lecture 24: biological therapies for respiratory disease

Question 1

What is the definition of a biological therapy?

Answer 1

• biological therapy involves the use of living organisms, substances derived from living organisms, or laboratory-produced versions of such substances to treat disease
• some biological therapies for cancer use vaccines or bacteria to stimulate the body’s immune system to act against cancer cells
• these types of biological therapy, which are sometimes referred to collectively as “immunotherapy” or “biological response modifier therapy”, do not target cancer cells directly
• other biological therapies, such as antiboides or segments of genetic material (RNA or DNA), do not target cancer cells directly
• biological therapies that interfere with specific molecules involved in tumour growth and progression are also referred to as targeted therapies
• all pharmaceutically-based therapy other than small molecules
• broad definition

Question 2

How are nucleotide sequences targeted?

Answer 2

• anti-sense oligonucleotides
  
  • marketed therapies for CMV (cytomegalovirus) and ApoB (Parenteral administration)
• siRNA/shRNA -constructs
  
  • preclinical stage focused on packaging and delivery
  • selectivity can be very high
• (bio)synthetic exosomes - containing siRNA
• fomivirsen
• phosphates and charged elements

Question 3

What is the potential use of exosomes?

Answer 3

1. exosome production
2. ex vivo exosome modification
3. exosome delivery

• pathway for potenitial delivery of siRNA
• exosomes are produced by invagination of the plasma membrane
• in this process there are a number of transmembrane proteins that are are captured in these vesicles also some packaged contents
• can then be exocytosed
• perhaps developed as a means of communication between cells
• 30 to 200nm
• regularly transfer across vascular endothelium
• also thought to be able to cross respiratory barrier
• could be delivered systemically by inhalation
• factory producing exosomes ex vivo
• targeted to plasma membrane
• biosynthetic
• prospect for future delivery of nucleotide particles

Question 4

What is monoclonal antibody technology?

Answer 4

• curiosity-driven research
  
  • generation of variability?
  • basel institute facilitated free enquiry
• have had a huge impact on the pharmaceutical industry
• about 40% of all new registrations of new therapeutic agents with the FDA are of a biologic nature - vast majority related to mABs
• mouse immunised with an antigen
• take b cells
• fuse with myeloma cells to form hybridoma
• development of monoclonal antibody
• discovery took place in 1974/5
• first reached clinic 1986
• 1990 - variable binding regions
• humanised Mab
• progressive humanisation has improved efficacy and capacity to be used chronically
• use of Fab fragments and Fc fusion proteins are later developments
• confer longer half life

Question 5

What are novel protein scaffold technologies?

Answer 5

• anti-calin complexed to digitoxigenin (analogues of lipocalin)
• designed ankyrin repeat protein IN (DarpIN) bound to aminoglycoside phosphotransferase
• adnectin complexed to IL-23 (fragment of Fn)
• avimer scaffol
• VHH nanobody complexed to lysozyme (camelid)
• bivalent diabody Bispeficic T cell engager (Bite)-like structure → one end binds T cell receptor, other targets tumour cells
• whole IgG1 (anti HIV)
• whole IgG1 showing variable or antigen-binding domains
• non Ig scaffold
  
  • anticalin → lipocalins, 160-180 AA, 16 AA randomised n/4 loops, 0 to 1 S-S bridge
  • DARPin → AR proteins, [67 +33n], 7 AA on each n-repeat, oligomer, no S-S bridge
  • AdNectin → 10th FN3 (fibronectin, 94 AA, 3 loops, 15 AA randomization, no S-S bridge
  • Avimer → domain A/LDL receptor, [~35AA]n, 4 loops, 3-S-S bridges + Ca2+ ion, oligomer
• Ig based scaffold
  
  • nanobody → VHH (camelid Ig), ~100 AA, 3CDRs + FRs
  • dAb → VH or VL antibody domain, 100-130 AA, 3CDRs + FRs
  • BiTE → bispecific diabody, ~55kDa, 2 natural VHs and VLs and optimised linker
  • TandAb → dimerized, bispecific diabody, ~110kDa, spontaneous dimerization of bispecific diabody → 4 x VHs and 4 x VLs
• typically have shorter half lives than whole immunoglobulin
• but might have properties that allow them to be used in target tissues where Igs have trouble penetrating
• used by inhalation
  
  • energy of nebulisation does not destroy these smaller molecules where it might destroy Igs

Question 6

What are recombinant proteins?

