Antibodies often termed immunoglobulin. They are glycoprotein that bind antigens with high specificity and affinity

They are molecules originally identified in serum

Heavy chain

Light chain

220 a.a

~500 a.a

NH2

IMMUNOGLOBULIN

Antigen Binding Site

Variable

Regions

Constant Regions

Effector

Functions

Antibody engineering services , involves most forms of recombinant antibody gene manipulation. These include v-region discovery and sequencing ,isotype switching, discovery of enhanced binding variants through in vitro affinity maturation

Fully integrated work packages involving

HumanizationProduction of cell line development Production of the purified antibody

Antibody engineering has been an extremely intensive research

area for many years

Recent achievements in 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.

• 2. New structural network ,in particular using NMR

• 3. The cloning of essentially the complete set of human VH genes

• 4. The use of antibodies to catalyze complicated chemical

reactions

• 5. Novel antibody fusion proteins to potentiate immune therapy

• 6. New development is the replacement of antibodies with more stable protein scaffold for many future biotechnological

Applications

RECOMBINANT DNA TECHNOLOGY FOR ANTIBODY ENGINEERING

• The work of Cesar Milstein has paved the way to design and constructgenes that encode Ig molecule in which the variable region genes comefrom one species to and constant gene come from another

• By this technique molecular hybrids or chimers can be made to delivera powerful toxin to a particulate antigenic target such as tumour cell

• Transplanted Ig genes have endured receiver to respond to immunizationby 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

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)

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 lymphomaUnlike the major therapies used in treatment of lymphomas

• By this grafting entire B cell population including b lymphomas cellsWere cleared from the body in a way that other cell population are spared.

• 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

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

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

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

CHIMERIC HUMAN/MOUSE

FR1 FR2 FR3 FR4CDR1 CDR2 CDR3

CONSTANT

CONSTANT

CONSTANTFR1 FR2 FR3 FR4CDR1 CDR2 CDR3

Mouse

Human

Chimeric

Human Mouse

Chimeric

HUMANIZED ANTIBODIES

FR1 FR2 FR3 FR4

CDR1 CDR2 CDR3

CONSTANT

CONSTANT

CONSTANTFR1 FR2 FR3 FR4CDR1 CDR2 CDR3

Mouse

Human

Humanized

Human Mouse

Humanized

CHIMERIC/HUMANIZED

.Reduce the HAMA response.

.Maintain effector functions.

.Increase the half life of the antibody.

.Maintain binding affinity.

.Easy to construct.

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

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 cells3. Monoclonal antibodies can generate an anti mouse antibody response in patients4. It rapidly clearing the injected antibodies from the blood and Rendering it useless5. Then depending upon the manner in which it will be used6. Therapeutic antibody may need a stronger or weaker effector Function, increased or decreased antibody affinity ,and / or reduction in size , for maximum efficacy

I. IMMUNOGENICITYII. EFFECTOR FUNCTIONIII. ANTIBODY SIZE & STRUCTUREIV. AFFINITY

IMMUNOGENICITY• Most mAbs are originally produced in a mouse cell system, because it 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

EFFECTOR FUNCTIONGenetic 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

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

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

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

ANTIBODY V-REGION SEQUENCING

Production of antibodies by genetic engineering

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

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