Monoclonal Antibodies Flashcards
What is the difference between Mono and Polyclonal Abs, and what does Polyclonal Ab require for production?
-Polyclonal Abs are produced by multiple immune cells –> bind to many epitopes on an antigen, monoclonal Abs are produced by identical immune cells (clones from the parent cell) binding to a single epitope
-Production of polyclonal Abs require live-animals
Advantages and Disadvantages of Polyclonal Abs
Advantage: bind to multiple epitopes -> highly effective in neutralizing antigens or toxins
Disadvantages: immunogenicity, serum sickness, antibodies may vary from batch to batch
What are the clinical uses of polyclonal antibodies?
-passive immunization
-post-exposure to toxins or virus -> to neutralize toxins or virus
What are the difficulties a patient might suffer from multiple polyclonal treatments?
-Serum sickness
-Deposition of immun-complexes in joints -> Pain
-in severe cases: Anaphylactic shock
What is the purpose of the hybridoma technique?
-creating immortal monoclonal antibody-producing cells
-by fusing B-cells from the spleen (from mouse, producing Abs) and cancer cells (human myeloma, immortal) –> Hybridoma cells
Why was PEG used in the hybridoma technique?
Becuase of the solvent property, binding to lipid membrans
-it fuses B-cell and myeloma cells
What is the purpose of the selection HAT medium?
-To separate the preferred hybridoma cells from unhybridized B-cells and cancer cells
-the cancer cells carry a mutation for Thymidine kinase, so they cant use the salvage pathway to survive + HAT medium contains aminopterin (antimetabolite) preventing the cancer cells to use the de novo pathway –> cancer cells die in the HAT media
-B-cells are mortal and die after some cell cycles
ONLY HYBRIDOMA cells survive
Why do only Hybridoma cells survive in the HAT medium?
Because they are hybrids
-gained the ability to use the thymidine kinase from B-cells to use the Salvage pathway
-gained immortality from cancer cells
->even though the HAT media blocks the de novo pathway, they survive by using the Salvage pathway
How to check if the hybridoma technique worked?
Selection for hybridoma positive cells
-Grow the cells on Selection HAT medium and on Screening plates (ELISA) containing antigens that are specific for the produced antibodies
-> Only hybridoma cells will stick to the antigens -> These cells can be recultured and produce multiple monoclonal antibodies
What are the advantages of using hybridoma cells instead of live animals to produce antibodies?
-hybridoma cells are immortal and can keep on producing endless amounts of Abs
-constant amounts of Abs
-> live-animals will have variability because they might not produce the same amounts depending on the conditions
What are the disadvantages of using hybridoma cells?
-they are murine (mouse-origin) -> can be immunogenic
-antibodies can only be tested against one antigenic protein per clone
-it is a slow process, takes time to find the Ab that really works for an antigen
How does the Phage expression system work?
-creating predominantly human antibodies
- multiple human antigen-binding fragment genes (Fab) were expressed in a phage (virus) -> the human Fabs were expressed in the viral coat
- the viruses that contain the human Fab of interest were reproduced in E-coli to generate a large number of predominantly human antibodies
Why is the Phage expression system useful?
-many different monoclonal Ab can be created and screened at once
-it predominantly produces human antibodies (the Fab part is human, the Fc region might be from an animal) with lower immunogenic response in patients
Which drug was first approved using the Phage expression system?
Adalimumab (HUMIRA)
How is a Xenomouse used to produce human monoclonal Abs?
-using Knockout mice
-Deactivation of the mouse gene for Ab -> insertion of human Ab heavy and light chain genes
-the mouse produces human antibodies
Which Immunoglobin Isotpe is mostly used for therapeutic use?
IgG
Which part of the Antibody can contain Glycolysation?
The Fc region
Why are most of the Antibodies soluble?
Because of Glycolysation on the Fc region of the Ab
Which region of the Ab is involved in Ag binding?
CDR = complementary determining region on the Variable part of the light and heavy chain
-highly variable, and adaptable binding to different antigens
What is the meaning of a bivalent IgG antibody?
An IgG antibody can bind to 2 identical antigens (same antigen)
What are functions of the Fc region?
-can be Glycolyzed
-provides a longer half-life compared to Ab lacking the Fc region
-it binds to Fc-γ receptors on NK cells to mediate the killing of an infected cell (ADCC - Antibody-dependent cellular cytotoxicity)
-complement-fixation (killing by complement system)
Nomenclature of monoclonal antibodies
EXAM !!!
Mouse (o): 0% human - momab
Chimeric (xi): 60% human - ximab
Humanized (zu): 90% human only CDR is animal - zumab
Fully Human (u): 100% human from Xenomouse - umab
Advantages of whole monoclonal Abs
-highly specific -> minimal off-target effects
-human mAB have little immunogenicity
-Fc fragment help attract immune cells/complement fixation
-Fc fragment prolongs half-life
-Fc fragment contains glycosylation -> increase solubility
Disadvantages of monoclonal Abs
-monospecific (bind to only 1 type of Ag)
-binding to more than 2 Ag is not possible (not bivalent)
-Extensive glycosylation make production complex (f.e. Ecoli cant make PTMs)
-hard to penetrate tissues bc of the large size
