Biology 6 - Molecular Biology Therapy (Keith Spriggs)

Question 1

What are gene therapies?

Answer 1

Individualised therapy for genetic disease - sounds like the ideal solution;
gene repair for correcting mutations,
pro-drug metabolising enzyme to sensitise cancer cells to cytotoxic drugs,
viruses that selectively target cancers,
modification of the tumour envrionment (hypoxia),
drug resistance gene therapy for non-cancerous cells, immunotherapy with GM effector T cells (APCs).

Question 2

What are the commercial barriers to cancer gene therapy?

Answer 2

Costs for materials are high
Individualised gene therapy is required; difficult for late phase studies (efficacy) and potentially a small market of suitable patients. Will R&D be profitable?
Costs for patents and license

Question 3

What are the biological barriers to cancer gene therapy?

Answer 3

Many genes can be mutated
Variation between tumours and patients
Requires the majority of cancer cells to be affected

Question 4

What are miRNA therapies?

Answer 4

Use anti-miRs (antagomirs) to block oncomiR action. They act as complementary ‘false targets’ for oncomiRs.

OR

Upregulate tumour suppressor mIRs -oligonucleotides to mimic TS miRs.

Question 5

What are the barriers to successful miRNA therapy?

Answer 5

Stability of miRNA
Excretion (readily excreted, lots of degrading enzymes)
Cellular uptake and targeting
Off target effects

Question 6

How can future pro-drug metabolising enzyme act as a therapy?

Answer 6

Herpess simplex TK (thymidine kinase)
Phosphorylates pro-drugs such as valaciclovir to toxic nucleosides
Target to diving cells using gamma retroviral vector or by targeting cell surface antigen

Question 7

How can future viral oncolysis / virotherapy be used in cancer gene therapy?

Answer 7

Viruses can be engineered to only replicate in cancer cells.
Adenovirus dl1520 required defective p53 pathway (China license)
Other viruses trialled with promising results but not yet licensed.

Question 8

How can future therapies target the tumour microenvironment in cancer gene therapy?

Answer 8

Prevent angiogenesis by modifying normal cells
Doesn’t require high efficiency of transduction.
Modify immune response and metastatic potential.

Question 9

What is transduction?

Answer 9

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. An example is the viral transfer of DNA from one bacterium to another.

Question 10

How can gene therapies target a reduction in toxicity?

Answer 10

MGMT gene ; Tx causes overexpression in healthy cells to protect them from alkylating agents
MGMT gene confers resistance to alkylating agents.

Question 11

How can T cells be genetically engineered for treatment?

Answer 11

Donor T cells from healthy volunteers, genetically modified to attack CD19+ cells and be resistant to immunotherapy which would otherwise kill all T-cells.
Following therapy:
Transplant of healthy cells restores the patients immune system which then kills all GM T cells and the patient is free of cancer and donor GM cells.

Question 12

What are monoclonal antibodies?

Answer 12

Proteins produced by the immune system to bind specifically to foreign antigens
Monoclonal antibodies come from single clone B cells and target a single epitope

Question 13

Where are mabs produced?

Answer 13

Produced by B lymphocytes

Question 14

What is the difference between monoclonal and polyclonal antibodies?

Answer 14

Monoclonal antibodies come from a single clone of B cells and target a single epitope

Polyclonal antibodies come from many clones of B cells and target multiple epitopes of the antigen

Question 15

What is an epitope?

Answer 15

The part of an antigen that the antibody binds itself to

Question 16

What is the basic structure of an antibody like?

Answer 16

Heavy chain, constant region
Y shape
Variable regions attached via disulphide bond and contain an antigen binding site (Fab)

Question 17

What are MAbs used for?

Answer 17

Make cells visible to the immune system e.g. against cancer cell makers
Stop cells dividing e.g. against cancer cell growth factors
Target therapies e.g. attach drug to antibody to target site (either conjugated to a drug or radioisotope.)
Diagnosis e.g. testing for expression for hormone receptrors (HER2+)

Question 18

What is Rituximabs mechanism of action?

