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Biotechnology

Monoclonal antibodies

Dr. sarah I bukhariAssistant Professor of pharmaceutical

microbiology Microbiology

Microbiology section

THE IMMUNE SYSTEM

DEFINITION: - The integrated body system of organs, tissues, cells & cell products that differentiates self from non – self & neutralizes potentially pathogenic organisms.(The American Heritage Stedman's Medical Dictionary)

The Latin term “IMMUNIS” means EXEMPT, referring to protection against foreign agents.

The Immune System consists of

1. Innate Immunity Primary Response

2. Acquired Immunity Secondary Response

                                                            

ANATOMY OF THE IMMUNE SYSTEM

CELLS OF THE IMMUNE SYSTEM

BECOMES

FUNCTIONING OF THE IMMUNE SYSTEMHUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE CELL MEDIATED IMMUNE RESPONSE

ANTIGEN (1ST EXPOSURE)

ANTIGENS DISPLAYED BY INFECTED CELLS ACTIVATE

CYTOTOXIC T CELL

GIVES RISE TO

ACTIVE CYTOTOXIC T CELL

ENGULFED BY

STIMULATES

MACROPHAGE

APC

HELPER T CELLS STIMULATES

MEMORY HELPER T CELLS

MEMORY T CELLS

MEMORY B CELLS

PLASMA CELLS

STIMULATES STIMULATES

B CELLS

FREE ANTIGENS DIRECTLY ACTIVATE

STIMULATES

GIVES RISE TO

SECRETE ANTIBODIES

STIMULATES

ANTIGEN (2nd EXPOSURE)

STIMULATES

Defend against extracellular pathogens by binding to antigens and making them easier targets for phagocytes and complement

Defend against intracellular pathogens and cancer by binding and lysing the infected cells or cancer cells

IMMUNOTHERAPY

Treatment of the disease by Inducing, Enhancing or Suppressing the Immune System.

Active Immunotherapy: -

It stimulates the body’s own immune system to fight the disease.

Passive Immunotherapy: -

It does not rely on the body to attack the disease, instead they use the immune system components ( such as antibodies) created outside the body.

Enhancing the immune system

by

HISTORY OF IMMUNOLOGY

• 1862 Ernst Haeckel, Recognition of phagocytosis

• 1877 Paul Erlich, recognition of mast cells

• 1879 Louis Pasteur, Attenuated chicken cholera vaccine development

• 1883 Elie Metchnikoff Cellular theory of vaccination

• 1885 Louis Pasteur, Rabies vaccination development

• 1888 Pierre Roux & Alexandre Yersin, Bacterial toxins

• 1888 George Nuttall, Bactericidal action of blood

• 1891 Robert Koch, Delayed type hypersensitivity

• 1894 Richard Pfeiffer, Bacteriolysis

• 1895 Jules Bordet, Complement and antibody activity in bacteriolysis

• 1900 Paul Erlich, Antibody formation theory

• 1901 Karl Landsteiner, A, B and O blood groupings

• 1901-8 Carl Jensen & Leo Loeb, Transplantable tumors

• 1902 Paul Portier & Charles Richet, Anaphylaxis

• 1903 Almroth Wright & Stewart Douglas, Opsonization reactions

• 1906 Clemens von Pirquet, coined the word allergy

• 1907 Svante Arrhenius, coined the term immunochemistry

• 1910 Emil von Dungern, & Ludwik Hirszfeld, Inheritance of ABO blood groups

• 1910 Peyton Rous, Viral immunology theory

• 1914 Clarence Little, Genetics theory of tumor transplantation

• 1915-20 Leonell Strong & Clarence Little, Inbred mouse strains

• 1917 Karl Landsteiner, Haptens

• 1921 Carl Prausnitz & Heinz Kustner, Cutaneous reactions

• 1924 L Aschoff, Reticuloendothelial system

• 1926 Lloyd Felton & GH Bailey, Isolation of pure antibody preparation

• 1934-8 John Marrack, Antigen-antibody binding hypothesis

• 1936 Peter Gorer, Identification of the H-2 antigen in mice

• 1940 Karl Lansteiner & Alexander Weiner, Identification of the Rh antigens

• 1941 Albert Coons, Immunofluorescence technique

• 1942 Jules Freund & Katherine McDermott, Adjuvants

• 1942 Karl Landsteiner & Merill Chase, Cellular transfer of sensitivity in guinea pigs (anaphylaxis)

• 1944 Peter Medwar, Immunological hypothesis of allograft rejection

• 1948 Astrid Fagraeus, Demonstration of antibody production in plasma B cells

• 1948 George Snell, Congenic mouse lines

• 1949 Macfarlane Burnet & Frank Fenner, Immunological tolerance hypothesis

• 1950 Richard Gershon and K Kondo, Discovery of suppressor T cells

• 1952 Ogden and Bruton, discovery of agammagobulinemia (antibody immunodeficiency)

• 1953 Morton Simonsen and WJ Dempster, Graft-versus-host reaction

• 1953 James Riley & Geoffrey West, Discovery of histamine in mast cells

• 1953 Rupert Billingham, Leslie Brent, Peter Medwar, & Milan Hasek, Immunological tolerance hypothesis

• 1955-1959 Niels Jerne, David Talmage, Macfarlane Burnet, Clonal selection theory

• 1957 Ernest Witebsky et al., Induction of autoimmunity in animals

• 1957 Alick Isaacs & Jean Lindemann, Discovery of interferon (cytokine)

• 1958-62 Jean Dausset et al., Human leukocyte antigens

• 1959-62 Rodney Porter et al., Discovery of antibody structure

• 1959 James Gowans, Lymphocyte circulation

• 1961-62 Jaques Miller et al., Discovery of thymus involvement in cellular immunity

