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Flashcards in Biologics I Deck (39)
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What are the importance of biologics for the drug industry?

- revolutionary treatment
- more difficult to manufacture (most mAbs made from immunoglobulins, 150,000 da - size/properties)
- 37% of the drug market is made up of biologics
- blockbuster drugs
- strongest growth for pharmaceutical companies
- quicker to get a biologic to the market than a small drug molecule
- lower failure rates than small drug molecules because it's not a traditional agonist/antagonist
- smaller companies successful with biologics
- biologics used to replace diseased tissue functionality
- biologics have highly specific binding, only small changes to the CDR region of the antibody needed


What are the comparisons drawn between biologics and small drug molecules?

- versatility - biologics replace AND modify diseased tissue
- specificity - small drug molecules tend to bind to other target sites that can cause toxicity
- duration of actions - not an issue with mAb because their half life is extremely long anyway
- structures - mAb structures largely stay the same (IgG) its just the CDR region that is modified to target different things
- biologics product immunogenic effects (due to the complexity of the molecule aggregating and causing an immunoresponse)


What are the different types of biologics available on the market?

- peptides
- protein fragments
- mAbs (the MAJOR biologic on the market)
- ADCs (Antibody Drug Conjugate), mAb with a cytotoxic drug on
- viruses
- vaccines
- new modalities (e.g. lipid nanoparticles)


What are the various steps of the bioprocessing of biologics?

- Immunisation
- Preparation of Myeloma Cells
- Fusion
- Clone Screening and Picking
- Functional Characterisation
- Scale Up and Wean
- Expansion


What does immunisation involve?

isolate the spleenocytes (the white blood cell):
- mice injected with an antigen
- mice produce the specific antibody for that antigen
- isolate the antigen for hybridoma production


What is a hybridoma?

the spleeoncyte fused to a myeloma cell (cell growth) in order to produce the antibodies


What does fusion involve?

the formation of the hybridoma from spleenocytes and myeloma cells

- use PEG in order to change the osmolarity and draw in water to break cell membranes


What are the issues with mouse antibodies?

- they produce immunogenic reactions
- their Fc region is not recycled in humans in order to prolong the half life, so is rapidly cleared from the body
- recognised as a foreign body


What are the different types of recombinant engineering of antibodies?

- chimeric (human except for the Fv region which is the mouse, where the antigen binds)
- humanised (human except for the CDR loops)
- fully human antibodies produced vi a mouse


How to antibodies work, what are their therapeutic functions?

- Complement Dependent Cytotoxicity
- Conjugates
- Apoptosis Induction
- Receptor/Ligand Blockage
- Antibody Dependent Cell-Mediated Toxicity


What is complement dependent cytotoxicity?

- antibody binds to the antigen of a cell
- complement molecules attach to the antigen to then induce cytotoxicity


What is conjugation?

- antibody binds to the antigen of a cell
- has attached to it toxins, cytokines to kill the cell


What is apoptosis induction?

- where the antibody binds to a specific receptor on a cell to induce apoptosis


What is receptor/ligand blockage?

- cell blocking
- where the antibody blocks the reception site so that ligands cannot binds and exert their effect


What is ADCC, antibody dependent cell-mediate cytotoxicity?

- antibody binds to the antigen of a cell
- uses the Fc receptors to bind to an NK/Neutrophil
- this then induces the cytotoxicity


What is the structure of an antibody?

- two chains, in a Y formation
- Fab Region (at the top of the Y)
- Glycan Receptor
- Fc Region (at the bottom of the Y)


What is the purpose of the Fab region of an antibody?

Fab - antibody binding region
- there is a constant domain and a variable domain - this is where the Fv is
- within the Fv is where the binding loops are (CDRs) - the variable part you change of the antibody
- region has many charges and is affected by the isoelectric points of molecules, causing off-target binding


What is the purpose of the glycan receptor of an antibody?

- this its where chains of sugars can bind which can influence the clearance/distribution of the antibody


What is the purpose of the Fc region

- binds to Fc receptors on other cells of the body (NK/Neutrophils/Monocytes/Endothelial Cells) to either induce killing or be recycled


How are mAbs absorbed?

- administer via IV, SC or IM injections
- absorption for SC is variable (20 - 95%) and is facilitated by the lymph system
- rate of absorption is slow for SC/IM injections (max plasma concentration between 1 - 8 days)


How are mAbs eliminated?

- proteolytic catabolism (breaking molecules down to smaller molecules) - broken down into amino acids in lysosomes
- target mediated clearance (where the antigen binds to its target, and then destroyed - the complex is then cleared from the body through the reticulo-endothelial system (RES))
- Fc gamma receptor mediated clearance
- Non-Specific Pinocytosis (type of endocytosis)


What is the main aspect that affects PK/PD of mAbs?

unique to the biology of the antigen you are targeting, the pharmacokinetics/dynamics depend on the antigen


What is the main aspect that gives mAbs a long half-life?

mAb recycling
- is one of the Critical Quality Attributes of a mAb
- want a mAb to have good recycling (the correct affinity for the FcRn receptor) in order to have a longer half life


What receptor is responsible for recycling albumin and IgG?

- Neonatal Receptor/Brambell Receptor (FcRn)
- recycles both albumin and IgG because they have an Fc region


How does mAb recycling occur?

- Fc region of the IgG (or albumin) binds to the FcRn on a monocyte/endothelial cell through endocytic mechanisms
- transported in an endocytic vesicle away from the cell surface (pH = 7 - 7.4) into the cell
- the endosome then becomes acidified (pH = 6)
- Fc region has high affinity at a more acidic pH, so will bind to the FcRn receptors within the endosome
- the endosome then moves back up to the cell surface, where the pH then returns to normal, and the IgG then has a lower affinity and is released back into the blood (RECIRCULATED)
- whatever is not bound to the FcRn receptors within the endosome is then sorted into lysosomes to be degraded


What factors can influence the mAb recycling?

- Fc region, modify the amino acid sequence to get the correct affinity
- Fv region has lots of charges associated and may result in a stronger affinity, or weaker affinity, changing the half life


What are the implications of antigen-antibody complexes being recycled through the FcRn pathway?

- the antigen (as well as the antibody) may also bind to the FcRn receptor
- this results in the recycling of the antigen, forming an accumulation in the blood
- this is not ideal, you would want them to be degraded in the lysosomes to be removed from the blood


What are the half lives of the various IgG types?

1, 2, 4 - half life of 21 days
3 - half life of 7 days


What is an Fc Fusion Protein?

an Fc region linked to a protein/peptide
- or an albumin fusion protein (both recycled by the FcRn pathway to extend the half life of the protein you're using)

- the addition of a target binding structure (protein/peptide) may alter the Fc domain or statically hinder it so that there is less binding to the FcRn and the half life is lower


How does glycosylation impact on a mAb's pharmacokinetics?

- all IgGs are glycoslyated (all possess a sugar chain)
- glycosylation is not required for a longer half-life for mAbs
- BUT it IS important however for an Fc Fusion Protein, their shorter half-life is because of:
1. lower binding affinity for the FcRn (not recycled as well)
2. glycan mediated disposition
3. receptor mediated disposition