Lecture 23

Question 1

How are monoclonal antibodies made for HER2

Answer 1

Immunise mice with HER2 and then isolate the spleen cells containing the antibody-producing B memory cells. These cells are fused with myeloma immortal cells. This creates an immortal cell line that will continue to make huge amounts of the antibody

Question 2

What process can be used to determine the copy number of a gene that has been amplified in tumorigenesis

Answer 2

Fluorescent in situ hybridisation (FISH)

Question 3

Different combinations of receptors stimulate different signalling pathways and can lead to cell proliferation, survival and a prevention of apoptosis, T or F

Answer 3

T

Question 4

HER2 is a negative prognostic marker for several cancers, what is meant by this

Answer 4

Overexpression of HER2 correlates with poor survival rates of breast cancer patients

Question 5

What are the different types of HER receptor

Answer 5

HER1, HER2, HER3 and HER4

Question 6

On average, how many people will have cancer in their lifetime

Answer 6

1 in 3 to 1 in 4

Question 7

Explain how immunoglobulins are humanised

Answer 7

The hypervariable domain involved in antigen binding is swapped into human antibodies. This enables the antibodies to be used in human patients without causing massive immune responses. It is achieved by cloning the murine heavy and light chain cDNA encoding 4D5 into the human IgG1 heavy chain and light chain plasmids. The humanised antibody is then made by transfecting Chinese Hamster Ovary (CHO) cells with light and heavy chain plasmids

Question 8

What is unusual about the HER2 receptor

Answer 8

Unlike the other members of the HER receptor family, HER lacks the extracellular ligand binding domain. This means that it doesn’t require ligand binding to dimerise and hence is constitutively active

Question 9

What is the most common type of cancer

Answer 9

Lung cancer

Question 10

What causes HER proteins to become oncogenic

Answer 10

Overexpression or mutations

Question 11

Up to what percentage of breast cancers are believed to be HER2 positive

Answer 11

0.25

Question 12

What is the problem with using mice to produce HER2 antibodies

Answer 12

Murine antibodies are not well tolerated in humans, the immune response from these will kill

Question 13

What are the three main causes of cancer

Answer 13

Physical carcinogen – UV and ionising radiation, chemical carcinogens – asbestos and tobacco smoke, biological carcinogens – infection from certain viruses, bacteria or parasites

Question 14

Which conformations of HER receptor dimers signal the strongest and why

Answer 14

Homodimers don’t signal as strongly as heterodimers. The HER2:HER3 heterodimer signals the strongest as it activates both the Ras/MAPK pathway and the PI-3 Kinase pathway

Question 15

How do the median survival rates of HER2 positive and HER2 negative breast cancers compare

Answer 15

HER2 positive – 3 years, HER2 negative – 6-7 years

Question 16

What happens to the HER receptors (apart from HER2) following ligand binding

Answer 16

HER proteins undergo a conformational change upon ligand binding that is essential for dimerization and signalling. Once the ligand binds, the receptor can either homo or heterodimerise which stimulates the receptor to signal

Question 17

What was the three different pieces of evidence for the use of the 4D5 monoclonal antibody in the treatment of HER2 positive breast cancer

Answer 17

4D5 binds to the extracellular domain of HER2 and leads to an inhibition of their proliferation. Injecting mice with the anti-HER2 monoclonal antibody supresses tumour growth in mice. Finally, injection of a radiolabelled 4D5 targets HER2 positive breast cancer cells in women

Question 18

Outline the HER pathway of activation

Answer 18

Growth factor binding to a HER receptor allows dimerisation with other HER receptors. Dimerisation of the HER receptors allows for phosphorylation of the intracellular tyrosine kinase domain. This creates binding sites for signalling proteins such as PTEN PDK etc. This in-turn leads to the activation of PI-3 Kinase pathway which prevent the apoptosis of cancer cells. HER receptors can also signal through the activation of the MAP-K pathway to increase cancer cell proliferation

Question 19

Describe Herceptin’s mechanism of action

Answer 19

Binds to extracellular domain of the HER2 receptor and inhibits the proliferation of human tumour cells through multiple mechanisms of action. Firstly, it blocks downstream signalling of HER2 receptor but it also marks cells for degradation by the immune system. Finally, it is believed to also exploit the internalisation of HER2 receptors

Question 20

What family of receptors do the human epidermal growth factor receptors belong to

Answer 20

Receptor tyrosine kinases

Question 21

HER proteins are tumour suppressor genes, T or F

Answer 21

F – they are proto-oncogenes

Question 22

New versions of HER2 inhibitors also inhibit receptor dimerisation, T or F

Answer 22

T

Question 23

What is unique about the HER3 receptor

Answer 23

The HER3 receptor has lost its intracellular tyrosine kinase domain and so cannot phosphorylate target proteins

Question 24

Which mutant form of HER2 was first discovered in rats the promote cancer aetiology

Answer 24

Neu

Question 25

What is the other name for the hypervariable domain involved in antigen recognition by immunoglobulins

Answer 25

Complementary determining region (CDR)

Question 26

Normal breast tissue contains around 20,000 copies of the HER2 receptor, how many copies can be found in cancerous tissues

Answer 26

2 million copies – 10-fold increase

Question 27

Herceptin is a monoclonal antibody used to target HER2 overexpression, what is meant by a monoclonal antibody

Answer 27

A massive population of antibodies derived from a single cell and that all specifically recognise a single epitope

Question 28

Explain the serious complications that can occur because of Herceptin treatment

Answer 28

Because HER2 receptors are present in the heart, the inhibition as a result of Herceptin treatment can lead to decreased cardiac viability

Question 29

What is the best way of using Herceptin to treat HER2 positive breast cancer

Answer 29

Combination with surgery, chemotherapy – 85% of patient’s cancer free for 5 years

Question 30

How is the problem of immune response to foreign antibodies overcome

Answer 30

Humanisation