Lecture 18 Molecular Biology and Cancer Flashcards
Cancer is the … common cause of death in the developed world
2nd
Most common cancer
Lung
Causes of cancer
Physical, chemical and biological
Physical carcinogens
UV
Ionising radiation
Chemical carcinogens
asbestos
tobacco smoke
Biological
infections from viruses, bacteria, parasites
What is now believed to be causing cancer again
viruses 10%
HER stands for
Human Epidermal GF Receptors
HER is what type of receptor
• Tyrosine kinase receptors
3 domains of the receptor
IC tyrosine kinase domain, EC ligand BD, TMD
Name the 4 HER encoded by the human genome
HER1,2,3,4
HER are a family of
structurally-related cell surface proteins
HER proteins undergo… upon ligand binding
undergo a conformational change upon ligand binding
What is the conformation shape change of HER proteins essential for
essential for dimerization and signalling
Type of dimers
Once ligand binds, the receptor can either homo (e.g. HER3:HER3) or heterodimerise (HER2:HER3).
Dimerisation activates…
(via phosphorylation) the tyrosine kinase domain
Different combinations of receptors stimulate different downstream signalling pathways e.g.
differentiation, migration, proliferation and cell survival
Which dimer has strongest mitogenic signal
HER2:HER3
How does HER2 cause cancer
changing expression level of receptor
Describe conformation of HER2
In conformation which promotes dimerisation so always in conformation to promote signalling
HERs can be described as
proto-oncogenes
How do HERs become oncogenic
- Overexpression
- Mutation
- HER2 mutant form in rats (neu) causes cancer
Breast cancer samples what occurs to the HER2 gene
Frequently amplified so get hundreds of copies of the gene = gene amplification
Normal breast cancer cells
diploid, 2n, 2 copies of gene shown by 2 bright spots
Mutant breast cancer cells
Mutant – many bright spots, gene amplification
The copy number of a gene can be determined using a technique called
Fluorescence in situ hybridisation (FISH)
Result of gene amplification of HER2
increased levels of the receptor
elevated signalling
cancer
Number of copies of HEr2 per cell in normal breast tissue and cancerous tissue
• Normal breast tissue 20,000 copies of the HER2 per cell
Cancerous tissue 2 million copies per cell
HER2 is a … for several cancers
negative prognostic marker
Over expression of HER2 correlates with
poor survival rate
Median survival for breast cancer patients without treatment
(HER2 positive 3 years)/(HER2 normal 6-7 years).
What is there strong evidence of if HER2 is overexpressed
Strong evidence overexpression of Her2 promotes overexpression of cancer
AB interfere with… and target…
Antibody binding can interfere with receptor signalling.
Antibody binding can target cells for destruction by the bodies immune system.
What is the first step in making a therapeutic antibody
making a monoclonal antibody
Monoclonal antibodies recognise
a single epitope on an antigen
How can vast quantities of AB be made?
Through a cell line
How would you make an AB?
Inject mice with Her2 receptor, take spleen cells and fuse with myeloma cells, makes hybridomas which are immortal.
What is the issue with murine AB?
Immune reaction - not well tolerated in humans
Evidence: Targeting HER2 over expressing cancer cells with 4D5 inhibits their growth
1) Treating HER2 over expressing cells with the monoclonal antibody (4D5) which binds the extracellular domain of HER2 inhibits their proliferation.
2) Injecting mice with the anti-HER2 monoclonal (4D5) antibody suppresses tumor growth in mice.
3) Injected radio labeled 4D5 targets HER2 positive breast cancer cells in women.
Structure of IgG1
2 heavy chains and 2 light chains
Hypervariable region of AB
Involved in antigen binding i.e. bind to HER2
These regions are also called complementary determining regions (CDRs)
Up to…CDRs can be involved in binding an antigen
• Up to six CDRs can be involved in binding an antigen
Humanised version of 4D5 Herceptin (Trastuzumab) - production
1) Clone the murine heavy and light chain cDNA encoding anti-HER2 monoclonal (4D5) antibody
2) Clone the murine CDRs of 4D5 into human IgG1 heavy chain and light chain plasmids.
3) The humanised antibody is then made by transfecting CHO cells with light chain and heavy chain plasmids.
Phase II clinical trials of Herceptin showed
- Study shows that - –Herceptin is active on women with HER2 positive breast cancer
However, it did not show any benefit on women who had normal levels of HER2.
Genentech and DAKO develop a diagnostic test for determining HER2 expression levels
Phase III clinical trials showed
Showed women with HER2 positive breast cancer gain extra 5 months disease free survival
However, Herceptin can cause serious complications as needed for normal heart function
Cancer returns in the majority of the women.
Why were more clinical trials carried out
• To investigate the best way to use Herceptin
- Herceptin is most effective if used in combination with surgery or chemotherapy for 1 year
- 85% cancer free after 5 years vs 66% with just Herceptin
• To determine if Herceptin works on other HER2 positive cancers (indications).
- It can be used in metastatic gastric cancer
How does Herceptin work
Herceptin binds to the EC domain of the HER2 receptor and:
• Interferes with IC signalling, hence inhibiting proliferation of the receptor
• Targets cells for antibody-dependent cell-mediated cytotoxicity (ADCC) which flags the receptor for destruction
• Inhibits EC domain shedding
Cancer returns in …%
15-25%
Cancer returning is… to Herceptin/chemotherapy
resistant
Future direction of treatment
1) HER2 dimerisation inhibitors (in combination with chemo/Herceptin)
2) Antibody drug conjugates - Trastuzumab emtansine delivers toxic drug to HER2 positive tumors
3) Targeting other HER family members
4) Drug combinations
- Giving Herceptin plus tyrosine kinase inhibitor (Tyverb) got rid of HER2 positive tumors within 11 days
HER2 screening is a good example of
personalised medicine in action