Answer 18

Targets CD20 on B cells which causes antibody dependent cell mediated cytotoxicity (ADCC) and complement mediated cytotoxicity (CDC).
Kills B cells in lymphomas and leukemia (including healthy B cells).

Question 19

What is Ibritumomab tiuxetan used for in cancer therapy?

Answer 19

Used in conjunction with yttrium-90 or indium-111 for non-hodgkin lymphoma
ADEPT - antibody directed enzyme pro-drug therapy
MAb+enzyme binds to target; prodrug only activated near target cells (Trials)

Question 20

What are the criteria for MAbs?

Answer 20

Good specificity, large quantities, well defined purity

Question 21

Where were early MAbs derived from?

Answer 21

Animal spleen cells fused with cancer cells

But were recognised as foreign by patient immune system.

Question 22

What are recombitant antibodies?

Answer 22

Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than using mice. Advances in molecular biology have lead to the ability to synthesize antibodies de novo in vitro – completely without the use of animals.

Question 23

What are the two main reasons that recombinant antibodies are useful?

Answer 23

Reduction in immune response problems

Reduction in size and complexity of antibodies (bits that are not needed are removed).

Question 24

How can we avoid human immune response to antibodies?

Answer 24

Use human antibody but mouse Fv region = chimeric antibody
Use human antibody but with mouse CDR (complimentarity-determining region) = reshaped antibody (needs reshaping / adjustment to the antibody framework, difficult to do but better results).

Question 25

What region of antibodies is commonly recognised as foreign by immune system?

Answer 25

Fc (constant region)

Question 26

Can human antibodies alone work as a gene therapy for cancer?

Answer 26

Use human lymphocytes rather than mouse/rat
(-) low yield, unreliable, vulnerable to contamination, ethically limited

Genetically modified mice to produce entirely human antibodies
Panitumumab against EGFR

Recombinant antibodies e.g. using micro-organisms to synthesise MAbs (phage display - using bacteria)

Question 27

What is the size/specificity needed for recombinant antibodies?

Answer 27

Fc region - non specific binding and activation of the immune system
IgG very large complex molecule ; could be smaller and simpler

Question 28

How could the development of antibody fragments be useful?

Answer 28

Smaller molecules extravasate more readily and distribute around the body quickly
Smaller molecules penetrate through tissues and tumours faster (removal of Fc functions can remove immunological reactions though so a double edged sword / balance needed).
Easier to produce by recombinant DNA technology (and cheaper), easier to use E.coli or yeast than using mammalian cells.
Can be used in constructs which contain additional functionality e.g ADEPT (antibody directed enzyme prodrug therapy).

Question 29

What are recombinant antibody fragments based on?

Answer 29

Either a Fab fragment or an Fv fragment of antibody.

Question 30

What are the problems associated with recombinant antibody fragments?

Answer 30

Reduced circulation half-life; controlled through Fc region and sugar residues + can improve lifespan by modifying the fragment

Loss of immune effector functions e.g. phagocytes, NK cells

Reduced binding activity - reduction in binding site number / effectiveness

Question 31

What are third generation molecules? 3Gs?

Answer 31

Miniture MAbs that have non essential regions removed.
SMIPs (small modular immunopharmaceuticals)
Retain effector function (e.g. phagocytes / NK cells)
Increased tumour penetration

Question 32

Is there a possibility for cancer vaccines?

Answer 32

A tumour associated antigen (TAA) to induce immune response specifically against tumour cells

Induction of cytotoxic T lymphocytes
TAA expressed on APCs
Modify tumour cells to act as APCs (antigen-presenting cells)

Question 33

What is Provenge?

Answer 33

Licensed in US, EU, UK but not currently recommended by NICE.
Sipuleucel-T (Provenge)
Metastatic hormone refractory prostate cancer