Cancer Bio (Dan Lecture 5 - Antibodies for cancer therapy)

Question 1

What are the 3 main ways that tumour specific antibodies (TSA) can be utilised against tumours?

Answer 1

1) TSA on its own
- binds to tumor cell
- NK cells with Fc receptors (CD16) are activated to kill tumor cells

2) TSA with conjugated toxin
- Binding
- Conjugates internalised, killing the cell

3) TSA conjugated to radioisotope
- Binding
- Radiation kills tumor cell and neighboring cells

4) TSA conjugated to enzyme
- Binding
- Enzyme converts pro-drug to active drug in vicinity of tumor cells

Question 2

Give some examples of antigens expressed y tumors that can be potential drug targets

Answer 2

1) CD20 and CD22
- overexpressed in pre-B cell and B cell Non-Hogkins lymphoma

2) CEA
- majority of NSCLC’s and colorectal cancers, 50% of breast cancers

3) EGFR
- Many solid tumours

Question 3

What are the potential advantages and disadvantages of using antibody fragments instead of intact antibodies

Answer 3

Advantages:

• Ease of production
• simple protein enginnering (e.g epitope tagging for purification)

Disadvantages:
- Unfavourable pharmacokinetics and biodistribution: Impaired antigen binding

Question 4

What are the features of the single-chain antibody (scFv)?

Answer 4

• Consists of the variable light (Vl) chain of an antibody joined via a linker to the variable heavy (Vh) domain
• Linker typically consists of flexible/soluble peptide
• Maintains binding specificity (but not always affinity) of parent antibody

Question 5

Describe the features of beta emitters in treating solid tumours

Answer 5

Tumor cells grouped together

Beta emitters have mm range and moderate energy

mm range means multiple tumor cells will be killed

Question 6

Describe the features of alpha emitters in treating single cell tumours

Answer 6

Tumour cells on their own amongst healthy cells e.g leukemia

Alpha emitters have µm range, high energy

Short range required to radiation only kills single targeted cell

Beta emitters would cause too much damage to healthy cells

Question 7

What is Linear Energy Transfer (LET)?

Answer 7

A function of emission energy and emission transit distance and path

Question 8

Increasing emission energy and decreasing path length does what to the LET?

Answer 8

Increases LET

Question 9

When designing a radioimmunotherapy agent, what must be considered when selecting an antibody?

Answer 9

1) Selection of target antigen
2) Antibody format (Intact or fragment)
3) Source species (mouse or human)

Question 10

When designing a radioimmunotherapy agent, what must be considered when selecting a radionuclide?

Answer 10

1) Emission properties (alpha or beta)
2) Availability and half life
3) Safety
4) Conjugation stability and chemistry

Question 11

What are the most commonly used immunotoxins?

Answer 11

Bacterial origin:
- diphtheria toxin DT

Plant origin:
- ribosome inactivating proteins (RIPs)

Question 12

What is the mechanism of action of diphtheria toxin?

Answer 12

• Antibody binding
• Endocytosis
• Taken into an acidified endosome
• Linker between antibody and toxin cleaved
• DT inhibits elongation-factor 2 (EF-2), resulting in protein sysnthesis inhibition

Question 13

What is the mechanism of action of ribosome inactivating protein (RIP)?

Answer 13

• Binding
• Endocytosis
• Localisation in trans-golgi/ER
• Linker cleavage
• Inhibits ribosome function (inhibits protein synthesis)

Question 14

How do these toxins bind to healthy cells and how is these dealt with so they are more tumor specific?

Answer 14

• All toxins bind to ubiquitous receptors on normal cells

- Binding domain neutralised or removed before use

Question 15

What is ADEPT?

Answer 15

A 2 step targeted anti-cancer therapy

Question 16

What are the steps in ADEPT?

Answer 16

1) Administration of an antibody-enzyme conjugate
- Targets and binds to tumor antigen

2) Administration of a pro-drug (nitrogen mustard-based drug)
- Cleavage of glutamate by enzyme to form chemo agent at tumor site
- Occurs extracellularly, so drug kills bound cell plus neighbouring cells (the bystander effect)

Question 17

Name some ways that ADEPT needs to be improved

Answer 17

Immunogenicity of enzyme component currently derived from a bacterial source.

Alternative oncology targets that are more susceptible to killing with nitrogen mustard-based drugs.

A better understanding of the role of DNA repair in tumour resistance to nitrogen mustard-based drugs.

Improved expression technologies to provide adequate yields of clinical grade fusion proteins with suitable post-translational modifications (e.g. “humanised” glycosylation profiles).