Antibody engineering by R.S.Priyengha

BY-Priyengha.R.S
M.Sc.,Microbiology,
15KUSM6014
RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE
DEPT OF MICROBIOLOGY
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Introduction
Antibodies
Structure and its function
Methods of Antibody Engineering
Hybridoma Method
Chimeric Method
Humanised Method
Applications
Conclusion

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 In recent years, antibodies have become
increasingly accepted as therapeutics for human
diseases, particularly for cancer, viral infection
and autoimmune disorders.
 Monoclonal antibodies (Mabs) have been used as
diagnostic and analytical reagents since
hybridoma technology was invented in 1975.
 “man-made antibodies.” was named by Cesar
Milstein, who was one of the inventors of
monoclonal antibody technology.
 Until the late 1980’s, antibody technology relied
primarily on animal immunization and the
expression of engineered antibodies.

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Antibodies Are Made Up Of:
 2 Light Chains (identical) ~25 KDa
 2 Heavy Chains (identical) ~50 KDa
 Each Light Chain Bound To Heavy Chain By Disulfide (H-L)
 Heavy Chain Bound to Heavy Chain (H-H)
 First 100 a/a Of Amino Terminal Vary of Both H and L Chain Are
Variable
 Referred To As VL , VH, CH And CL
 CDR (Complementarity Determining Regions) Are What Bind Ag
 Remaining Regions Are Very Similar Within Same Class

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 Repeating Domains of ~110 a/a
 Intrachain disulfide bonds within each domain
 Heavy chains
 1 VH and either 3 or 4 CH (CH1, CH2, CH3, CH4)
 Light chains
 1 VL and 1 CL
 Hinge Region
 Rich in proline residues (flexible)
 Hinge found in IgG, IgA and IgD
 Proline residues are target for proteolytic digestion (papain and pepsin)
 Rich in cysteine residues (disulfide bonds)
 IgM and IgE lack hinge region
 They instead have extra CH4 Domain

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 Digestion With Papain Yields
 3 Fragments
 2 identical Fab and 1 Fc
 Fab- Fragment That is Antigen Binding
 Fc - Found To Crystallize In Cold Storage
 Pepsin Digestion
 F(ab`)2
 No Fc Recovery, Digested Entirely
 Mercaptoethanol Reduction (Eliminates Disulfide Bonds) And
Alkylation Showed

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 Hybridoma development
 Novel mAb development technology
 Purification
 Antibody heterogeneity
 Recombinant
 Chimeric antibodies
 Human antibodies

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 Several monoclonal antibody technologies had been
developed recently, such as phage display, single B cell
culture,single cell amplification from various B cell
populations and single plasma cell interrogation
technologies.
 Different from traditional hybridoma technology, the
newer technologies use molecular biology techniques to
amplify the heavy and light chains of the antibody
genes by PCR and produce in either bacterial or
mammalian systems with recombinant technology.
 One of the advantages of the new technologies is
applicable to multiple animals, such as rabbit, llama,
chicken and other common experimental animals

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 The production of recombinant monoclonal
antibodies involves repertoire cloning or phage
display/yeast display technologies. Recombinant
antibody engineering involves antibody production
by the use of viruses or yeast, rather than mice.
 These techniques rely on rapid cloning of
immunoglobulin gene segments to create libraries
of antibodies with slightly different amino
acid sequences from which antibodies with desired
specificities can be selected

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 Production of chimeric mouse-human monoclonal antibodies.
 Chimeric mouse-human heavy- and light-chain expression vectors are
produced.These vectors are transfected into Ab myeloma cells.
 Culture in ampicillin medium selects for transfected myeloma cells that
secrete the chimeric antibody.
 While structurally similar, differences between mouse and human
antibodies were sufficient to invoke an immune response
when Murine monoclonal antibodies were injected into humans,
resulting in their rapid removal from the blood, as well as systemic
inflammatory effects and the production of Human Anti-Mouse
Antibodies (HAMA).
 Recombinant DNA has been explored since the late 1980s to increase
residence times. In one approach, mouse DNA encoding the binding
portion of a monoclonal antibody was merged with human antibody-
producing DNA in living cells. The expression of this "chimeric" or
"humanised" DNA through cell culture yielded part-mouse, part-human
antibodies.

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 Ever since the discovery that monoclonal
antibodies could be generated, scientists have
targeted the creation of "fully" human products to
reduce the side effects of humanised or chimeric
antibodies. Two successful approaches have been
identified: transgenic mice and phage display.
 As of November 2016, 13/19 cells "fully" human
monoclonal antibody therapeutics on the market
were derived from transgenic mice technology.
 These antibodies have been approved to
treat cancer, cardiovascular disease, inflammatory
diseases, macular degeneration, transplant
rejection, multiple sclerosis and viral infection.

HUMANIZED ANTIBODIES
FR1 FR2 FR3 FR4
CDR1 CDR2 CDR3
CONSTANT
CONSTANT
CONSTANT
FR1 FR2 FR3 FR4
CDR1 CDR2 CDR3
Mouse
Human
Humanized
16/22RAMAIAH COLLEGE OF ARTS,SCIENCE AND COMMERCE

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 Reduce the HAMA
response.
 Maintain effector
functions.
 Increase the half life
of the antibody.
Maintain
binding affinity.
Easy to
construct.

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 Immunology – Kuby 5th Edition.
 Roitt’s Essential immunology by lvan M. Roitt, Peter J.
Delves, 10th edition.
 Essentials of Medical Pharmacology by KD Tripathi
 Antibody Engineering Textbook by Carl
A.K.Borrebaeck.
 Antibody Engineering Article By – Hyo Jeong Hong and
Sun Taek Kim in Biotechnology and Bioprocess
Engineering 2002,7:150- 154
 AntibodyEngineering for the development of
Therapeutics Antibody –Molecules and cells @ KSMCB
2005

By
Priyengha.R.S
II M.Sc., MB.,III sem
15KUSM6014

Antibody engineering by R.S.Priyengha

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