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3152 IC7-8 Proteins > Strategies to improve PK profile of protein therapeutics > Flashcards

Strategies to improve PK profile of protein therapeutics Flashcards

1
Q

What are the 3 common strategies to improve PK profile of protein therapeutics?

A
  1. Glycosylation of proteins
  2. PEGylation of proteins
  3. Increase in MW by means of fusion proteins
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2
Q

[GLYCOSYLATION]

What is glycosylation?

A

It is a PTM - enzyme reaction that adds glycans (carbohydrates) to specific amino acids in a protein

*Glycosylation pattern can vary - straight vs branched chain)

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3
Q

[GLYCOSYLATION]

How does the pattern and amount of glycosylation affect the protein drug?

A
  1. May affect activity
  • Glycans may enhance receptor binding to the protein
  1. Increase half-life of the protein
  • Increase MW, >50kDa, not renally filtered
  • Increase MW can hinder approaching proteases, poorer substrate of proteolysis
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4
Q

[GLYCOSYLATION]

Why is N-linked glycosylation used? Give an example.

A

N-linked glycosylation increases circulation half-life (by increasing MW) + modifies binding to glycoprotein receptor

E.g., Darbepoetin - addition of 2 N-linked CHO chain increases its MW and hence residence time, also hinders approaching proteases

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5
Q

[GLYCOSYLATION]

What makes glycosylation a safe and effective strategy?

A

Glycosylation already happens naturally in mammals (PTM)
Hence, compatible and safe strategy

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6
Q

[GLYCOSYLATION]

List two examples whereby glycosylation is NOT beneficial

A
  1. Naturally occuring fucosylated antibodies (N-linked glycosylated with fucose)
  • Fc domain of defucosylated Abs have greater affinity for binding to Fc receptor on effector cell, allow greater ADCC induction, increase efficacy of the antibodies
  • Can use mutant CHO cells deficient in the enzyme that adds fucose, to produce defucosylated antibodies
  1. Antibodies containing high mannose glycans
  • Found to be rapidly eliminated, short half-life (*can be good or bad)
  • Mannose and asialoglycoprotein receptors believed to be responsible for the rapid removal
  • Mannose receptor is a cell surface pattern recognition receptor (PRR) expressed on phagocytes, allow phagocytes to directly recognize structure of pathogen
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7
Q

[PEGylation]

What are the properties and structure of polyethylene glycol?

A

Amphiphilic, chemically inert polymers made up of repeating units of ethylene oxide

Generally hydrophilic, compatible with water, can increase solubility in water

Non-toxic, inert, non-immunogenic

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8
Q

[PEGylation]

What are the 2 types of PEG?

A
  1. PEG with free hydroxyl at both ends.
  2. mPEG (methoxylated PEG) with methoxylated hydroxyl at one or both ends
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9
Q

[PEGylation]

What is PEG conjugation?

A

PEG conjugation is when a reactive functional group of activated PEG is attached to sites (usually an amino acid - lysine, sulhydryl SH in cysteine, nucleophilic grps on amino acids)

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10
Q

[PEGylation]

What are the 2 types of configurations of PEG polymers and how do they affect the circulation half-life of the protein drug?

A
  1. Linear - less effective in extending half-life
  2. Branched - more effective in extending half-life, protect from proteolytic degradation
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11
Q

[PEGylation]

How does the size of the protein drug determine the effectiveness of PEGylation on extending circulation half-life?

A

Small proteins like cytokines shown to have bigger enhancement in half-life compared to large proteins

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12
Q

[PEGylation]

How does PEGylation lead to increased circulation half-life?

A
  1. Increase size of conjugated protein
  • PEG molecule MW 40-50kDa, retard glomerular filtration
  1. Decrease elimination by proteolysis
  • Protective layer on surface of protein molecules, dcr accessibility for proteolytic enzymes
  • Esp branched chain, protect from proteolytic degradation
  1. Decrease elimination by action of antibodies and activated immune cells
  • Protective layer on surface of protein molecules, dcr accessibility for antibodies and activated immune cells, protects from recognition by them
  • Also confers reduced immunogenicity and antigenicity
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13
Q

[PEGylation]

Other than increased circulation half-life what are some advantages of PEGylation?

