Monoclonal antibodies are identical antibodies produced by a single clone of B cells that specifically bind to target cells. They can be used for cancer therapy by triggering immune system attacks on cancer cells, blocking growth signals, or preventing new blood vessel formation. Monoclonal antibodies are produced through hybridoma technology, fusing B cells with myeloma cells. This produces immortal clones that secrete identical antibodies. Monoclonal antibodies have applications in cancer diagnosis and treatment, with therapeutic antibodies targeting tumors through mechanisms like antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity.

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MONOCLONAL ANTIBODIES IN CANCER THERAPY
Introduction
Monoclonal Antibodies that are identical because they were produced by one
type of immune cell (B cell), all clones of a single parent cell.
specifically bind to target cells. This may then stimulate the patient's immune
system to attack those cells.
It is possible to create a MAb specific to almost any extracellular/ cell surface
target, and thus there is a large amountof research and development currently
being undergone to create monoclonals for numerous serious diseases (such
as rheumatoid arthritis, multiple sclerosis and different types of cancers)
When monoclonal antibodies enter the body and attach to cancer cells, they
function in several different ways: first, they can trigger the immune system to
attack and kill that cancer cell; second, they can block the growth signals; third,
they can prevent the formation of new blood vessels.

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Discovery
The idea of a "magic bullet" was first proposed by Paul Ehrlich, who, at the
beginning of the 20th century, postulated that, a compound can be made that
selectively targeted a disease-causing agent.
Production of monoclonalantibodie involving human–mousehybrid cells was
described by Jerrold Schwaber in 1973.
In 1988, Greg Winter and his team pioneered the techniques to humanize
monoclonal antibodies.

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Production of monoclonal antibodies
 By Hybridoma Technique -
A hybridoma, is produced by the injection of a specific antigen into a
mouse, procuring the antigen-specific plasma cells (antibody-producing
cell) fromthe mouse's spleen and the subsequentfusion of this cell with
a cancerous immune cell called a myeloma cell.

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Once splenocytes areisolated from the mammal’s spleen, the B cells are
fused with immortalised myeloma cells. The hybrid cell, which is thus
produced, can be cloned to produce many identical daughter clones.
These daughter clones then secrete the immune cell product. Since
these antibodies come from only one type of cell (the hybridoma cell)
they are called monoclonalantibodies.The advantage of this process is
that it can combine the qualities of the two different types of cells; the
ability to grow continually, and to producelarge amounts of pure
antibody.
HAT medium (HypoxanthineAminopetrin Thymidine) is used for
preparation of monoclonal antibodies. Laboratory animals (eg. mice) are
firstexposed to an antigen to which we are interested in isolating an
antibody against.
Once splenocytes are isolated from the mammal, the B cells are fused
with immortalized myeloma cells - which lack the HGPRT(hypoxanthine-
guanine phosphoribosyltransferase) gene - using polyethylene glycol or
the Sendai virus.
Fused cells are incubated in the HAT (HypoxanthineAminopetrin
Thymidine) medium. Aminopterin in the myeloma cells die, as they
cannot produce nucleotides by the de novo or salvagemedium blocks
the pathway that allows for nucleotide synthesis. Hence, unfused D cell
die.
Unfused B cells die as they havea shortlife span. Only the cell-myeloma
hybrids survive, sincethe HGPRTgene coming from the B cells is
functional. These cells produceantibodies (a property of B cells) and are
immortal (a property of myeloma cells)
The incubated medium is then diluted into multiwell plates to such an
extent that each well contains only 1 cell.Then the supernatantin each
well can be checked for desired antibody.
Since the antibodies in a well are produced by the sameB cell, they will
be directed towards the sameepitope, and are known as monoclonal
antibodies

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Fig- Production of monoclonalantibodies
 Types of Monoclonal Antibodies

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

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Chimeric antibodies
 Chimeric antibodies are composed of
murine variable regions fused onto
human constant regions. Affinity and
specificity unchanged. Also cause human
antichimeric antibody response (30%
murine resource)
 Antibodies are approximately 65%
human.
 This reduces immunogenicity and thus
increases serum half-life.

