Antibodies can be engineered using genetic techniques to alter their properties for therapeutic applications. Key characteristics that can be modified include immunogenicity, effector function, size and affinity. Techniques like generating chimeric or humanized antibodies can reduce immunogenicity. The effector function can be enhanced or reduced depending on the intended use of the antibody. Size can be decreased by removing nonessential components. Affinity can be increased by altering amino acids in the binding site. Antibody engineering also includes techniques like phage display which can be used to isolate antibodies with desired properties.

2. Antibodies often termed immunoglobulin. They
are glycoprotein that bind antigens with high
specificity and affinity
They are molecules originally identified in
serum

3. Heavy chain
Light chain
220 a.a
~500 a.a
NH2
IMMUNOGLOBULIN

4. Antigen
Binding Site
Variable
Regions
Constant
Regions
Effector
Functions

5. Antibodyengineeringservices, involvesmost formsof recombinant antibodygene
manipulation. These include v-region discovery and sequencing,isotype switching, discovery
of enhancedbindingvariantsthroughin vitroaffinitymaturation
Fullyintegrated workpackages involving
Humanization
Productionof cell line development
Productionof the purified antibody
Antibodyengineeringhas been an extremelyintensive research
areafor manyyears

6. Recent achievementsin this field includes
• 1 . significant improvement in the field of selection of antigen-specific
Antibody fragment on bacteriophages
Production of
antibody
fragments.
Reduce the
size of
Antibody.
Simplifying the
expression/production
of antibodies.

7. •2. New structural network ,in particularusingNMR
• 3. The cloningof essentiallythe completeset of human VHgenes
• 4. The use of antibodiesto catalyze complicatedchemical
reactions
• 5. Novelantibodyfusion proteins to potentiateimmunetherapy
• 6. Newdevelopment is the replacement of antibodies withmore stable protein scaffold
for manyfuture biotechnological
Applications

8. RECOMBINANT DNA TECHNOLOGY FOR ANTIBODY
ENGINEERING
•The work of Cesar Milstein has paved the way to design and construct
genes that encode Ig molecule in which the variable region genes come
from one species to and constant gene come from another
•By this technique molecular hybrids or chimers can be made to deliver
a powerful toxin to a particulate antigenic target such as tumour cell
•Transplanted Ig genes have endured receiver to respond to immunization
by producing antibodies encoded that of the donor
•The entire antibodies repertoire of individuals may be reconstructed
by the selected L & M chain variable region genes

9. ADDITIONAL APPRORACHES TO r -DNA TECHNOLOGY
1. HUMANIZED CHIMERIC ANTIBODIES
2. BISPECIFIC ANTIBODIES ( HETEROCONJUGATES)
3. DERIVATION AND USE OF IG –GENE COMBINATORIAL
LIBRARIES ( H & L LIBRARIES)

10. HUMANIZED CHIMERIC ANTIBODIES
• Cloning the recombinant DNA from mouse containing the promoter
leader and variable region sequences, from a human mouse antibody
gene and the constant region exons from a human antibody gene is
called humanized chimeric antibodies
• The humanized chimeric antibodies are potential to trigger human
competent activation or Fc receptor binding
•This technique has been used to treat patients of Hodgkin's lymphoma
Unlike the major therapies used in treatment of lymphomas
•By this grafting entire B cell population including b lymphomas cells
Were cleared from the body in a way that other cell population are
spared.

11. •The therapeutic agent used in the
humanized chimeric antibodies is
known as CD20 .
•A membrane bound phospho
protein
•The important issues with such a
treatment efficacy ,safety, and
immunogenicity are under
vigorous and intensive studies is
illustrative of application of CD20

12. BISPECIFIC ANTIBODIES ( HETEROCONJUGATES)
• Bispecific antibodies are
hybrids of two different
molecules
• Constructed by chemically
cross linking of two different
antibodies or synthesised
monoclonal antibody cells
• The destruction of tumour cells
begin when the effector cell is
cross linked to the tumour cells

13. DERIVATION AND USE OF Ig GENE
COMBINATORIAL LIBRARIES
• Producing gene libraries encoding Fab fragments is another approach
for generating monoclonal antibodies
• PCR is employed to amplify the DNA that encodes antibody H & L
chain Fab from hybridoma cells or plasma cells
• Amplified sequences are endowed with a promoter region and ECoR1
restriction site and resultant construct are then inserted in to
bacteriophage yielding separate H & L libraries
• It have specificities against a wide varieties of antigen can be achieved
from these gene libraries
• The immune response is important and highly potential ,in comparison
to gene libraries and they have different effective functions so they
high versatility and higher affinity