A
  • Enhanced thermal and physical stability
  • Slow-release effect, controlled drug release
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14
Q

[PEGylation]

Should vaccines be PEGylated to improve circulation half-life?

A

No. PEGylation reduces immunogenicity and antigenicity, which is not desirable for vaccines which should evoke the immune system and get recognized by antibodies.

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15
Q

[FcRn-mediated recycling]

What is the role of FcRn?

A
  • Can trigger activation of effector cells (like other Fc receptors)
  • Known more for its critical role in regulating IgG and albumin turnover

=> IgG homeostasis, mediate IgG recycling by allowing intracellular trafficking of antibodies (allow them to escape degradation in lysosomes)
=> same for serum albumin

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16
Q

[FcRn-mediated recycling]

FcRn shares structural similarity to _______

A

MHC Class I molecule - presents to CD8+ T cells

17
Q

[FcRn-mediated recycling]

Describe the process of cellular recycling of IgG and albumin
(Recycling back into the blood from endothelial cells)

A
  1. IgG and albumin dissolved in blood (pH 7.4), taken up by endothelial cells by pinocytosis to form early endosomes
  2. Acidic endosomes containing internalized FcRn fuse with endosomes containing IgG or albumin. At pH 5-6, forms FcRn-IgG complex / FcRn-albumin complex
  3. The complex gets recycled back to the cell surface via exocytosis into the bloodstream
  4. Neutral pH of blood (pH 7.4) disfavor binding, complex dissociates. IgG/albumin released/recycled back into bloodstream. FcRn recycled and reexpressed on cell surface or internalized into acidic endosomes.
  5. Proteins not bound to FcRn which were also taken up by pinocytosis, are sorted to lysosomes for degradation

=> IgG/albumin escape degradation in lysosomes, hence longer circulation half-life

18
Q

[FcRn-mediated recycling]

Describe the process of transcytosis of IgG and albumin
(transport across intestinal epithelial cells)

A
  1. At apical side of mucosal epithelial cells, acidic pH of lumen allows binding of IgG/albumin to cell surface FcRn
  2. Receptor-mediated endocytosis occurs
  3. Endosomes fuse with basolateral side of epithelial cells (transcytosis), leads to exposure of complex to neutral pH of interstitium/bloodstream, complex dissociated and IgG/albumin is released/recycled. FcRn recycled and reexpressed on cell surface.

=> IgG/albumin escape degradation in lysosomes, hence longer circulation half-life
*IgG/albumin can travel to hepatocytes, but proteins are not substrates of CYP enzymes and do not get metabolized in the liver, hence able to enter systemic circulation

19
Q

[FcRn-mediated recycling]

How is transcytosis of IgG important in pregnant or nursing mothers?

A
  1. High expression of FcRn on cells in the placenta lining, allow maternal IgG to be transported across placenta to the fetus
  2. High expression of FcRn on intestinal epithelial cells of the fetus, allow maternal IgG to be transported via breastmilk across intestines of fetus

=> Confers passive immunity to the fetus

20
Q

[FcRn-mediated recycling]

Utilization of FcRn fusion proteins can _________

A
  • Larger protein => slower clearance, longer half life
  • Fc domain of antibody or albumin allows utilization of FcRn mediated recycling to enhance circulation half-life
21
Q

[FcRn-mediated recycling]

What is an example of Fc-fusion protein therapeutics?

What is a possible downside of this method?

A

E.g., Etanercept (anti-TNFa for RA)

Structure: Recombinant protein fused with Fc domain of IgG

  • TNF receptor (to intercept circulating TNFa)
  • Fc domain of IgG (cellular recycling, incr half-life)

Downside:

  • Fc domain can bind to Fc receptor on effector cells and trigger unwanted effector functions, e.g., ADCC, CDC, causing immune and inflammatory response
22
Q

[FcRn-mediated recycling]

Why is albumin-based therapeutics not utilized?

A

Albumin is a plasma protein containing 3 binding domains, each of which binds to small endogenous and exogenous substances such as drugs, FFAs, ions etc.

Albumin-FcRn binding requires 2 albumin binding domains

  • In vivo, albumin domain may bind to other small molecules in the interstitial fluid and plasma, unable to ensure binding and utilization of FcRn-mediated recycling

3152 IC7-8 Proteins (4 decks)

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