Antibody engineering

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  • Created by: Biokid373
  • Created on: 22-05-22 15:36
How can we improve engineered antibodies?
Subtype switching, Species switching, reformatting, Isotype switching
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Why are bispecific antibodies an attractive format for therapeutic antibodies?
They can target multiple antigens at the same time
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How could you mutate Variable domain sequences?
Using display libraries and/or rational design
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Why might you want to mutate V domain sequences in a therapeutic antibody?
To alter binding affinity or specificity
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How would you mutate Fc sequence of antibodies?
Using display libraries or rationale design and by selecting IgG isotype
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Why would you want to mutate Fc sequence of antibodies
to alter antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and to alter half-life
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What would happen if an antibody fragment lacking an Fc was used?
Decrease half life, CDC, ADCC and ADCP
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Why might you alter the glycosylation of an antibody
to modify the FcgR and complement interactions
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What would aglycosylation of an antibody result in?
decreased ADCC, ADCP and CDC
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What would bisecting N-acetylglucosamine in the antibody result in?
Increased ADCC
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What would non-fucosylation of an antibody result in?
Increased ADCC
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is it more common to alter effector function or specificity?
effector function
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Why would you modify the effector function
modulate interaction with Fc receptors
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What is CDC
Complement dependent cytotoxicity
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How are ADDC/ADCP/CDC driven?
Interaction with Ch2 domain of effector function
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What domain is responsible for determining the half-life of antibodies?
Ch3
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What modulates half life of antibodies?
ability to bind to FcRn
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Why is glycosylation important in antibodies?
Important for interaction with receptors and stability
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by introducing a mutation Gly236Ala what happens?
Increase affinity for FcgRIIA - an activating receptor
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Why would you want to mutate the Ch2 domain of an antibody
To alter FcR binding to increase activation of effector cells by antibody
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What is the benefit of non-fucosylated variants of rituximab and trastuzumab?
Increased affinity for FcgRiiA and 100 fold more potent ADCC activity
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Why would you mutate the CH3 domain of an antibody?
to alter interaction with FcRn and modify half-life
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Why might you want to modify an antibody to have a less potent effector function?
if there are severe side effects associated with treatment of antibody
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what is OKT3 used for?
Depleting T cells
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What are the cons of using OKT3
Severe side effects
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What molecule does OKT3 target?
CD3
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How have humanised CD3-specific antibodies been improved by Fc engineering?
attenuate FcgR interactions resulting in less inflammatory responses, overcome mitogenic activity, aglycosylation
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Name two examples of therapeutic antibodies where effector functions are not desired
Natalizumab and Eculizumab
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What is Natalizumab used to treat?
MS
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What subclass lacks effector function?
IgG4
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Why does the IgG4 subclass not have effector function?
Avoids interaction with FcR so ADCC does not occur
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How does Natalizumab work?
Binds to integrin and blocks effector function of pathogenic autoimmune T cells
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What is interesting about IgG4?
It can exchange Fab arms in vivo to become functionally monovalent
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Mutations in the CH3 domain and a flexible hinge can be used for...
antibody engineering of bispecific antibodies
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what is Eculizumab?
antibody with hybrid IgG2-IgG4 constant region
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What is the Ch1 domain and hinge region of Eclizumab from?
Human IgG2
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What are the CH2 and CH3 domains of Eclizumab from?
Human IgG4
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What does Eclizumab lack?
The ability to bind FcgR and activate complement
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What is noteworthy about the IgG2 domain?
It lacks ability to bind FcgR
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What domain inhibits the activation of the complement pathway?
IgG4
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What is the terminal half life of Fab fragments
20 minutes
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Why might you want to alter the pharmacokinetic profile of an antibody for use in the clinic?
To increase half-life
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What mutation was engineered into Bevacizumab to extend serum half life by 3-4 fold in experimental models?
Met428leu:Asn434Ser
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What are FcRn-blocking antibodies potentially useful for?
reducing pathogenic IgG levels in antibody mediated autoimmune diseases or toxic effects of mABs by accelerating half-life