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Humanised Mab
• Humanised antibodies are produced by
grafting murine hypervariable amino
acid domains into human antibodies.
• contained only the CDRs of the rodent
variable region grafted onto human
variable region framework.
• antibodies have been genetically
engineered to replace all of the antibody
molecule with human counterparts
except the hypervariable regions directly
involved with antigen binding.
• This results in a molecule of
approximately 95% human origin.

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Human Monoclonal antibody
Human monoclonal antibodies are produced by transferring human
immunoglobulin genes ito the murine genome, after which the transgenic
mouse is vaccinated against the desired antigen, leading to the production of
monoclonal antibodies.
Humanised MAbs consist of mouse derived complementary determining
regions (CDR) which are bound to a human IgG framework.
Humanised MAbs contain 5-10% of mouse proteins unlike chimeric MAbs
which contains approximately 34% murine protein.
Humanized MAbs are ideally employed as part of a therapeutic regime against
diseases such as Cancers e.g. Non-Hodgkin’s Lymphoma (NHL), breast cancer
and renal cell carcinoma.

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Applications of Monoclonal Antibodies
 Diagnostic Applications
as research tools, scientists quickly
recognized their practical uses,
especially in diagnostic tests and in
therapy
 Detects protein of interest either by
blottingor immunofloroscence.
 In diagnosis, pregnancy can be detected
by assaying of hormones with
monoclonal.
 A monoclonal antibody can be used to detect
pregnancy only 14 days after conception.
Other monoclonal antibodies allow rapid
diagnosis of hepatitis, influenza, herpes,
streptococcal, and Chlamydia infections.
 They can be used to detect for the presence
and quantity of this substance, for instance in
a Western blot test (to detect a substance in a
solution) or an immunofluorescence test.
 Monoclonal antibodies can also be used to
purify a substance with techniques called
immunoprecipitation and affinity
chromatography
 Therapeutic Applications
 Transplant rejection
 Cancer
 Autoimmune disorders
 Inflammatory disease

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Antitumor MABs
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

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Mechanism of antitumor effect
• Antibody dependent cellular cytotoxicity (ADCC)
• Complement dependent cytotoxicity (CDC)
• Direct induction of apoptosis
• MAb may be conjugated with a toxin e.g. gemtuzumab-ozogamicin
• MAb can also be conjugated with radioisotope
A. MAb act directely when binding to a cancer specific antigens
and induce immunological response to cancer cells. Such as
inducing cancer cell apoptosis, inhibiting growth, or interfering
with a key function.
B. MAb was modified for delivery of a toxin, radioisotope,
cytokine or other active conjugates.
C. it is also possible to design bispecific antibodies that can bind
with their Fab regions both to target antigen and to a conjugate
or effector cell.

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ADEPT (Antibody Directed Enzyme Prodrug Therapy)
 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.
ADEPT is a system that aims to restrict the
action of a high concentration of a cytotoxic
drug to cancer sites. This is achieved by
using an antibody (or antibody fragment) to
deliver a non-human enzyme to cancer
sites.

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Immunoliposomes
• Immunoliposomes are antibody-conjugated liposomes.
• Liposomes can carry drugs or therapeutic nucleotides and when
conjugated with monoclonal antibodies.
• When conjugated with antibodies as targeting ligands,
immunoliposomes can target tumor cells with high specificity and
affinity, resulting in significantly improved antitumor activity over
untargeted liposomes.
• antitumor activity could be further ehanced for liposomes through
ligand-mediated targeting.
• For liposomes, the ligands would promote the selective binding and
facilitate the intracellular delivery. The most commonly used ligands
include MAbs or antibody fragments.

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Cetuximab, Panitumumab
• MAB that targets epidermal growth factor receptor (EGFR)
• Binding of cetuximab to EGFR inhibits tumor growth by variety of
mechanisms, including decrease in kinase activity, MMP activity, growth
factor production and increased apoptosis
• Approved for metastatic colorectal carcinoma whose tumor
overexpresses EGFR.
• Cetuximab 400 mg/kg loading followed by 200 mg/kg weekly IV alone or
in combination with irinotecan
Bevacizumab, Ranibizumab
• Humanized antibody against Vascular endothelial growth facto, thus
preventing angiogenesis
• Approved 5 mg/kg IV every 2 weekly ,For non–small-cell lung cancer.
• S/E – Since it is antiangiogenic, pt should be watched for hemorrhage, GI
perforations and wound healing problems.

