Molecular Biology & Cancer Therapies Flashcards
What are some examples of gene therapies?
- Gene repair; correction of mutations
- Pro-drug metabolising enzyme therapy to sensitise cancer cells to cytotoxics
- Viral oncolysis; viruses that selectively target cancers
- Modification of the tumour microenvironment
- Drug resistance therapy for non-cancerous cells (allow high dose cytotoxic use)
- Immunotherapy w/GM effector T cells, APCs
What barriers have there been to cancer gene therapy?
- Commercial
- Biological
What commercial barriers are there to gene therapy?
- Costs for materials high; R&D, tailor-made etc.
- Individualised therapies requires; difficult for late phase clinical studies
- Potentially small market of suitable patients; not fiscally attractive
- Costs of patents & licenses
What biological barriers are there to gene therapy?
- Many genes may be mutated
- Variation within tumours; different clones within a tumour
- Variation between patients
- Requires majority of cancer cells to be affected; unlike gene therapy for monogenic diseases, e.g. single mutation in an enzyme
Give an example of a proposed miRNA therapy targeting antagomiRs, and what barriers prevent its successful use.
- miRNA usually an important regulating molecule; binds to 3’ UTR of target mRNAs and regulate translation
- Proposed use of anti-miRs (antagomiRs) to block oncomiR action; antagomiR complementary to oncomiR sequence
Barriers:
• Stability
• Excretion
• Cellular uptake and targeting
• Transient inhibition (rapid mRNA turnover); repeated doses required
• Off target effects; turning off other targets
• RNA v. unstable (lots of RNA enzyme)
Give an example of miRNA therapy involving tumour suppressor miRs, and barriers preventing its successful use.
- To upregulate tumour suppressor miRs
- Proposed oligonucleotides that would mimic TS miRs (synthetic versions)
Barriers (similar to antagomiRs): • Stability • Excretion • Cellular uptake and targeting • Transient inhibition (rapid mRNA turnover); repeated doses required • Off target effects
What is an example of pro-drug metabolising therapy? Give an example.
E.g. Herpes simplex TK (thymidine kinase)
- Phosphorylates pro-drugs such as valaciclovir to toxic nucleosides
- Target to dividing cells using gamma retroviral vector (viruses) or by targeting to cell surface antigen
- Not yet licensed (promising results)
> Activate drug only in vicinity of tumour (reduce systemic effects)
> mAb is conjugated to the pro-drug thus therapy is only targeted at tumour cells
What is an example of viral oncolysis/virotherapy? What is it?
- Viruses which have been engineered only to replicate in cancer cells
E.g. Adenovirus dl1520, which requires defective p53 pathway; this adenovirus normally inhibits p53 as part of its life cycle
How is the tumour microenvironment targeted? Why is this an advantageous therapy?
- Prevent angiogenesis by modifying normal cells
- Modify immune response and metastatic potential
»> Doesn’t require high efficiency of transduction (other therapies target cancer cells themselves; which requires hitting most of them to be successful (success in animal models of viruses expressing angiostatin and endostatin)
»> Target host not the cancer
How can gene therapy be used to reduce toxicity?
- Effectively increase therapeutic index
- MGMT gene removes alkylating DNA modifications; which would confer resistance to alkylating agents
»> Overexpressing MGMT in healthy cells; spared from cytotoxic effect
Describe the new therapy recently developed to treat aggressive ALL (acute lymphoblastic lymphoma) which involves T-cells?
- Donor T-cells taken from a healthy volunteer (unrelated to patient)
- Genetically modified to attack CD19+ cells; marker of B cells of ALL
- GM-T cells modified to be resistant to immunotherapy which would otherwise kill T-cells
- All of patient’s T-cells thus killed but still has GM donor T-cells
»> Then after therapy own T cells kill GM T-cells (recognised as foreign), leaving patient with self-T cells and no cancer cells.
What are mAbs?
- Proteins produced by immune system to bind specifically to foreign antigens
- Produced by B lymphocytes
What’s the difference between monoclonal and polyclonal antibodies?
Monoclonal Abs:
- Come from a single clone of B lymphocytes, targeting a single epitope (part of the antigen where antibody attaches)
Polyclonal Abs:
- Come from many clones of B lymphocytes
- In turn can target multiple epitopes of the antigen (extracted from blood of immunised animals)
What are mAbs used for? Give examples.
- Make cells visible to the immune system
e. g. against cancer cell markers (rituximab) - Stop cells dividing
e. g. against cancer cell growth factor receptors (cetuximab) - Target therapies
e. g. conjugated to drug or radioisotope (so they can exert their effect in the vicinity of cancer cells only) - Diagnosis
e. g. testing for expression of hormone receptors (HER2)
How does Rituximab work? What is its mode of action an example of?
- mAb that targets CD20 on B cells; recruiting immune cells to cancer e.g. NK (natural killer) cells
- Causes ADCC (antibody dependent cell mediated cytotoxicity)
- Causes complement mediated cytotoxicity (CDC); leads to pore in cell wall = cell death
- Kills B cells in lymphomas/leukaemias (and health B cells that also express CD20; but recovery after)
»> Makes cells visible to the immune system.