• 1961-62 Noel Warner et al., Distinction of cellular and humoral immune responses

• 1963 Jaques Oudin et al., antibody idiotypes

• 1964-8 Anthony Davis et al., T and B cell cooperation in immune response

• 1965 Thomas Tomasi et al., Secretory immunoglobulin antibodies

• 1967 Kimishige Ishizaka et al., Identification of IgE as the reaginic antibody

• 1971 Donald Bailey, Recombinent inbred mouse strains

• 1974 Rolf Zinkernagel & Peter Doherty, MHC restriction

• 1975 Kohler and Milstein, Monoclonal antibodies used in genetic analysis

• 1984 Robert Good, Failed treatment of severe combined immunodeficiency (SCID, David the bubble boy) by bone marrow grafting. 1985 Tonegawa, Hood et al., Identification of immunoglobulin genes

• 1985-7 Leroy Hood et al., Identification of genes for the T cell receptor

• 1990 Yamamoto et al., Molecular differences between the genes for blood groups O and A and between those for A and B

• 1990 NIH team, Gene therapy for SCID using cultured T cells.

• 1993 NIH team, Treatment of SCID using genetically altered umbilical cord cells.

• 1985-onwards Rapid identification of genes for immune cells, antibodies, cytokines and other immunological structures.

• 1798 Edward Jenner, Smallpox vaccination

• 1975 Kohler and Milstein, Monoclonal antibodies used in genetic analysis

• 1798 Edward Jennr, Smallpox vaccination

Immunoglobulins

GlycoproteinsBind specifically to antigens that induced

their formationPresent in gamma globulin fraction of

plasma proteinsPresent in extravascular compartmentFive classes: IgG IgA IgM IgE IgDSubclasses also present: IgG1, IgG2, IgG3,

IgG4

Antibody Structure

Two identical light chains

Two identical heavy chains

Linked by S-S bonds

Light Chains

200 amino acids each

MW: 25kDaMay be kappa (κ)

or lambda (λ)Both types found

among all isotypes One type only per

antibody

Heavy chains

400 amino acidsMW: 50 – 75 kDaDetermine isotype of

antibody:◦Gamma (γ) → IgG◦Alpha (α) → IgA◦Mu (μ) → IgM◦Delta (δ) → IgD◦Epsilon (ε) → IgE

2 heavy chains joined by

S-S bonds in hinge region

Domains

Variable domains: widely variable

amino acid sequence

Constant domains: uniform amino acid sequence

Light chain: one VL + one CLHeavy chain: one VH + 3-4 CH

The structure of antibodies

http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html

Hypervariable Regions

Variability of amino acid sequence in VH and VL domains is restricted to short segments

Hypervariable regions of H and L chains are folded together single → hypervariable surface: PARATOPE

Paratope = antigen-

binding site

Paratope complementary to epitope

Proteolytic cleavage of antibody gives:

I. Two identical Fab

fragments: (fragment antigen binding)

II. One Fc fragment: (fragment crystallizable)

Biological activities:• Complement fixation• Attachment to various

cells• Placental transfer

NOTE:Different isotypes →different Fc fragments→ different functions

Structure of Antibody

EPITOPE = ANTIGEN-BINDING

SITE

POLYCLONAL vsMONOCLONAL ANTIBODIES

• Protein Antigens have many epitopes

• B cells make Ab to a single epitope

• Different clones of B cells make Ab to different epitopes

Polyclonal and MonoclonalAntibodies

Pure single Ab

After immunization, the mouse spleen contains B cells producing specific antibodies.Each B cell produces only one kind of antibody, which binds to its specific antigen. Conventional antiserum is the mixture of all antibodies produced by B cells from spleen.If a single B cell was picked up and cultured, then it will produce only one kind of antibody. But B cell can not survive well in the culture.Myeloma cell can be cultured in the test tube, but can not produce useful antibody.Each hybridoma line can produce pure single antibody, called monoclonal antibody.If B cell is fused with myeloma, the fused cell might be cultured and produce antibody.

Adapted from Milstein (1980) Scientific American, Oct. p.58

1

23

4

mm

mm

1

2 34

1 2 3 4

Monoclonalantibodies

Cell fusion

Spleen cells

+Myeloma

x

Antiseum

Antigen

Immunization

A mixture of all Ab

1 23 4

BALB/c

1 2 3 4

1 23 4

B cell

ANTIBODIES

Derived from different B Lymphocytes cell lines

POLYCLONAL. MONOCLONAL.

Derived from a single B cell clone

Batch to Batch variation affecting Ab reactivity &

titre

mAb offer Reproducible, Predictable & Potentially

inexhaustible supply of Ab with exquisite specificity

Enable the development of secure immunoassay

systems.

NOT Powerful tools for clinical diagnostic tests

1. Antibodies neutralize bacterial toxins and complete virus particles and bacterial cells.

2. Antibodies coating an antigen render it recognizable as foreign by phagocytes (macrophages and neutrophils), which then ingest and destroy it; this is called opsonization.

3. Antibodies activate of the complement system by coating a bacterial cell.

4. Antibody-dependent cellular cytotoxicity (ADCC)

Fig. 1.24 Antibodies can participate in host defense in three main ways.

Functions of antibodies

1- Neutralization

Prevents attachment to cell receptors

→ prevents infectivity or toxicity

2- Opsonisation

Binding of antibody-coated pathogen to Fc receptor on phagocyte

→ better phagocytosis and killing

3- Complement Activation

This may lead to:

Opsonization

Lysis of bacterial cell

Complement activation followed by opsonization

Complement activation leading to lysis

4- Antibody-dependent cellular cytotoxicity (ADCC)

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