Immunotechnology

1. COMMERCIAL PRODUCTION OF
MONOCLONAL ANTIBODIES

2. Monoclonal Antibody Production
Method
• Monoclonal Antibody Production technology was developed in 1975. Since its
development it has been very important in the modern medical science with the
diagnosis, therapy, research and even basic science today. It is still largely
dependent upon animal testing however. Because it requires immunization of
mice in order for them to create the antibodies to be grown.
• Monoclonal Antibody Production or mAb is produced by cell lines or clones
obtained from the immunized animals with the substance to be studied. Cell lines
are produced by fusing B cells from the immunized animal with myeloma cells. To
produce the desired mAb, the cells must be grown in either of two ways: by
injection into the peritoneal cavity of a suitably prepared mouse (the in vivo, or
mouse ascites, method) or by in vitro tissue culture.
• The in vitro tissue culture is the method used when the cells are places in culture
outside the mouse's body in a flask.

3. • Large Amounts Of Antibody Can Be Produced
Using Animals
– Prime Balb/c animal with IFA
– Inject clone i.p
– Collect peritoneal ascites
• Alternatively Bioreactors Are Used
– Cells are cultured in hollow fibers
– Fresh media and waste are recirculated
– High concentrations of Ab produced in cell
compartment
– Collected at different time points
Antibody Production

4. Why this method is used!!
• This method is used because antibodies must be formed from the immunization of
the substance being studied. So antibodies must be produced. Once the
antibodies are produced the animal aspect of the study can be eliminated and
tissue culture can then be used.
• When using live mice researchers have found that it is the better option because in
vitro doesn’t always produce adequate cell lines that are adaptive to tissue
culture. Protein denaturation can occur from purification techniques and antibody
activity is decreased with normal activity not represented. Also cell lines could
possibly become contaminated when using in vitro technique.

5. Problems with using mouse mAb
• The therapeutic use of murine monoclonal antibodies in humans
is limited by their immunogenic, short circulating half-life, and
inability to efficiently trigger human effectors mechanisms.
• This is due to differences between the mouse and humans.
• Also severe allergic response in human when mouse mAb are
introduced to a patients.
• Main difficulty is that mouse antibodies are "seen" by the
human immune system as foreign, and the human patient
mounts an immune response against them, producing HAMA
("human anti-mouse antibodies")
• Also constant region of murine mAb are not effective in
interacting with human effectors molecules.

6. MONOCLONAL ANTIBODIES PRODUCED
BY RECOMBINANT DNA TECHNOLOGY
•With the emergence of recombinant DNA technology,
monoclonal antibodies with greater amounts of human
sequences are now being developed and used.
•One recombinant DNA method by which this can be
accomplished is by isolating the variable region genes from a
murine hybridoma secreting an antibody that binds the
desired target, then amplifying these genes using polymerase
chain reaction (PCR).
•This initial product is a copy DNA (cDNA) of the murine
variable region (V-cDNA) The V-cDNA can then be ligated into
a plasmid.

7. • In a parallel sequence, cDNA for human heavy chain
constant regions is also amplified and ligated into a
separate plasmid producing heavy chain copy DNA (HC-
cDNA).
• At this point, the V-cDNA and HC-cDNA can be brought
together into a common host cell using co-transfection.
• This elaborate method provides the desired fusion protein,
intact chimeric antibody.
• Because bacteria do not correctly glycosylate human
proteins, the antibodies are not usually secreted from the
bacteria.
• Instead, the chimeric antibody is normally expressed in
inclusion bodies in the bacteria cells and must be extracted
and purified.

8. ENGINEERED MONOCLONAL
ANTIBODIES

9. Chimeric mAb
• Chimeric mAb are genetically engineered antibodies produced by using
a molecular approach.
• Chimeric Ab are obtained by genetically fusing the mouse variable
domains to human constant domains.
• Variable regions are Isolated using polymerase chain reaction (PCR).
CHIMERIC MONOCLONAL ANTIBODIES
Abciximab (ReoPro®) Platelet aggregation inhibitor. Used with coronary artery
procedures
Infliximab (Remicade®) Binds tumor necrosis factor. Used to treat autoimmune
diseases
Cetuximab (Erbitux®) Inhibits epidural growth factor. Used to slow growth of
metastatic disease
Rituximab (Rituxan®) Destroys B cells. Used to treat non-Hodgkins lymphoma

10. Chimeric mAb

11. CHIMERIC HUMAN/MOUSE
FR1 FR2 FR3 FR4
CDR1 CDR2 CDR3
CONSTANT
CONSTANT
CONSTANT
FR1 FR2 FR3 FR4
CDR1 CDR2 CDR3
Mouse
Human
Chimeric

12. Human Mouse
Chimeric

13. Issues with Chimeric mAb
• Sometimes the body may elicit an anti-chimeric Ab
in the present of these genetically engineered Ab.
• The human against chimeric antibody (HACA) is an
immune response to the murine portion(variable
region) of the antibody (30% murine resource).

14. Humanized mAb
• To address this problem, the complementarity
determining regions (CDRs), which are responsible for
antigen binding within the variable regions, have been
transferred to human frameworks creating ‘‘CDR-
grafted’’ or ‘‘humanized’’ antibodies. This is, in essence
a human Ab with small segments containing mouse Ab
genes.
HUMANIZED MONOCLONAL ANTIBODIES
Palivizumab (Synagis®) Prevention of respiratory syncytial virus
(RSV) infections in infants.
Trastuzumab (Herceptin®) Treatment of HER2-positive metastatic
breast cancer.
Alemtuzumab (Campath®)
Treatment of chronic lymphocytic leukemia (CLL), cutaneous T-
cell lymphoma (CTCL) and T-cell lymphoma.

15. HUMANIZED ANTIBODIES
FR1 FR2 FR3 FR4
CDR1 CDR2 CDR3
CONSTANT
CONSTANT
CONSTANT
FR1 FR2 FR3 FR4
CDR1 CDR2 CDR3
Mouse
Human
Humanized

16. Human Mouse
Humanized

17. Chimeric mAbs
Chimeric mouse human
mAbs
Chimers combine the human
constant regions with the intact
mouse variable regions. Affinity
and specificity unchanged.
Humanized mAbs
or
Grafted CDRs
Contained only the CDRs
of the rodent variable
region grafted onto
human variable region
framework.

18. ENGINEERED MONOCLONAL
ANTIBODIES
Chimeric mAbs Hybrid mAbs

19. Hybrid mAb