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why could controlled Fab arm exchange be beneficial in antibody engineering?
To make duobodies and bispecific antibdoies
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What are the benefits of bispecific antibodies
Can bind antigen on target cell, bind molecule on T cell and Fc region can bind accessory cell
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How many combinations are possible in bispecific antibodies when you combine 2 antibodies together?
16
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Quadroma - how are bispecific antibodies generated?
combining light and heavy chains of two different monoclonal antibodies
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what is the knob into hole method of generating bispecific antibodies
Specific pairing of heavy chains to avoid heavy chain mispairing
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With knob into hole method of specific pairing of heavy chains, how many possible products are formed?
4
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How many possible combinations are there with the quadroma approach?
10
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What problem did the crossMAb method of generating bispecific antibodies solve
the problem of light chain mispairing
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Name a key barrier preventing antibody being able to engage with target
Blood brain barrier
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Why is treatment of neurodegenerative diseases with therapeutic antibodies difficult?
Blood brain barrier is designed to not let through large proteins
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What is the amyloid hypothesis?
Increased deposition of amyloids leads to tau phosphorylation and inflammation leading to synaptic loss and neurodegeneration
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Why is it hard to diagnose and treat Alzheimer's
Lack of biomarkers
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what is Solanezumab?
a humanised IgG1 mAb directed against the mid domain of the amyloid beta peptide
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what form of Amyloid beta does Solanezumab recognise
Soluble monomeric
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Mechanism of action of Solanezumab
removes small soluble species of amyloidbeta that are directly toxic to synaptic function
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drawback of Solanezumab
no acucmulation over time and depends on constant high exposure as it binds abundant soluble form of amyloidbeta
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What is Aducanumab?
humanized IgG1 mAb directed against the N-terminal domain of amyloidbeta peptide
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Mechanism of action of Aducanumab
Recognises aggregated forms of Amyloid beta peptide
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How does the mechanism of Aducanumab treat alzheimers
Removes plaques and prevents their formation
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Drawback of Aducanumab
Induces ARIA
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What is Bapineuzumab?
Humanised IgG1 mAb directed against the N-terminal domain of alpha beta peptide
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What form does Bapineuzumab recognise amyloidbeta peptide in?
aggregated and soluble forms of Ab peptide
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How does Bapineuzumab treat alzheimers?
removes plaque or prevents formation
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drawback of Bapineuzumab
Induces ARIA
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What is ARIA
Antibody related imaging adverse events
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What does ARIA cause?
inflammatory lesions around the blood vessel wall in the meninges
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How was aducanumab generated?
reverse engineering from amyloid positive patient who didnt have Alzheimers
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What was found to reduce amyloid plaque burden in AD transgenic mouse models?
active immunisation against full length amyloidbeta in combination with an adjuvant
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What animals with amyloid plaques did not respond to bapineuzumab antibodies
Those with a diet reducing phytomineB causing inflammation around blood vessels
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How can you get an antibody across the blood brain barrier?
Make a bispecific antibody
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how can a brain shuttle be used to transport an antibody against amyloid beta across the blood brain barrier
fab fragment interacts with TfR on endothelial cells of BBB used to transport ions in the brain and piggyback on that mechanism
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What happens when tfr binds antibody?
tfr shields fc interaction with inflammatory cell causing no effector function in the periphery
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When antibody binds amyloid after being shuttled in via tfr what happens?
conformation of transferrin component and fc tail available for microglial fc receptors who can then phagocytose amyloid
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ApoE is another potential target for treating alzheimers - why is this?
interacts with receptors on microglial cells and is a risk factor for vascular inflammation
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How could TNF-alpha be a target for reducing progression of Alzheimers
Neutralise TNF in periphery and blocking inflammatory comorbidities
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Why might you use enzymes to modify glycosylation patterns of antibodies used to treat alzheimers?
to enhance entry to the brain
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Other cards in this set

Card 2

Front

Why are bispecific antibodies an attractive format for therapeutic antibodies?

Back

They can target multiple antigens at the same time

Card 3

Front

How could you mutate Variable domain sequences?

Back

Preview of the front of card 3

Card 4

Front

Why might you want to mutate V domain sequences in a therapeutic antibody?

Back

Preview of the front of card 4

Card 5

Front

How would you mutate Fc sequence of antibodies?

Back

Preview of the front of card 5
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