Monoclonal antibody

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-The Secret of Monoclonal antibody-The Secret of Monoclonal antibody

Introduction
Antibodies have important uses beyond fighting infections in
the body. Production of long-lasting monoclonal antibodies is a
recent invention and it is used in both medicine and research.
Monoclonal Antibody: a stable antibody which can be used over
a period of time. monospecific antibodies that are identical
because they are produced by one type of immune cell (a
single parent cell).
Antibodies produced from a single clone of B cells. Produced
by fusing a B cell secreting the desired antibody with a
myeloma cell capable of growing indefinitely in tissue culture.
Monoclonal antibodies all have identical antigen-binding sites.
i.e. they all bind to the same epitope with the same affinity.
They are all of the same antibody class (isotype).

• Early antibodies displayed insufficient
activation of human effector functions (i.e. the
antibodies did not kill the infecting organism or
cell)
• The early antibodies were of murine (mouse)
origin, and thus triggered the production of
human anti-mouse antibodies (HAMA).
• Antigen distribution of malignant cells is highly
heterogeneous, so some cells may express
tumor antigens, while others do not.
• Tumor blood flow is not always optimal
• High interstitial pressure within the tumor can
prevent the passive monoclonal antibody from
binding.

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.

History of Mab Development
• 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.
• 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.

Evolution of Therapeutic Antibodies

• ‘Naked’ means these antibodies are not fused to a
toxin.
• Naked Monoclonal antibodies can kill cells via a
variety of mechanisms, including: Antibody-
Dependent Cellular Cytotoxicity (ADCC),
Complement-Dependent Cytotoxicity (CDC), and
direct induction of apoptosis.
• However, the precise clinical mechanisms often
remain uncertain
‘Naked’ Monoclonal Antibodies

The types of mAb Designed
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.

Producing Monoclonal Antibodies
1. Inject a mouse with a specific antigen to stimulate its
immune system to produce necessary antibodies.
2. Extract mouse spleen cells (containing B-lymphocytes) and
culture them in the lab.
3. Extract mouse tumour cells, which grow continuously, and
culture them in the lab.
4. Mix spleen cells and tumour cells on the same plate and
culture.
5. Add polyethylene glycol – this causes some B-lymphocytes
to fuse with tumour cells to produce a hybrid cell called a
hybridoma.
6. Grow the cells under conditions that allow only hybridoma
cells to survive.
7. Extract the cells, culture them separately and test the
medium around each cell for the specific antibody of
interest.
8. Culture the cells making the desired antibody and use as
needed.

Cotaminants:
 Media components: hormones, growth factors, transferrins etc.
 Viral, bacterial,endotoxins….etc
Methods of purification:
1. Filteration for larger particles.
2. Ultrafilteration esp. for low concentration samples.
3. Chromatography: Ion exchange chromatography
(either cation or anion) can be used (Most impurities are usually
anions).
1. Size exclusion chromatography.
Purification of monoclonal Antibodies

mAbs Development
1. Phage display library: construction of VH and VL gene
libaries and expression of them on a filamentous
bacterophage. The phage expressing an antigen-bonding
domain specific for a particular antigen to screen the mAbs.
2. Transgenic plants: transgenic tobacco plants to produce
IgA.
3. Transgenic animals: transgenic mouse to make humanized
IgG. (Abgenix,CA).

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

Disadvantages
• Many patients develop immune response to
monoclonal antibodies produced in mice, as
these are foreign proteins.
• Genetically engineered antibodies are being
perfected to avoid triggering immune response.

Application
• Treatment of Cancer.
• Diagnosis of HIV Infection.
• Pregnancy Tests: When pregnant woman’s
urine travels up the pregnancy test, HCG will
bind to monoclonal antibodies in reaction
region.
• mAbs was modified for delivery of a toxin,
radioisotope, cytokine or other active
conjugates.
• Purification of drugs, Imaging the target.
• Fight against Bioterrorism
• Detecting small quantities of metastatic cancer
and MDR

Uses
1. Measuring protein and drug levels in serum.
2. Typing tissue and blood.
3. Identifying infectious agents.
4. Identifying clusters of differentiation (CDs) for
5. Classification and follow-up therapy of leukemias
and lymphomas.
6. Identifying tumor metastasis.
7. Identifying and quantifying hormones.
8. Immunoaffinity Purification.

Conclusion
Antibodies have important uses beyond fighting infections
in the body. Production of long-lasting monoclonal
antibodies is a recent invention and it is used in both
medicine and research. Monoclonal Antibody: a stable
antibody which can be used over a period of time. 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 were produced by one type of immune cell,
all clones of a single parent cell. This has become an
important tool in biochemistry, molecular biology and
medicine.

References
1. Manshouri T, Do KA, Wang X, Giles FJ, O'Brien SM, et al.
(2003) Circulating CD20 is detectable in the plasma of
patients with chronic lymphocytic leukemia and is of
prognostic significance. 101:2507-2513.
2. Lundin J, Osterborg A (2004) Advances in the use of
monoclonal antibodies in the therapy of chronic
lymphocytic leukemia. SeminHematol. 41:234-245.
3. Golay J, Lazzari M, Facchinetti V, Bernasconi S, Borleri G, et
al. (2001) CD20 levels determine the in vitro susceptibility
to rituximab and complement of B-cell chronic lymphocytic
leukemia: further regulation by CD55 and CD59. Blood.
98:3383-3389.
4. Huhn D, von Schilling C, Wilhelm M, Ho AD, Hallek M, et al.
(2001) Rituximab therapy of patients with B-cell chronic
lymphocytic leukemia. Blood. 98:1326-1331.

5. Tsai PC, Hernandez-Ilizaliturri FJ, Bangia N, Olejniczak SH,
Czuczman MS (2012) Regulation of CD20 in rituximab-resistant
cell lines and B-cell non-Hodgkin lymphoma. Clin Cancer
Res.18:1039-1050.
6. Singh V, Gupta D, Arora R, Tripathi RP, Almasan A, et al
(2014) Surface levels of CD20 determine anti-CD20 antibodies
mediated cell death in vitro. PLoS One. 9: e111113.
7. Hofmeister JK, Cooney D, Coggeshall KM (2000) Clustered
CD20 induced apoptosis: src-family kinase, the proximal
regulator of tyrosine phosphorylation, calcium influx, and
caspase 3-dependent apoptosis. Blood Cells Mol Dis.26:133-43.
8.

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