Antibody engineering part II

Question 1

What is the target of rituximab? And what cell population is it on?

Answer 1

CD20 is the target expressed specifically on B cells, but not memory cells.

Question 2

where does CD20 localise to upon Ab binding? Might this have benefits?

Answer 2

Upon binding CD20 pulled into lipid rafts. Could potentially induce apoptotic signalling here.

Question 3

Mechanisms by which rituximab depletes B cells.

Answer 3

1. Ab opsonisation and complement activation, MAC formation and lysis.
2. rituximab binds to Fc receptors on phagocytic cells.
3. Binds to Fc receptors on NK cells (ADCC)
4. direct apoptosis through lipid raft signalling?

Question 4

cocktail of chemotherapeutic agents rituximab can synergise with?

Answer 4

CHOP

CHOP+ rituximab + better survival but NOT cure.

Question 5

What other antibodies that target CD20 might be more effective than rituximab and why?

Answer 5

GA101, which is glycosylated to increase Fc receptor binding.

Question 6

3 general reasons why resistance might develop in rituximab treatment of patients with B cell leukaemia?

Answer 6

1. Loss of CD20 expression.
2. resistance to complement activation.
3. Altered TME.

Question 7

What mechanisms can lead to CD20 depletino in rituximab treatment?

Answer 7

Ab binding induces endocytosis of CD20.
Ab bniidng and FCR macrophage binding and phagocytosis may rip off CD20 too.
Tumour evasion of CD20 expression?

Question 8

What mechanism can tumours avoid rituximab mediated complement activation?

Answer 8

resistance via increased expression of regulatory complement components.

Question 9

How can TME alterations change rituximab efficiency?

Answer 9

Can prevent the infiltration of immune cells e.g. macrophages, and may downregulate their expression of FcR.

Question 10

How can addition of anti-CD27 help overcome resistance to rituximab?

Answer 10

anti CD27 stimulates T cell and NK production of inflammatory cytokines and chemokines that increased myeloid cell activation of FcR and their infiltration.
enhanced phagocytic capability.

Question 11

What 2 other methods can you try and keep rituximab functional?

Answer 11

1. Modify the TME e.g. with microenviornment.

2. Increasie potenty e.g. with glycosylation.

Question 12

Rituximab is helpful in autoimmunity, what mechanisms in RA?

Answer 12

anti CD20 can deplete B cells and prevent autoantibody producing B cell formation.
May also impact autoreactive T cells by inhibiting T-B cell interactions.
Indirectly may expand Tregs by moidying inflammatory environment.
Long lasting effects post antibody.

Question 13

what chimeric antibody and humanised antibody targets TNF-a?

Answer 13

infliximab and humira.

Question 14

Symptoms of RA?

Answer 14

Inflammation in joints and bone erosion (type II/III and type IV reaction).

Question 15

Why is TNF-a an effective target?

Answer 15

Because it is involved early in inflammatory cascade in disease.

Question 16

What cells can TNF-a activate in RA?

Answer 16

endothelial cells (increases infiltration), monocytes, synovial fibroblasts (damaging and inflammatory), osteoclasts.

Question 17

Why might patients infected with TB not be recommended TNFa?

Answer 17

TNF-a importatn for granuloma formation to contain mycobacteria. Would increase incidicnce of TB without TB blockers.

Question 18

Why might TNF-a not be beneficial in other autoimmune inflammatory diseases?

Answer 18

Becuase it may have anti-inflammatory effects in disease.

Question 19

How to improve mouse anitbodies?

Answer 19

Make them species specific (mouse/ rat/human)

Question 20

How to improve Fc region for specific purposes in mAb?

Answer 20

Isotype switching, as well as subclass switching (e.g. IgG4 has low effector function).

Question 21

Other ways of formatting mAb?

Answer 21

Use components of Ab, e.g. Fab region(s) coupled with PEG.
engineer Fc binding portion.
make Antibodies bispecific.

Question 22

How might you engineer Fab binidng affinity?

Answer 22

Make mutations to give a hihger affinity.

Question 23

How might you engineer Fc portion? What effects might this have?

Answer 23

May alter amino acid sequence to affect Fc capacity to bind FcRs.
Or modify glycosylation and fuxosylaiton patterns to change affinity to bind FcRs.
Will effect the effector functions such as complement activation and ADCC and ADCP.
Increasing the affinity for FcnR (neonatal FcR) can improve half life of the antibody.

Question 24

What two categories of Fc receptors are there?

Answer 24

inhibitory and activatory.

Question 25

What are non-fucosylated variants of rituximab supposed to do and how?

Answer 25

They are tailored to bind to the stimulatory FcyRIIA to improve ADCC by 100 fold.

Question 26

why might you want to decrease effector function of IgG? Example?

Answer 26

reduced FcR interactions and inflammation and side effects. Teplizumab: IgG1 isotype with mutations to minimise fCyR binding/

Question 27

How has Eculizumab been modified to lack effector function?

Answer 27

Has a hybrid IgG2 and IgG4 constatn cahin. Lacks ability to activate FcyRs (IgG2) and complement (IgG4).

Question 28

What antibodies can you use to reduce Ab levels? When might you want to do this?

Answer 28

Abdeg, to reduce pathogenic effects of autoantibdies and mAbs.

Question 29

Whats special about IgG4? And what can we do o take advantage of this?

Answer 29

IgG4 can exchange Fab domains with other Abs.
We can utilise this to create bi-specific antiboides which have IgG1 effector functions. Can target e..g tumour and t cell and accessory cell.

Question 30

What is the target of many therapeutic antibodies in Alzheimers?

Answer 30

target amyloid plaques to deplete them, clearnace by microglial cells.

Question 31

5 reasons why theraputic antibodies have failed so far.

Answer 31

Target is wrong. Dose and target engagement not high enough. But high doses give side effects.
Too late in treatment to be effective.
Poor stratification of patients.
Comorbidities induced which can have worse side effects.

Question 32

Problems with therapeutic antibodies that may indicate they would work better earlier in disease? Solution?

Answer 32

They can uptake soluble amyloid well, but if late, all bound to soluble amyloid and don’t tackle plaque. May build up causing an immune response.
Solution to target plaques and not soluble amyloid specifically.

Question 33

How might bispecific antibodies be useful in Alzheimers?

Answer 33

they can bind to transferrin, and cross blood-brain barrier via transendocytosis.

Question 34

Problems with bi specific antibodies binding to transferrin?

Answer 34

Bind too strongly and they iduce endocytosis and degradation of the antibody.

Question 35

What can glycosylation effect in terms of transport (4G8 Ab)

Answer 35

they can effect influx and efflux of antibody at sites.

Question 36

Three other targets in alzeihmars and nueroinflammatory conditions?

Answer 36

Tau, ApoE and cytokines.