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Alemtuzumab
• Humanised anti CD52 antiboby that binds to normal and malignant B
and T cells, NK cells
• Approved 30 mg IV once weekly for treatment of B-cell CLL failed to
respond to alkylating agents
• S/E – neutropenia, thrombocytopenia, pt should be closely monitored
for opportunistic infections
Some others-
• Gemtuzumab
• Rituximab
• Ofatumumab
• Trastuzumab
• Denileukin diftitox

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MABs conjugated with radioisotopes
• Provide targeted delivery of radionucleotides to tumor cells
• generally preferred because it is readily available, inexpensive and easily
conjugated
• Can be used for diagnosis as well as therapeutic purpose
Ibritumomab
• Anti CD20 murine antibody coupled with isotopic yttrium (90
Y)
• Approved for refractory B cell non hodgkins lymphoma, not even
responding to rituximab therapy
• S/E – common to both ibritumomab and tositumomab are severe
cytopenias
Tositumumab
• Used for refractory non hodgkins lymphoma notresponding to rituximab
and chemotherapy agents.

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Drugs in pipeline-
• Ibalizumab: an anti-CD4 monoclonal antibody for the treatment of HIV-1
infection
• HN125: A Novel Immunoadhesin Targeting MUC16 with Potential for
ovarian cancer Therapy.
• AMU32901 - Anti-phospholipid human monoclonal antibodies inhibit
CCR5-tropic HIV-1.
Possible side effects can include:-
• Vomiting
• Fever
• Diarrhea
• Chills
• Low blood pressure
• Weakness
• Rashes
• Headache
• Nausea

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Conclusion
 MAb are Antigen specific, can be produced against any type of antigen,
hence vast diagnostic applications
 Target specificity, a novel therapeutic approach particularly in cancer.
• 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.

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References
• Adams, G.P. And Weiner, L.M. (2005) Monoclonal Antibody Therapy of
Cancer, Nature Biotechnology , 23,1147-1157
• Douglas Lake, Immunopharmacology; Bertram G. Katzung, Basic and
clinical pharmacology:11:963-85;2009
• Bruce A. Chamber, Jeol Neal et.al, Targeted therapies, Tyrosine kinase
inhibitors, monoclonal antibodies; Goodman and Gilmans,
Pharmacological basis of therapeutics; 12;1731-50; 2010.
• Ronald V.M.,Immunomodulation; Harrisons Principle of internal
medicine; 17; 1600-20;2009.
• HL Sharma and KK Sharma, Immunomodulation and immunotherapy,
Principles of Pharmacology;2;871-91;2010
• Tripathi K.D.,Immunotherapy; Essentials to medical pharmacology;
6;710-18;2009.
• Raben D, Helfrich B,The effects of cetuximab alone and in combination
with radiation and/or chemotherapy in lung cancer, PubMed; 2010
• Uehara K, Ishiguro S, Bevacizumab for Locally Advanced Rectal Cancer,
Pub Med; 2009
• Bruno CJ, Jacobson JM, Ibalizumab: an anti-CD4 monoclonal antibody for
the treatment of HIV-1 infection; J Antimicrob Chemother;1839-41; 2010
• Kishimoto, T., Narazaki, M. & Tanaka, T. 2011. Anti-interleukin-6
receptor antibody, Tocilizumab, for the treatment of autoimmune
diseases. FEB letters, 585, p.3699-3709

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• Mehren, M.V., Adams, G.P., Weiner, L.M., (2003) Monoclonal Antibody
Therapy For Cancer, Annual Review of Medicine, 54, 343-369
• Burris III, H.A., Holden, S.N., Lewis-Phillips, G.D., Sliwkowski, M.X. &
Tibbitts, J. 2011. Trastuzumab Emtansine (T-DM1): A Novel Agent for
Targeting HER2+ Breast Cancer. Clinical Breast Cancer, 11(5), p.275-82
• Albanell, J., Baselga, J., Clinton, G.M., García-Conde, J., García-Conde, J.,
Keenan, E.J., Lluch, A., Molina, M.A., Ramsey, E.E., Rojo, F. & Sáez, R.
2006. p95HER-2 predicts worse outcome in patients with HER-2-positive
breast cancer. Clinical Cancer Research, 12(2), p.424-31.