MONOCLONAL ANTIBODIES

AND ANTIBODY ENGINEERING

PRESENTED BY :-ARUNDHATI MEHTAB.Sc. BIOTECH VI SEMESTER© Arundhati Mehta 2016

WHAT ARE ANTIBODIES ?An antibody is a protein ( Antigen binding ) used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. These are present on the B-cell membrane and secreted byplasma cells.

STRUCTURE OF ANTIBODY

2 Identical Light chains (~ 220 amino acid long) Variable domain : VL Constant domain: CL

2 Identical Heavy chains (~440 amino acid long) Variable domain : VH 3 Constant domain : CH1, CH2, CH3

Covalent Disulphde bonds between Cysteine residues.

Flexible “ Hinge region ”

BEGINNING OF MONOCLONAL ERA In 1890, Von Behring & Kitasato discovered antibodies. In1900, Ehrlich proposed the “ side-chain theory.” In 1955, Jerne postulated “Natural selection theory.” which F.M. Burnet expended. In the same same time (1955) , Porter isolated Fragment of antigen binding (Fab) & Fragment crystalline (Fc) frm rabbit y-globulin.

In 1975,Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells i.e., Hybridoma Technology.

In 1964, Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymidine (HAT) selection media.

In 1986-1990, the first monoclonal antibodies reached the market - Muromonab- CD3 ( produced by Milstein). In 1988, Greg Winter et al pioneered the techniques to humanise monoclonal antibodies. Paul Ehrlich at the beginning of the 20th century theorized that a cell under threat grew additional side-chains to bind the toxin, and that these additional side chains broke off to become the antibodies that are circulated through the body. It was these antibodies that Ehrlich first described as the "magic bullets" in search of toxins. In 2003, First Fully Human monoclonal antibody – Adalimumab

NOBEL PRIZE IN MEDICINE AND PHYSIOLOGY WAS AWARDED IN 1984

Prof. Niels K. Jerne Prof. Georges J.F. Köhler Prof. César Milstein

"for theories concerning the specificity in development and control of the immune system and the discovery of the principle for

production of monoclonal antibodies".

POLYCLONAL ANTIBODIES Polyclonal antibodies are a mixture of antibodies with different antigen binding sites that may bind to different epitopes or antigens of the immunizing agents with varying affinity.

It is produced by immunizing an animal with the appropriate antigen- wide array of B cells will be stimulated to produce anti- protein antibodies.

The serum obtained from immunized animal referred to as “Polyclonal Serum”

MONOCLONAL ANTIBODIES Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. These are a class of highly specific antibodies produced by the clones of a single hybrid cell.They all have identical antigen- binding sites.

Bind to the same epitope with same affinity. same antibody class ( isotope)

ANTIBODIESCHARACTERIS

TICS POLYCLONAL MONOCLONAL

PRODUCED BY :

Many B cell clones A single B cell clone

BIND TO : Multiple epitopes of all antigens used in the

immunization

A single epitope of a single antigen

ANTIBODY CLASS :

A mixture of different Ab classes (isotopes)

All of a single Ab class

ANTIGEN - BINDING SITES :

Different antigen-binding sites

All antibodies have the same antigen

binding sites

CHARACTERISTICS

POLYCLONAL MONOCLONAL

COST Less expensive More expensive

YIELD Limited supply Infinite supply

EASEEasily

Rapidly producedTime consuming

More technical skill

POTENTIAL FOR CROSS-

REACTIVITYHigh Low

HYBRIDOMA CREATES MONOCLONAL ANTIBODIES

Monoclonal antibodies (mAb) are directed against a specific epitope (antigen, antigenic determinant) . Typically made by fusing myeloma cells with the spleen B cells from a mouse that has been immunized with the desired antigen or a single Hybridoma cell line.

PRODUCTION OF MONOCLONAL ANTIBODIES

HYBRIDOMA TECHNOLOGY STEP 1 :- Immunization of Mice & Selection of Mouse

Donor for generation of Hybridoma Cells.

ANTIGEN (intact cell/ whole cell

membrane/microorganisms) +

ADJUVANT (emulsification)

Antibody titre reached in Serum

Spleen removed ( source of cells )

STEP 2 :- Screening of Mice for Antibody Production

After several weeks of immunization

Serum Antibody Titre Determined( Technique :- ELISA / Flow cytometry )

Titre too low

BOOST(Pure Antigen)

Titre high

BOOST(Pure Antigen)

STEP 3 :- Preparation of Myeloma Cells

8 - AzaguanineMyeloma cells

Immortal Tumour of Lymphocytes

Myeloma cellsHGPRT ¯

High Viability & Rapid Growth

STEP 4 :- Fusion of Myeloma cells with Immune Spleen Cells & Selection of Hybridoma

Cells

Spleen Cells Myeloma Cells

HYBRIDOMA CELLS

ELISA PLATEHAT Medium

Feeder cellsGrowth Medium

STEP 4 :- Cloning of Hybridoma Cell Lines by “Limiting Dilution” or Expansion

A.Clone each positive Culture .

