#humanasation antibody

1. HUMANISATION ANTIBODY
THERAPY
BY: CHAUDHARI KRINA

2. HUMANISATION antibody:
Definition ◦ Humanized antibodies are antibodies made from non-
human species whose protein sequences have been modified to
increase their similarity to antibody variants produced naturally in
humans.
It can be necessary when the
process of developing a
specific antibody
involves generation in a non-human immune system

3. Structure of antibodies :
Fab and Fc are antigen binding and effector portions
respectively.
The constant regions are given in brown while variable regions
are in light green.
The constant regions of the heavy chains are subdivided into
CH1, CH2, and CH3 (and CH4 in case of IgM and IgE).
Each light chain has V, J and C segments.

6. Era of humanized antibody:
1. MURIN
2. CHIMERIC
3. HUMANIZED
4. HUMAN OR FULLY HUMAN

10. HUMAN OR FULLY HUMAN
•A human antibody is one of which both chain type,
and the J chain in the case of polymeric antibodies,are
of human origin.

11. PRODUCE:
• Using recombinant DNA technology to create
constructs capable of expression in mammalian
cell culture.

12. PRODUCTION :
• Synthesized by crafting murine CDRs to a human antibody.
• Using rDNA human immunoglobulin light & heavy chain genes can
be amplified by PCR.

13. Technologies
used
Phage display
Primarily
human system
Transgenic
mice

14. Transgenic mice:

15. Phage display:
• The genes for the V regions of antibody heavy and light
chains can be inserted separately into these phages, and
they display the proteins on the surface.
• Gene isolated from;
• (a) Naïve B-cells
• (b) Immunized, antigen-specific memory B- cells.

17. selection:
The proteins may be selected by screening against the antigen
of choice, and the desirable proteins may be produced and
secreted by bacteria infected by the specific phage.

19. Primarily human system:
The best approach so far for producing antibody .
Strategies used ;
Mouse myeloma cell line transfected with human
immunoglobulin genes Fusion of human B cells with a
mouse-human hybrid 'heteromyeloma'

20. MAJOR APPLICATIONS:
(1) DiagnosticApplications
• Biochemical analysis
• Diagnostic Imaging
(2) TherapeuticApplications
• Direct use of MAbs as therapeutic agents
• MAbs as targeting agents
(3) Protein Purification

21. Applications in therapy and Diagnosis Monoclonal antibodies are widely
used as diagnostic and research reagents as well as in human therapy.
In some in vivo applications, the antibody itself is sufficient. Once bound to
its target, it triggers the normal effector mechanisms of the body
In other cases, the monoclonal antibody is coupled to another molecule, for
example a fluorescent molecule to aid in imaging the target a strongly-
radioactive atom, such as Iodine-131 to aid in killing the target Diagnostic
application The diagnostic applications of monoclonal antibodies are by far
the most advanced, especially for tests that are performed on body fluids
such as blood and urine sample
MAb is used to detect pregnancy as early as a week or two after conception
by reacting with human chorionic gonadotrophin, a hormone secreted by
the placenta and found in the urine of pregnant women.
To detect the presence of the substance/antigen, MAbs can be used.

22. Methods of humanisation of antibody
therapy :
Chimeric antibodies are produced by
i) grafting complementarity determining regions (CDRs) and
ii) producing chimeric antibodies by splicing the mouse variable
regions onto human constant regions.
Fully human antibodies can also be generated by
i) the selection of human antibody fragments from phage
libraries,
ii) transgenic mice and
iii) through selection from human hybridomas

23. CDR grafting (antibody reshaping)
CDR grafting is the most advanced approach in
which the CDR residues from the variable region
of a mouse mAb are transferred to human
constant and variable domain frameworks that
have high sequence homology with the mouse
counterparts.

24. Memory B cell immortalisation
This technique involves isolation of human memory B
cells from peripheral blood mononuclear cells (PBMCs)
of infected patients.
These transformed cells are capable of producing a
human monoclonal antibody with desired antigen
specificity

25. Hybridoma technology using transgenic
mouse :
This technology is a slightly modified version of
traditional hybridoma technology.
Here, the hybridoma is produced from the spleen cells
of transgenic mice in which the immunoglobulin genes
are knockedout and replaced with human counterparts.
This is followed by the antigen immunisation.

26. Human Antibody e segments responsible for
Fv/Fab are amplified by antibody library :
Cloned for display on the surface of the phage
Fusion product will be incorporated into mature phage coat
Connection between ligand genotype and phenotype established
Antibody library construction and display onto the phage surface
Biopanning which involves severalwashes
Non specific antibodies washed away
Phage with specific antibody is eluted followed by DNA amplification
Selecting library against specific antigen targets(Panning)

27. Recombinant antibodies by cloning v-region
genes:
The functional structure of the antigen-binding site is
determined by genes of both heavy (H) and light (L) variable
(V) domains.
Cloning of mouse variable genes into human constant-region
genes generates chimeric as well as humanised antibodies
depending on the size of the clone

28. Advantage of humanised
Antibodies :
It includes:
- Reduction in the immunogenicity of an
antibody
- Therapeutic value of an antibody
increases after Humanization.
-Improved pharmacokinetics due to
prolonged plasma half life.

29. DRAWBACKS:
• Ideal CDR to be slightly altered in its three – dimensional
conformation.
• Humanized antibodies are less likely to an immune response than
murine & chimeric monoclonal antibodies.