces cell lines that at later in vitro and in
vivo stages show enhanced hardiness and optimal antibody
production.
Cloning of Hybridoma Cell
Lines by “Limiting Dilution”

18. Selection in HAT medium
*DNA synthesis in mammalian cells proceed through a main (de novo)
pathway which requires glutamine and aspartate respectively as initial
substrates for a series of reactions for the synthesis of purine-type (dATP and
dGTP) and pyrimidine-type (dCTP and dTTP) dNTPs.
*Several of the reactions involved can be blocked by aminopterin, an
analogue of dihydrofolate (another one is methotrexate) that binds with very
high affinity and blocks the enzyme dihydrofolate reductase.
*As a result, de novo synthesis of dATP, dGTP, dCTP and dTTP is blocked.

19. *Mammalian cells, however, survive culture in the presence of aminopterin
because they can utilise two salvage pathways. The first converts
hypoxanthine, a reaction catalysed by the enzyme hypoxanthine-guanine
phosporybosyl transferase (HGPRT).
*The second converts thymidine in dTMP, a reaction catalysed by thymidine
kinase (TK). Thus a mutation in either the HGPRT or the TK gene would
lead to normal growth in standard culture medium but to death in
Littlefield's HAT medium (aminopterin, hypoxanthine and thymidine).
*The hybrid produced between such myeloma line and a B cell, however,
would survive because it would utilise the normal HGPRT or TK gene of the
B cell.
Selection in HAT medium

22. Analytical applications
*Antibody may be used to detect an antigen in forensic applications, in
microbiological testing of foodstuffs and in diagnostic testing of
blood samples for toxins or infectious organisms.
*The antibody-based test for the presence of antigen may be rendered
quantitative, providing an assay for antigen.
*Antibody-based assays are widely used in medicine to determine
levels of growth factors, hormones, blood cells or malignant cells; the
applications are essentially unlimited.

23. *Still in analytical mode, monoclonal antibodies may be used to locate
antigen.
*Antibodies are widely used in conjunction with color forming labels
and microscopy to localize antigens in tissue sections. This is known
as Immunohistochemistry.
Analytical applications

24. *Antibodies may also be used preparatively to purify molecules or
cells from crude mixtures.
*Immunoaffinity based preparative techniques are very powerful
compared to more traditional biochemical purification methods.
*A successful purification procedure will combine both affinity-based
and conventional methods.
Preparative applications

25. *Antibody-based purification methods are useful from the laboratory
scale, where the aim is to purify nanogram to milligram quantities of
biological substances from complex mixtures such as serum, to
production of therapeutic substances, such as the blood-clotting
factor VIII from large volumes of blood.
*The use of antibody to identify particular cell types in complex
mixtures has been extended to preparative methods to purify these
cells
Preparative applications

26. *One approach is to link antibody to magnetic particles.
*A magnet is then used to physically separate cells that bear the
antigen from cells that do not.
*A more powerful technique uses the Fluorescence Activated Cell
Sorter (FACS) , in which a cell may be identified on the basis of
antibody tagged with fluorescent dye and physically separated from
the other cells.
Preparative applications

27. *Potential therapeutic applications of antibodies include the
neutralisation of toxins, the removal of infectious agents from the
circulation and the destruction of body cells mediating disease,
including autoimmune cells and cancer cells.
Therapeutic applications

28. *Polyclonal antisera against human lymphocytes were developed in the 1970s
to treat patients who were rejecting organ grafts.
*The principle was that the host immune response was responsible for the
organ graft rejection;
*Antibodies against key components of the immune system should suppress
the rejection.
*These antisera have largely been superseded by a monoclonal antibody
called OKT3, directed against a molecule expressed on human T cells and
*Involved in T-cell function.
Therapeutic applications

29. *OKT3 has been highly successful in reversing rejection episodes. The
injected antibody is a mouse protein and is immunogenic, but the
response is muted because the patients are immunosuppressed, both
by the antibody itself and by other immunosuppressive therapy used
to reduce the graft response.
*Nevertheless, OKT3, successful though it has been, has been
perceived as limited in effectiveness partly by its immunogenicity.
Therapeutic applications

30. *There were highly promising results in animal models, where
antibodies can destroy established tumours, induce a state of
permanent tolerance to transplanted organs, reverse autoimmune
disease and rescue animals from acute toxicity caused by, for
example, bacterial endotoxin.
Therapeutic applications

31. Antibodies have been developed against :
*CD20 in B-cell malignancies and arthritis.
*against the cell-surface receptor human epidermal growth factor
receptor 2 (HER2) in breast cancer and
*against the inflammatory cytokine tumour necrosis factor (TNF) in
autoimmune disease.
Therapeutic applications

32. *Immunogenicity;
*Difficulty and cost of production on an adequate scale.
*Unwanted biological activity – due, for example, to direct effects on
cells of the immune system.
*Limited binding affinity, which necessitates the injection of large
amounts of antibody in order to achieve a therapeutic effect.
*Lack of direct functional action, requiring conjugation of drugs or
other biologically active materials and
*Limited penetration into the target tissue – especially dense, poorly
vascularised tumour tissue.
Limitations of Monoclonal
Antibodies