B. Test each Supernatent for Antibodies

C. Expand positive clones

The “ HAT Trick ”HYBRIDOMA SELECTION

NOMENCLATURE OF MONOCLONAL ANTIBODY

TYPES OF MAB’S

MURINEThese are derived

from Mice. Patients treated

with murine mAbs develop Human

Anti-Mouse Antibody (HAMA)

response

Eg:90 Y-ibritumomab

CHIMERICThey combine the Antigen binding parts (variable

region) of mouse with effector parts (constant region)

of human.

Eg:Infliximab

HUMANISEDThese are human

antibody with complementary

determining region (CDR) or

hypervariable region from non human source

(rodent) grafted onto human

variable region.

Eg:Daclizumab

SPECIFITYThe product of a single

hybridoma reacts with the same epitope on antigen

IMMUNIZING ANTIGENNeed not to be pure or characterized and is

ultimately not needed to produce large quantities.

SELECTION It is possible to select for

specific epitope specificities and generate antibodies against a wider range of antigenic determinants.

ANTIBODY PRODUCTIONUnlimited quantities of a

single well-defined monospecific reagent.

ADVANTAGES OF MAB’S

DISADVANTAGES OF MAB’SAFFINITY

Average affinity of Mabs are generally lower than polyclonal

antibodies

EFFECTOR FUNCTIONSIt may not produce the

desired biological response

SPECIFICITYMonoclonals against

conformational epitopes on native proteins may lose reactivity wth antigens.

CROSS REACTIONSAntibodies sometimes

display unexpected crossreactions with unrelated antigens.

TIME & EFFORT COMMITMENT

Very large

APPLICATIONS OF MAB’STHERAPEUTIC USES- Immunosuppression, Malgnancies, Asthama, auto immune disease,Cancer, etc

RESEARCH TOOL – In western blot, ELISA, RIA, flow cytometry, Immunodot blot ,etc.

DIAGNOSTIC APPLICATONS – In HIV, hepatitis, influenza, herpes, pregnancy detection ec.

ADEPT- antibody directed enzyme prodrug therapy;

ADCC- antibody dependent cell-

mediated cytotoxicity;

CDC- complement dependent

cytotoxicity; MAb- monoclonal antibody;

ScFv- single-chain Fv fragment

RECOMBINANT MONOCLONAL ANTIBODIES

The growing knowledge of antibody gene structure and regulation has made possible what Cesar Milstein, one of the inventors of monoclonal antibody technology, has called “man-made antibodies.” It is now possible to design and construct genes that encode immunoglobulin molecules in which the variable regions come from one species and the constant regions come from another. New genes have been created that link nucleotide sequences coding nonantibody proteins with sequences that encode antibody variable regionsspecific for particular antigens. Finally, by replacement of the immunoglobulin loci of one species with that of another, animals of one species have been endowed with the capacity to respond to immunization by producing antibodies encoded by the donor’s genetically transplanted Ig genes.

Chimeric and Hybrid Monoclonal Antibodies for HUMANISED ANTIBODY production

Engineering an antibody to clone recombinant DNA containing the promoter, leader, and variable region sequences from a mouse antibody gene and the constant-region exons from a human antibody gene.

Production of chimeric mouse-human monoclonalantibodies. Chimeric mouse-human heavy- and light-chain expressionvectors are produced. These vectors are transfected into Abmyeloma cells. Culture in ampicillin medium selects for transfectedmyeloma cells that secrete the chimeric antibody.

Heteroconjugates, or Bispecific AntibodiesThese are hybrids of two different antibody molecules which can be constructed by chemically cross linking two different antibodies or by synthesizing them in hybridomas consisting of two different monoclonal-antibody-producing cell lines that have been fused.

A CHIMERIC IMMUNOTOXIN is chimeric monoclonal antibody in which the terminal Fc domain is replaced by toxin chains (white). A HETEROCONJUGATE in which onehalf of the mouse antibody molecule is specific for a tumor antigen and the other half is specific for the CD3/T-cell receptor complex.

Mab Construction from Ig - Gene LibraresGenerating monoclonal antibodies employing the polymerase chain reaction (PCR) to amplify the DNA that encodes antibody heavy-chain and light-chain Fab fragments from hybridoma cells or plasma cells.

Mab Construction by Grafting heavy- & light- chain miniloci into mice

The capacity of mice to rearrange Ig heavy- and lightchaingene segments was disabled by knocking out the C and C loci. The antibody-producing capacity of these mice was reconstituted by introducing long stretches of DNA incorporating a largepart of the human germ-line and heavy-chain loci (miniloci). Chimeric mice were then bred to establish a line of transgenic mice bearing both heavy- and light-chain human miniloci. Immunization of these mice results in the production of humanantibody specific for the target antigen.

SIDE - EFFECTS OF MONOCLONAL ANTIBODIES

Monoclonal antibodies are given intravenously (injected into a vein). These are often more like an allergic reaction & are more common while the drug is first being given. Possible side effects can include :

mAbs

Anaphylaxis

Malignancy

Fever & chills

Nausea &

vomiting

Low blood

pressure

Rashes & hypersensiti

vity

FDA APPROVED MONOCLONAL ANTIBODIES

The First approved mAbs was OKT-3 [1986] which is a murine IgGa2 protein to deplete T cells in patients with acute rejection of renal allotransplant. Until Feb 24, 2013, 312 mAbs were approved by FDA, which were applied in the treatment of organ transplant, Cancer, Asthma, Hematopoietic malignancies and psoriasis.