Biologics I Flashcards
(39 cards)
1
Q
What are the importance of biologics for the drug industry?
A
- 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
2
Q
What are the comparisons drawn between biologics and small drug molecules?
A
- 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)
3
Q
What are the different types of biologics available on the market?
A
- 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)
4
Q
What are the various steps of the bioprocessing of biologics?
A
- Immunisation
- Preparation of Myeloma Cells
- Fusion
- Clone Screening and Picking
- Functional Characterisation
- Scale Up and Wean
- Expansion
5
Q
What does immunisation involve?
A
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
6
Q
What is a hybridoma?
A
the spleeoncyte fused to a myeloma cell (cell growth) in order to produce the antibodies
7
Q
What does fusion involve?
A
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
8
Q
What are the issues with mouse antibodies?
A
- 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
9
Q
What are the different types of recombinant engineering of antibodies?
A
- 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
10
Q
How to antibodies work, what are their therapeutic functions?
A
- Complement Dependent Cytotoxicity
- Conjugates
- Apoptosis Induction
- Receptor/Ligand Blockage
- Antibody Dependent Cell-Mediated Toxicity
11
Q
What is complement dependent cytotoxicity?
A
- antibody binds to the antigen of a cell
- complement molecules attach to the antigen to then induce cytotoxicity
12
Q
What is conjugation?
A
- antibody binds to the antigen of a cell
- has attached to it toxins, cytokines to kill the cell
13
Q
What is apoptosis induction?
A
- where the antibody binds to a specific receptor on a cell to induce apoptosis
14
Q
What is receptor/ligand blockage?
A
- cell blocking
- where the antibody blocks the reception site so that ligands cannot binds and exert their effect
15
Q
What is ADCC, antibody dependent cell-mediate cytotoxicity?
A
- antibody binds to the antigen of a cell
- uses the Fc receptors to bind to an NK/Neutrophil
- this then induces the cytotoxicity
16
Q
What is the structure of an antibody?
A
- two chains, in a Y formation
- Fab Region (at the top of the Y)
- Glycan Receptor
- Fc Region (at the bottom of the Y)
17
Q
What is the purpose of the Fab region of an antibody?
A
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
18
Q
What is the purpose of the glycan receptor of an antibody?
A
- this its where chains of sugars can bind which can influence the clearance/distribution of the antibody
19
Q
What is the purpose of the Fc region
A
- binds to Fc receptors on other cells of the body (NK/Neutrophils/Monocytes/Endothelial Cells) to either induce killing or be recycled
20
Q
How are mAbs absorbed?
A
- 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)
21
Q
How are mAbs eliminated?
A
- 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)
22
Q
What is the main aspect that affects PK/PD of mAbs?
A
unique to the biology of the antigen you are targeting, the pharmacokinetics/dynamics depend on the antigen
23
Q
What is the main aspect that gives mAbs a long half-life?
A
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
24
Q
What receptor is responsible for recycling albumin and IgG?
A
- Neonatal Receptor/Brambell Receptor (FcRn)
- recycles both albumin and IgG because they have an Fc region
25
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
26
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
27
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
28
What are the half lives of the various IgG types?
1, 2, 4 - half life of 21 days
| 3 - half life of 7 days
29
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
30
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
31
How does charge and isoelectric points (PI) affect a mAb's pharmacokinetics?
- surfaces of cells are negatively charged
- having a charged Fab region may result in off-target binding
- increasing the PI will mean the mAb is constantly charged and will therefore NOT be affected by the changes of pH in the endosomes and therefore not bind to the FcRn in order to be recycled
- lower clearance of a mAb if it has a lower isoelectric point
32
What is the isoelectric point?
the pH at which a molecule carries NO charge
33
What does the formation of ADA's (antidrug-antibody) cause?
- these are antibodies that are produced in response to the administration of the drug (the mAb)
- they will bind to the drug and form immune complexes
- this may cause hypersensitivity responses
immunogenicity issues
graphs:
- if there is some level of ADA present in the blood, and then the level declines - due to the fact that it is then binding to the drug to form complexes for it to be cleared and results in reduced levels of the drug being unbound to the ADA
34
How do ADA's affect the mAb?
Neutralising - binds to epitopes on the mAb that are needed for the activity
Non-neutralising - binds to epitopes on the mAb that are not needed for the activity
35
Why does immunogenicity to a mAb vary?
- varies across species (mouse - human)
- different human fraction of the mAb (chimeric/humanised)
- different types of mAb
- hard to predict what will happen in humans (not reflected in what happens in mice)
there is no evidence to say that fully humanised mAbs will cause less immunogenic responses than partially humanised mAbs
36
What is the distribution of mAbs?
- limited to the blood and interstitial spaces
| - partitioning to tissues is only 5 - 15%
37
What are the newer (novel) formats of antibodies?
- antibody fragments (parts of the mAb)
- fusion protein
- ADCs
- bispecific antibodies (where there are 2 binding sites, making it more specific as a reaction will only occur when the two are occupied)
- multispecific antibodies (same as above but with more than 2 binding sites)
NOVEL FORMATS ARE MORE COMPLICATED THAN A MAB
- decrease in the conformational stability and decreased solubility because they are so synthetic
38
Antibody Drug Conjugates
- mAb used in order to delivery drugs (chemotherapy, immunotoxins, cytokines, radioisotopes)
- cleavable linker in either Lys or Cys residues to release the drug
39
What is Kd?
dissociation constant of the mAb to the FcRn receptors in the endosomes of cells
- concentration of the complex / concentration of the unbound molecules
- higher the Kd = the lower the affinity