14. Antibody engineering is useful in large scale production
Of antibodies ,in production of active antibody fragments, bifunctional,
Single domain and catalytic antibodies ,and has lead to the production
of novel expression systems useful in may other areas
it allows production of new antibody conjugates
E.G : antibody –toxin or antibody –enzyme linked proteins
Engineered antibodies have many potential application
E.G : imaging, therapy and biosensors
mouse antibodies can be humanized ,resulting in chimeric or reshaped
antibodies ,engineered antibody molecules ,and their fragments, are
being increasingly exploited as scientific and clinical tools
one factor that can be limit the applicability of this technology is the
ability to express large amount of active protein

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

16. Human Mouse
Chimeric

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

18. Human Mouse
Humanized

19. CHIMERIC/HUMANIZED
.Reduce the HAMA
response.
.Maintain effector
functions.
.Increase the half
life of the antibody.
.Maintain binding
affinity.
.Easy to construct.

20. Antibodies are other proteins have been engineered using a
variety of immunogenicity evaluation strategies and some have
advanced in to clinical development
The characteristics of an antibody that may prevent its use , or
reduce its efficacy as an immunotherapy, can be altered through
genetic engineering
Thus the immunogenicity of an antibody can be reduced or
eliminated and the effector function ,size, structure, and affinity of
the antibody can be altered

21. Four key characteristics of relating to the efficacy
of therapeutic antibody
1. The antibodies that are used in research and as cancer therapies
are monoclonal antibodies.
2. Which are identical antibodies derived originally from mouse cells
3. Monoclonal antibodies can generate an anti mouse antibody
response in patients
4. It rapidly clearing the injected antibodies from the blood and
Rendering it useless
5. Then depending upon the manner in which it will be used
6. Therapeutic antibody may need a stronger or weaker effector
Function, increased or decreased antibody affinity ,and / or
reduction in size , for maximum efficacy

23. IMMUNOGENICITY
• Most mAbs are originally produced in a mouse cell system, because i
produces large amount of antibodies
• The human immune system may recognize the mAbs as foreign
even though mouse and human mAbs are structurally identical
• A mAb be found to be immunogenic during clinical characterisation
• Genetic engineering techniques substitutes mouse gene sequences
With the corresponding human gene sequence, by altering the
Protein sequence of immunogenic mouse derive mAbs
• the risk that the patients own immune system will reject the therapy
can be reduced or eliminated .Alternatively scientists may generate
fully human antibody that targets the same antigen as the mouse
mAbs

24. EFFECTOR FUNCTION
Genetic engineering methods to enhance or reduce the effector
function of an antibody in order to suit the strategy for its
clinical application
A strong effector function is required for a naked antibody therapy,
in which the antibody is designed to be cytotoxic
The antibody can be engineered to enhance the effector function so
that the cytotoxicity is via complement dependent cytotoxicity ,
which is a part of humoral response, or via immune response

25. ANTIBODY SIZE AND STRUCTURE
The size of an antibody has direct impact on its ability to penetrate
the tumour and other tissues, as well as pharmacokinetics
Scientist reduce the size of antibody by removing non essential
components through genetic engineering ,to increase its tumour
penetration and improve its pharmacokinetics
Therapies that utilize the delivery of a toxin or radioisotope have
comprised of an antibody with a conjugated toxin or radio isotope
Scientist now fuse the toxin and radioisotope directly to the antibody
structure ,minimizing the potential for loss of the toxin from the
antibody and thus maximizing the delivery of antibody –toxin
conjugate to the tumour

26. AFFINITY
The affinity of an antibody for its
antigen can also be increased or
decreased through genetic engineering
,by altering the amino acids within the
antibody’s binding site

27. V –region discovery is a critical step in the commencement
of an antibody engineering project
Capture of the correct v- region sequences from a starting
hybridoma can lead to a number of project outcomes depending
On the study objectives
These may include the development of a lead therapeutic
candidate with enhanced properties such as
Better potency
Productivity
Reduced antigenicity

28. 1. Short primers to consensus sequences in heavy and light chain
variable regions of immunoglobulin genes are used to generate
a library of heavy and light chain v-region DNA’S by PCR. with
spleen DNA as the starting material .
2. These heavy and light chain v region genes are cloned randomly
into a filamentous phage such that each phage expresses one
heavy chain and one light chain v region as a surface fusion
protein with antibody like properties

29. 3. The resulting phage display library is multiplied in bacteria, and
the phage are then bound to a surface coated with antigen. The
bounded phages are washed away , the bound phage are recovered
,multiplied in bacteria, and again bound to antigen
4. After a few cycles, only specific high affinity antigen binding
phage are left .these can be used like antibody molecules or their
v genes can be recovered and engineered into antibody genes to
produce genetically engineered antibody molecule
5. this technology may replace the hybridoma technology for
producing mAbs and has advantage that humans can be use as
the source of DNA