[Seminar] Drug Delivery Stratagies in Immunology Notes

Immunology and pharmaceutics meet here, looking at two primary topics: Transplantation and Cancer.

Transplantation - need to lower immune response to tolerate organ
Cancer - The immunosome is important in checking against cancer cells.  This can be observed when the loss of functional t cells in aids leads to more cancer

Focus on Cancer:
Look at using oligonucleotides, polymeric particles, antibodies, and injectable films

A tumor starts out as uncontrolled cell growth, but once it picks up a mutation that allows for a phenotype that moves to other tissues, it is malignant.

Most standard therapy- non-specific actions- kill cells that are proliferating.  Very toxic.

20 years ago- idea- exploit immune system to kill cancer cells when at low numbers.
Maybe activate to a greater extent (like a vaccine, but not preventive so much as curative)
Called "cancer vaccines" (misnomer)
Results are really disappointing.  Tumors survive though increased immune response is detected.
Disconnect here.
Last 8-10 years- deconstruct to find problem.

Ideas:
Immunology of cancer.  Active # of helper t cells responding to tumor.  Tumor is not a passive bunch of cells waiting to die.  Especially (epithelial cancers), turn t-cell to regulatory-t cells for their own use and protection.  Helper- INF-GAMMA Regulatory- FOXP3

Turn back to anti-cancer phenotype?
FOXP3 If you shut down, turn back into helper t cells.
T cells already localized, now.  This is very specific to hit the tumor.

New drugs in the post-genomic era:

• Monoclonal antibodies Fc fusion proteins
• Antisense oligonucleotides
• Small interference RNA siRNA

20% drugs now are biologics, not drug discovery

Trying to get a molecule to be a drug- delivery is rate limiting step.
Ideal drug therapy- optimal spatiotemporal pharmacokinetics and pharmacodynamics
Get the right drug to the right place at the right time.
Biologics inherent instability is major barrier.  Need hours-days. Less than that not useful.  Get to tumor, but also inside it!
Use endosome for endocyosis, macropinocytosis, etc...
Needs to get outside the endosome.

Strategy
Make RNA molecules compact and neutralize charge.
Synthetic polymers for nucleic acid delivery- good in dish- not in vivo, because proteins in  living system compete against the complex, which dissociates back to original product.
Polyester-polycation hybrid carriers
Complex to a particle, can get substantial advantage in terms of stability.
Use with siRNA to shut down FoxP3- this group did.
How is it in a dish? T-cells will pick up these particles (important because t-cells don't pick up that much like macrophages or dendrocites)

Results
Tumor in mouse, injection, looking for delay in tumor growth.
Decreased Foxp3, but didn't much to cancer growth.  Ahh! Disaster.

Considerations for Improvement
Maybe not getting to right place?
Inject directly into the tumor.
Need to ensure endosome escape is possible.   Histadines as buffer.  More water in with buffers, more burst, payload release- need more histadine buffer.
Maybe use different types siRNA, maybe use more, etc, maybe didn't think about getting it at the right time.
Particles stay around for some time- a day or less.  T-cells move around.  Most other cells stay in one place.  Tumors change t-cells, but then they come and go throuh lymph system.  Dont spend a whole lot of time in the tumor. 
If we can trap t-cells- better chance to change.
Reverse drug delivery.

Solution Applied
Platform
Probability for connection with RNA loaded particle, and target t-cell low with moving target.   Need a trap!
Ab to surface marker on t-cell.  Anti-H6 ab h6 domain.
What is both injectable and can be used as a trap?
20 years ago, distinct --++ charge zuoin, a putative z-dna binding protein in saccharomyces cerevisiae
EAK: a stimuli-response gelling material.
Soluble in water.  Sodium and potassium- stack beta sheetss-fibers-crosslinnked network-membrane-like structure.
Could be platform for trap. 

Specificity
Problem is how to get t-cells (only) to stick.
Functionalize EAK film with his-tag.
This changes conformation from bsheet to ahelix, no sheet anymore.  Mix original and his tag at 4:1- can get his tag into cross linked network.
Can line up when packing sheets such that his tag sticks out on the end.
Are these two peptides really integrated or non-specifically adsorbed- will leave first chance it gets.
Evidence of integration Chem signature and conformational change.  Peroxidase with Ni (binds his), proof of accessible his tag.

Results AgainCan last at least 7 days.
Add antiH6-IgG-something-something- IgG
Can clustered ab bind antigens?
Yes,  remain functional - paper
Does it work in a living system?
An injectable system to concentrate antibodies in small murine footpad tumors. 
By 72 hours its gone normally, but still high in animal with this system.
Difficult because tumors have high interstitial pressure.
Using these hooked up to FAP fluorogen activating signal.  Flouescent once they get into binding groove.

These are notes from a talk given by Dr. Wilson S Meng on April 20, 2012 at Duquesne University, and my own thoughts, comments, and connections while listening. Some notes may be inaccurate and/or misspelled.