Answer 6

• GM-CSF autoantibodies and neutrophil dysfunction in pulmonary alveolar proteinosis
  
  • impact of lack of GM-CSF parallels the impact of pulmonary alveolar proteinosis in regards to neutrophul dysfunction
  • identifed autoantibodies
  • presence of autoantibodies can mop up exogenous GM-CSF and flatten their expected up regulation of proteins
• pulmonary alveolar proteinosis
  
  • rare autoimmune disease causing deficiency of GM-CSF activity
  • reduced macrophage/neutrophil clearance of surfactant protein leads to fluid accumulation and impaired gas exchange
  • treated by lung lavage
  • now may be treated with recombinant GM-CSF
  • inhaled version being trialled

Question 7

What are cell therapies for respiratory disease?

Answer 7

• mesenchymal stem cells - repair and anti-inflammatory action
  
  • sources of IL-10, TGFBeta1, IL-6
• vaccines/adjuvants
  
  • exacerbation of asthma and COPD
  • respiratory syncytial virus
  • rhinovirus
  • influenza
  • challenges: logistics, numbers of serotypes
• viruses/immunostimulants
  
  • pseudo-typed lentivirus in preclinical phase for cystic fibrosis
  • e.g. use of influenza M2 coat protein to facilitate uptake

Question 8

What is cytokine targeting in asthma?

Answer 8

• serum periostin used as a biomarker of Il-13 activity on epithelium
• interest of Pharma/Biotech is in severe asthma
• not fully controlled by LABA/ICS, +/- CysLT1 blocker
• therapies should have either a “steroid-sparing” profile or activity that complements steroid actions
• Lebrikizumab directed against IL-13
• Fab fragments co-crystallised with Il-13
• mutations changing single amino acids - influence on binding
• overlay shows IL-13 unable to bind IL-4Ra and Fab at same time
• studies useful target has multiple binding partners

Question 9

What was seen with cytokine targeting?

Answer 9

• at baseline:
  
  • FEV1 65% predicted (high periostin 73%) on 580µg ICS/80% LABA
  • high periostin +12% *
  • low periostin + 6% NS
• treat: lebrikizumab
• 250mg sc/month x 6
• no effect on mild exacerbations, symptoms, rescue therapy use
• possible effect on severe exacerbations (high periostin)
• asthma control not complete
• decred FeNO
• increased blood eosinophils (fewer in airways?)
• asthma control not complete, but probably improved
• trial design sought to establish an effect on FEV1 so primary endpoint met
• treatment delivered on top of standard care LABA/ICS +/- CysLT1 blocker

Question 10

What is dupilumab?

Answer 10

• blocking IL-4 receptor alpha subunit blocks both IL-4 and IL-13 binding
• we enrolled patients with persistent, moderate-to-severe asthma and a blood eosinophil count of at least 300 cells per microliter or a sputum eosiniphil level of at least 3% who used medium-dose to high-dose inhaled glucocorticosteroids plus LABAs
• we administered dupilumab (300mg) or placebo subcutaneously once weekly
• patients were instructed to discontinue LABAs at week 4 and to taper and discontinue inhaled glucocorticoids during weeks 6 through 9
• patients received the study drug for 12 weeks or until a protocol-defined asthma exacerbation occured
• the primary endpoint was the occurence of an asthma exacerbation; secondary end points included a range of measures of asthma control
• effects on various type 2 helper T-cell (TH2)-associated biomarkers and safety and tolerability were also evaluated
• 87% reduction
• increased time to exacerbation

Question 11

summary of cytokine targeting in asthma

Answer 11

• scientific proof of importance of IL-13/IL-4 signalling - ✔✔✔
• normalise airway function in mild to moderate asthma ✖
• some patients will be fully controlled ✔
  
  • new phenotypes defined by therapeutic response
• advantages
  
  • selecitivity, potentially lesser burdern of adverse effects
  • shorter development time, more predictable development success
  • long T1/2 (long dose interval)
• disadvantages
  
  • high cost of production (cost of goods)
  • parenteral administration
  • many targets are intracellular