Lec 15- Immunotherapy III (mAbs) Flashcards
History
1890
- Discovery of Abs (anti-toxic activity)
- Against diphtheria and tetanus
- Von Behring and Kitasato
- Serum sickness
- Limited use of serotherapy
1905
-Paul Ehrlich= magic bullet
1930s-40
-Development of chemotherapy/antibiotics
1950-60
- Abs structure elucidated
- Porter, Nisenoff and Edelman
1975
- Kohler and Milstein Cambridge
- Developed mAb technology
Ab= antibody
mAb= monoclonal antibody
B cell and Ab development
1) Stem cell Maturation into mature Agetically committed B cells
2) Mature B cells
3) Ag binds to B cell Ag-dependant Proliferation and differentiation into plasma and memory cells
4) Plasma and memory cells produced
mAb technology
- Take a B cell, grow it and harvest the mAb
- They die by apoptosis
1) Take a mouse and place Ag in it, The Ag will have multiple epitopes
NB-If we just isolate the serum and use a polyclonal anti-serum (has Ab for all epitopes) then this would lead to a higher level of serum sickness
2) Mouse has Ab for each epitope (4)
3) Isolate spleen cells
4) These cells contain a plasma cell(s) for each Ag
5) Hybridisation- Myeloma cells (cancer cells), this is because they never enter apoptosis, we then combine them with the plasma cells creating hybridomas
6) We then isolate the individual clones (hybridomas)- this is done by taking suspension with around 200 cells and split them into 1000 cells, this means that there is some wells with just one cell so is isolated
Structure/function of Abs
Fab (fragment of antigen binding)
- Ag binding
- Toxin neutralisation -Opsonisation Fc region
- Complement activation
- ADCC (Ab-dependant cell mediated cytotoxicity)
- Phagocytosis
mAb therapy
-A panacea (solution of lots of different diseases)
+Specific
+Targeted
+Effected
+Magic bullet
-Have problems
Problems with Ab therapy
1) Passive therapy
- No transfer of immunity to a recipient to give instant protection
- No long lasting memory cells (Unlike active immunisation)
2) Cost of production
3) Specificity- human anti-mouse antibodies (may trigger type III-like hypersensitivities
4) HAMA- Human anti-mouse- Ab
5) mAbs are Ag from mice -They elicit an immune response
- Serum sickness
Ab engineering- Chimaera
-We can from chimaera’s
+This is a 70:30 mix of human:mouse Ab
- Bottom part is a human Ab so it can activate our own immune system (Fc region)
- Top are (Fab region) this means that it is complementary to the Ag
Ab engineering- Humanised
- 95% human, 5% mouse
- CDR graft- CDR’s from mouse
- However this is hard because you have to be very specific is this is wrong (Take to much or to little) it can render the Ab useless
- Possible to choose the class or subclass of Ab we pick
- e.g. Transport across epithelium, Diffusion in extravascular site, complement activation
IgM, why is IgG better
-IgM will form big pentemeric rings will mean that it takes longer to get to the target area -Larger the molecules are the more unstable they are
Ab engineering
- Genetically modified mouse
- Xenomouse (Amgen)
+Knock-out of mouse Heavy and light chain loci
+Graft human Ig loci
+Immunise
+Mice produce human mAbs
-Phage Abs (these can be grown in bacteria)
+Human Ig genes in a library
+Ab-like molecules displayed on surface of phage
+Grown in bacteria
Ab engineering- phage Abs
- Phagemid
- Genetic material within bacteriophage
- We add genetic information for what we need V(light chain) V(heavy chain)
- We end up with half of an Ab
- We collect single chain variable fragments and Filamentous phage particle
- Low affinity reagents- No affinity maturation (not as good as Abs)
Enhanced Ab Therapy
1) Tumor-specific Abs (opsonise NK cells)
- Ab bind to the tutor cell
- NK cells with Fc receptors (CD16) are activated to kill the tumour cells
2) Tumor-specific Abs conjugated to toxin (chemotherapy)
- Ab-toxin conjugates bind to the tumour cells
- Conjugates are internalised killing the cells
3) Tumor-specific Abs conjugated to radioisotopes
- Radioactive Ab binds to tumour cell
- Radiation kills the tumour cell and neighbouring tumour cells
- Will also kill healthy cells in the vicinity
Enhanced Ab therapy 2
-Bispecific Abs
+Abs are bivalent
+Engineered to recognise 2 different Abs
+Anti-tumor arm and Anti-NK cell arm: this holds both the tumour and NK cell together until the NK cell has killed the tumour
Orthoclone OKT3
- Muromonab
- Fully murine mAb
- Mouse anti-human CD3 (a T cell Ag)
- Used to treat transplant rejection
- Depletes T cell
Remicade
- Infliximab
- li- Immune system target
- xi- Chimaeric mAb -Anti-TNF (a cytokine)
- Used to treat RA and other inflammatory conditions
