Molecular biology and the treatment of cancer (L9)

Question 1

What is the prevalence of cancer?

Answer 1

In the developed world, cancer is the second most common cause of death. In 2012, about 8 million worldwide died of cancer. The most common types of cancer:

1. Lung (1.59 mill deaths)
2. Liver (745000 deaths)
3. Stomach (723000 deaths)
4. colorectal (694000) deaths
5. breast (521000 deaths)
6. Oesophageal (400000)

Question 2

What is cancer caused by?

Answer 2

Cancer can be caused by physical carcinogens e.g. UV and ionising radiation, chemical carcinogens e.g. asbestos and tobacco smoke, or biological carcinogens e.g. viruses like HPV and Rouss sarcoma virus in chickens, bacteria or parasites.

Question 3

What are HERs and how do they relate to cancer?

Answer 3

Human Epidermal growth factor receptor. they are a family of structurally related cell surface proteins. There are 4 receptors in the family , HER1-4, sometimes also known as EGFRs or ERBs. They have 1 transmembrane domain and the intracellular domain has a tyrosine kinase. Ligand binding causes conformational change and activation is HER1,3 and 4. But Her2 is constitutively active. They can form homo/heterodimers by joining together once a ligand binds. This binding together stimulates the receptor to signal. The heterodimer of Her2/Her3 is the stringest and increases signalling the most.

Question 4

Explain Her signalling

Answer 4

Ligand binding causes a conformational change which leads to dimerisation. Dimerisation activates the tyrosine kinase domain. Her2 is in a conformation which promotes dimerisation. Different combinations of receptors stimulate different signalling pathways. HER pathways stimulate cell proliferation, survival and are anti-apoptotic.

Question 5

What is HER’s role in causing cancer?

Answer 5

HERs are proto-oncogenes. They can become oncogenic by over expression or mutation. It 1984 it was published that a mutant form of HER2 (neu) promotes cancer in rats. The Her2 gene is also shown to be frequently amplified in breast cancer cells. When DNA replication goes wrong the cell can end up with hundreds of copies of a gene (gene amplification). If the gene amplified is a proto-oncogene it can promote cancer. The copy of a gene can be determined using flourescence in situ hybridisation (FISH). Increased expression of HER2 gene leads to increased levels of Her2 receptor. Normal breast tissue has approximately 20,000 copies of the Her2 per cell, cancerous tissues can have up to 2 million per cell. You can identify using these tissues immunohistochemistry.

Question 6

How is Her used as a marker for cancer?

Answer 6

Her2 is a negative prognostic marker for severl cancers. Over expression of HER2 correlates with poor survival rates for breast and ovarian cancer. Median survival for breast cancer patients without treatment is 3 years for HER2 positive and 6-7 for HER2 normal patients

Question 7

How are monoclonal antibodies used to treat HER2 related cancers?

Answer 7

Genetech decided to target HER2 over-expressing breats cancer using therapeutic antibodies. Antibodies can be raised against nearly any protein and are very specific. The binding can interfere with receptor signalling and target cells for destruction by immune cells.. The first step in this process of making a therapeutic antibody is making a monoclonal antibody.

Question 8

Explain the process of making Monoclonal antibodies that target HER2

Answer 8

Process of making monoclonal antibodies was published in 1975. Monoclonal antibodies recognise a single epitope on an antigen. As you have a cell line which make the antibody, vast quantities can be made. It was thought that monoclonal antibodies would revelutionalise medicine. However, murine antibodies are not well tolerated in humans. This is solved by a process called humanisation. Also, myeloma cells are now used instead of B cells because they survive better. To make monoclonal antibodies you take an animal (usually mice) immunised with HER2, take a tissue from them (e.g. spleen) then are fused with myeloma cell. They are cultured in HAT medium and selected for positive cells. They are left to produce monoclonal antibodies which are then harvested and humanised.

Question 9

How are monoclonal antibodies humanised?

Answer 9

An IgG1 molecule consists of 2 heavy chain and 2 light chains. The hypervariable regions of the antibodies are involved in antigen binding. Also, called complementary determining regions (CDRs). Up to 6 CDRs can be involved in binding and antigen - so its very specific. Genetech generated a humanised version of 4D5 (the anti-her2 monoclonal) to reduce its destruction and make it safer. They cloned murine heavy and light chain cDNA encoding 4D5, then cloned the murine CDRs of 4D5 in human IgG1 heavy and light chain plasmids. The humanised antibody is then made by transfecting CHO or HEK cells with light chain and heavy chain plasmids (this makes Herceptin (Trauzamab))

Question 10

What different clinical trials did Herceptin undergo?

Answer 10

1990 - Phase 1 clinical trials
1993 - Phase II - study shows that Herceptin is activated in women with HER2 positive breast cancer. However, it didn’t show any benefit in women who had normal levels of Her2. Then, genetech and DAKO developed a diagnostic test to determine HER2 expression levels in patients. 1995-1997 - Phase III clinical trials - shows women with HER2 positive breast cancer gain an extra 5 months disease free. However, Her2 is in the heart so Herceptin can cause serious heart complication, so this must be monitored. - cancer returned in the majority of patients.
1998 - the FDA approved Herceptin for the treatment of women with metastatic breast cancer who have tumours that overexpress HER2. In 2000, the European Commission approve Herceptin too.
Many more clinical trials have been done on Herceptin to improve it. It has been used to treat over 1 million women worldwide. Herceptin is most effective if used in combination with surgery or chemo. Usually requires about a year of treatment. Herceptin has also now been approved for use in metastatic gastric cancer.

Question 11

What is the suggested action of Herceptin?

Answer 11

Herceptin is thought to interact with intracellular signalling, target cells for antibody-dependent cell mediated cytotoxicity and inhibit extracellular domain shedding.

Question 12

What are the future directions of Herceptin and other monoclonal antibodies treatment?

Answer 12

Cancer returns in around 15-20% of the women treated with Herceptin/chemotherapy. This cancer is resistant to Herceptin treatment. Potential use for other drugs affecting Her2.
1. HER2 dimerisation inhibitors pertuzumab gained FDA approval in 2012. used in combination with Herceptin and chemotherapy it increased diseases free survival in HER2 positive metastatic cancer by 6 months
Antibodydrug conjugates Trastuzumab emtansine gained FDA approval in 2013. it delivers toxic drugs to HER2 positive tumours. Increases survival by 6 months. Targeting other HER family members - therapeutic antibodies being developed against HER3.
Drug combinations giving Herceptin plus tyrosine kinase inhibitor (tyverb) got rid of HER2 positive tumours within 11 days.
There are around 70 other therapeutic antibodies currently in clinical use. with an estimated sale of over 120 billion dollars world wide.