Which mAb is used to conjugate radioisotopes for NHL (Non-Hodgkin’s lymphoma)?
• Ibritumomab tiuxetan
Conjugating:
- yttrium-90
- indium-111
What is ADEPT?
- Antibody directed enzyme pro-drug therapy
- mAb + enzyme binds to target; pro-drug only activated when near target cells
- mAb delivers enzyme to cancer cells
What characteristics are essential for therapeutic antibody use?
- Good specificity
- Large quantities
- Well defined purity; no contamination
What were the problems w/early mAbs derived from animal spleen cells?
- Full size antibodies
- Recognised as foreign by patient immune system
What are the advantages of using Recombinant mAbs?
- Reduction of immune response problems
- Reduction in size and complexity of antibodies; e.g. can rid of constant region (reducing immune detection)
What are the two possibilities of formulating recombinant mAbs to avoid immune response? What are these called?
A) Use human antibody but with mouse Fv (variable; light chain to recognise shit) region = Chimeric mAb
B) Use human antibody but with mouse CDR (Complementarity-determining region; most of light chain variable region is human but ‘tips’/business bit is mouse) = ‘Reshapes’ mAb
What are the advantages to using ‘reshaped’ human mAbs over Mouse Chimeric mAbs?
- Better
- But requires adjustment to antibody framework to retain OG antibody “fit”; more difficult to achieve than chimaeric
> > > Anti-idiotype responses cannot be removed by engineering
How can completely human mAbs be manufactured?
- Use human lymphocytes rather than mouse/rat to minimise foreign Ab exposure
- Use genetically modified mice to produce entirely human antibodies (e.g. Panitumumab against EGFR)
- Use recombinant antibodies .e.g micro-organisms to synthesise mAbs (phage display; viruses infect bacteria, which then generates large quantity of mAb
How is the use of human lymphocytes to generate mAbs of limited value?
- Low yield
- Unreliable
- Vulnerable to contamination (pathogen/viruses etc.)
- Ethically limited
What does the size/specificity of the Fc (constant) region of an mAb convey?
- Non-specific binding
- Activation of the immune system
> Removal of Fc (into antibody fragments) prevents immunological reactions
»> igG very large + complex molecule; could be smaller and simpler
What are the reasons for using antibody fragments? (e.g. removing Fc region/making mAb smaller)
- Smaller molecules extravasate more readily and distribute around the body more rapidly
- Smaller molecules penetrate through tissues and tumours faster
- Removal of Fc (constant) functions can remove immunological reactions (double edged sword; not necessarily what you want)
- Easier to produce by recombinant DNA technology
- Can reduce production costs; easier to use E. coli or yeast for production rather than mammalian cells (fewer PTMs - post translational modifications)
- Can be used in constructs which contain additional functionality e.g. ADEPT
What are recombinant mAb fragments based on?
- Fab fragment (one arm of variable region)
- Fv fragment (whole variable region; two arms)
What are the problems associated with fragments in mAb, and how are they overcome?
- Reduced circulation half-life
> Controlled through Fc region and sugar residues normally
> Can modify fragment (e.g. PEGylate) to improve lifespan - Loss of immune effector functions
> E.g. phagocytes, NK cells, complement binding sites - Reduced binding activity
> Reduction in binding site number, effectiveness
> Constant region (Fc) contributes to antigen binding
What are third generation molecules (3G) WRT mAbs, and what is their point?
- Minaturing mAbs by removing non-essential functions
- SMIPs (small modular immunopharmaceuticals)
- Retain effector functions (e.g. effector cells, complement binding)
»> Increased tumour penetration
e.g. TRU-015, anti-CD20
What do cancer vaccines entail? What’s the caveat? How do they work?
- Tumour-associated antigen (TAA) induces an immune response specifically against tumour cells
»> Difficult as host immune system often compromised (cytotoxic treatment)
• Induction of cytotoxic T lymphocytes
- TAA expressed on APCs e.g. Provenge
- Modify tumour cells to act as APCs (antigen presenting cells)
- CD40L (Ligand), CD80
- GM-CSF, lymphotactin, IL12, IL2 (proteins that recruit immune cells to cell vicinity)
How does Provenge (Sipuleucel-T) carry out its anticancer activity? What is it used to treat?
1) Immune cells harvested from patient with prostate CA
2) Centrifuged to separate into monocytes
3) Monocytes combined with PAP marker (prostatic acid phosphatase; cancer specific antigen), as well as GM-CSF (granulocyte-macrophage colony stimulating factor required for maturation of immune cells
4) Modified monocytes cultured for 36-44 hours
5) GM-CSF makes monocytes mature; which become activated APC (antigen presenting cells)
6) Activated APC mimics attenuated virus vibes; but fires off CD4/CD8 T-cells specific to PAP (cancer specific antigen)
7) Prostate tumour cell is murked by T-cells = cell lysis
