Chem 195

1
Chem 195

• Lecture 10
• Discovery and Development of Recombinant DNA
  Based Protein Drugs

2
Exam Summary

• Distribution of questions answered
• Everyone answered questions 2 and 5
• Only 6 people answered question 4 and only 4
  answered question 6
• So I did a good job teaching patents and the
  difference between in vitro and in vivo activity
• And a bad job teaching basic enzymology

3
Project Proposal

• 1 page summary - Why And How
• Rationale, mechanism/pathway, and target you
  would like to attack
• Unmet medical need, market
• Can work in pairs
• Other components as listed previously
• List presently defined projects

4
Why Protein Drugs?

• Agonists and Antagonists
• Costs and Technology
• Catalysts and Hormones vs. Antibodies
• Replacement therapy
• Insulin - human vs. porcine
• Growth Hormone - recombinant vs. cadavers
• Next generation therapeutic
• Tissue plasminogen activator vs. streptokinase
• Recombinant Factor VIIIC vs. F8 concentrate

5
Proteins as Drugs

• Pre-existing examples
• Insulin - from pig pancreas
• Plasma products - clotting factors, albumin
• Streptokinase - clot lysis - from bacteria
• Issues
• non-human source - antibodies?
• Availability - would there be shortages?

6
Protein Drugs

• New technology - 1970s
• Recombinant DNA plasmid expression vectors
• Contain promoter for transcription, signals for
  the initiation of translation, the human gene of
  interest, stop signals - so-called expression
  cassette
• Also, need origin of replication and selectable
  marker (usually antibiotic resistance)
• Initially bacterial expression but subsequently
  yeast, mammalian cells, and baculovirus infected
  insect cells

7
Protein Drugs

• Biosynthesis and Regulation of Insulin
• Pre-Pro-insulin
• Signal-Peptide-B-C-A -gt BA
• What is the pro-insulin convertase?
• How to get from B-C-A to BA?
• Dibasic specific proteases
• carboxypeptidase
• How to get 2 chains?

8
Insulins
9
Protein Drugs

• Genentech solution - E.coli expression
• Make two chains separately and put together
• Issues its tricky to get the right disulfide
  bonds
• Thermodynamics and kinetics
• Second generation - make proinsulin (Lilly)

10
Protein Drugs

• Chiron solution - yeast secretion
• Yeast alpha-mating factor processing
• Similarities to insulin (dibasic processing
  sites)
• Make 1 chain with a mini-C peptide
• Alternate processing enzymes (kex-2)
• Clue to furins - PI convertase family
• Steiner on proinsulin convertase

11
Protein Drugs

• Growth Hormone
• Secretion from E.coli
• Processing to eliminate N-terminal Methionine
• Identification of Methionine amino-peptidase
  (Cetus)
• Previously obtained from cadavers - issue with
  Creutzfeld-Jacobs disease (slow virus -
  resembling BSE)

12
Protein Drugs

• Hemophilia A
• X-linked trait
• Most common genetic disease
• Large fraction arises spontaneously but some
  inherited
• Known to be deficiency of FVIII but as of 1983
  the molecular structure of FVIII unknown
• Site of synthesis also unknown

13
Protein Drugs

• FVIIIC
• Monoclonal antibodies used to purify protein
  using a clotting assay in F8 deficient plasma as
  a detection scheme
• Protein sequencing followed by genomic library
  screening with degenerate probes using a 4X
  genome library
• Alternative approach to porcine FVIII

14
Protein Drugs

• FVIIIC
• Three way race, Genentech, Genetics Institute,
  Chiron
• Papers published in 1984/5
• Very complex molecule and very large gene (186
  kB, 9 kB cDNA)
• Proteolytic processing to activate (thrombin) and
  inactivate (protein C)

15
Protein Drugs

• Previous FVIII - pooled from many blood donors
• Led to a whole generation of hemophiliacs
  becoming HIV and HCV positive
• Scandals on lack of blood screening tests,
  especially in France

16
Protein Drugs

• New/Old Proteins with New Indications
• Interferon alpha and beta
• Interferon-alpha - originally described as
  antiviral substance
• First approval for treatment of hairy cell
  leukemia
• Later became major treatment for Hepatitis C
  (although it doesnt work that well)
• Interferon-beta used exclusively for treatment of
  multiple sclerosis
• IFN-alpha and beta work through the same receptor
  systems - still not clear why they work
  differently for different diseases.

17
Protein Drugs

• Two most profitable protein products
• Erythropoeitin - red blood cells
• Granulocyte Colony Stimulating Factor - immune
  cells
• Both stimulate blood cell production
• Used as adjunct to cancer chemotherapy
• EPO also used by athletes to promote oxygen
  capacity (illegal - blood doping)

18
Protein Products

• Deficiencies in First Generation Protein Products
• All agonists - all injectables
• How to improve properties?
• Increase potency
• Increase pharmacokinetics
• Decrease degradation

19
Protein Drugs - the Next Generation

• Next Generation Protein Therapeutic Agonists
• Aranesp - mutant EPO
• Glycosylation sites and receptor binding site are
  distinct
• Increased glycosylation leads to longer half-life
• Increased sialic acid glycosylation from 3 sites
  to 5
• Enabled Amgen to get a molecule which they didnt
  have to share with JJ

20
Protein Drugs - the Next Generation

• Next Generation Protein Therapeutic Agonists
• PEG-IntronA/Pegasys - pegylated IFN-alpha
• Pegylation increases half-life
• Thus, decreases dosing schedule
• Next generation tPA - TNK tPA
• 4 amino acid changes
• Moved glycosylation site by 2 mutations (T103N,
  N117Q) - increased in vivo half-life
• Mutated 4 residues KHRR 296-299 to AAAA
• 100 fold resistance to PAI-1 and improved fibrin
  selectivity

21
Protein Drugs

• Summary
• New technology (cloning and recombinant DNA based
  expression) enabled production of human proteins
• Agonists which replaced old products or were new
  ones
• Second generation products via protein
  engineering led to molecules with improved
  properties, especially in vivo half-life