Monoclonal Antibodies

Dr. Aws AlshamsanDepartment of Pharmaceutics

Office: AA87Tel: 4677363

aalshamsan@ksu.edu.sa

Objectives of this lecture

By the end of this lecture you will be able to:1. Define terms such as monoclonal, polyclonal,

isotype, idiotype, allotype, CDR, and hybridoma

2. Compare monoclonal-antibody production methods

3. Identify different mAb types4. List some applications of mAb in medicine

Antibody Response

Antibody StructureAntibody Structure

Complementarity Determining Regions

Antigen Antibody Interaction

Antigen Antibody Interaction

Ab

Isot

ypes

Ab Fragments

Ab Fragments

Ab

type

s di

ffere

nces

Polyclonal v.s. Monoclonal

Affinity and Avidity

Affinity: the strength of binding between a single binding site and a single ligand.

Avidity: the strength of binding between a molecule and a complex ligand, e.g. if there are multiple binding sites then the avidity may be increased by increasing the number of binding sites or by increasing the affinity of those binding sites.

Affinity and Avidity, continued

IgM is produced early in an immune response when the affinity for antigen often is low; as an immune response continues, antibody affinity is improved, this is combined by “class switching” to the use of smaller molecules (IgG, IgE and IgA). The increased affinity compensates for the decrease in number of binding sites in maintaining the overall avidity for antigen.

Polyclonal Response

• Polyclonal antibody– Antigens possess multiple epitopes– Serum antibodies are heterogeneous,

• To increase immune protection in vivo• To reduces the efficacy of antiserum for various in vitro uses

– To response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen

– To have clear advantages for the organism in vivo

• Monoclonal antibody– Derived from a single clone, specific for a single epitope– For most research, diagnostic, and therapeutic purposes

mAb Types

mAb nomenclature

Source stem

Suffix

o mAb

xi mAb

zu mAb

u mAb

Hybridoma Technology

1975, by Georges Köhler and Cesar Milstein- Be awarded a Nobel Prize in1984

(1) Immunisation of a mouse(2) Isolation of B cells from the spleen(3) Cultivation of myeloma cells

(4) Fusion of myeloma and B cells (using PEG)(5) Separation of cell lines

(6) Screening of suitable cell lines

(7) in vitro (a) or in vivo (b) multiplication(8) Harvesting

Selected by using HAT medium (Hypoxanthine-Aminopterin-Thymidine)

• Myeloma cells are unable to grow• B cells are able to survive, but can not live for

extended periods

HAT Selection

Two different pathways to synthesis nucleotide in mammalian cells

(Folic acid analog)

Phage Display - Introduction

• The display of functional foreign peptides or small proteins on the surface of bacteriophage particles.

• An important tool in protein engineering

• A powerful way to screen and select for peptides on the basis of binding or molecular recognition

Phage Display Principle

• More efficient than hybridoma system. • Cheaper to produce recombinant antibodies using

bacteria, rather than mammalian cell line. • Easier to maintain and grow bacterial cultures for

recombinant antibody production. • Bypass immunization in antibody selection. • Bypass the use of animal cells for production of antibodies. • Producing the combinatorial library (ideally with 108 to 109

members) of functional antibodies to generate a larger repertoire of antibodies than those available through conventional hybridoma technology.

Phage Display Advantages

Plantibodies• "plantibodies" are antibodies produced by genetically-engineered

corps e.g. corn, potatoes and tobacco plant• "plantibodies" are cheaper and arguably safer than mammalian

mAbs

Clinical Applications of Monoclonal AntibodiesA. Diagnosis

1. Pregnancy test2. ELISA3. Western Blot4. Flow Cytometry5. Radioimmunoimaging

B. TherapyA. Passive ImmunotherapyB. Active ImmunotherapyC. Drug Targeting

Pregnancy test

ELISA

Western Blot

Flow cytometry

Radioimmunoimaging

Passive immunotherapy

Passive immunotherapy

Active immunotherapy

Active immunotherapy using Bispecific mAb

Bispecific mAb

Drug targeting

You are now able to: Define terms such as monoclonal, polyclonal,

isotype, idiotype, allotype, CDR, and hybridoma

Compare monoclonal-antibody production methods

Identify different mAb types List some applications of mAb in medicine