Tumour-targeting antibodies and neutrophils

Question 1

What can monoclonal cancer antibodies do?

Answer 1

• Have direct effects by blocking necessary growth factors
• Induce complement-dependent cytotoxicity (CDC)
• Induce antibody-dependent cellular cytotoxicity (ADCC)
• Induce antibody-dependent phagocytosis
• Induce antibody-dependent killing (e.g. with neutrophils!)

Question 2

Why are neutrophils attractive effector cells for tumour killing?

Answer 2

• Effective induction of Antibody Dependent Cellular Cytotoxicity
• Are abundant in blood
• Are easy to mobilize (G-CSF)
• Secrete chemotactic stimuli attracting other immune cells

Question 3

What is trogocytosis?

Answer 3

Neutrophils can form tight synapses (integrin and Fc receptor are very important!) with tumour cells and start pulling at the tumour cells. This pulling is called trogocytosis. If trogocytosis leads to cell death, it’s called trogoptosis.

Question 4

What type of antibodies are all the antibodies currently in late stages of development?

Answer 4

All antibodies currently in late stages of development are IgG antibodies.

Question 5

What receptors do neutrophils have?

Answer 5

Neutrophils have the following receptors ordered on level of expression:

1. CD16 (Fc gamma receptor 3b) (a lot of expression)
2. CD32 (Fc gamma receptor2)
3. CD89(Fc alfa-receptor1)
4. CD64 (Fc gamma receptor1) (almost no expression)

Question 6

What is the most prevalent antibody at mucosal surfaces?

Answer 6

IgA!

Question 7

How is IgA produced and transported to the mucosa?

Answer 7

• Produced at lamina propria as dimeric molecule

* Transported through epithelial cells and released as secretory IgA

Question 8

What does IgA do at mucosal surfaces?

Answer 8

It forms anti-septic barrier in lumen by neutralizing bacteria

Question 9

What is the role of IgA in serum?

Answer 9

It is a pro-inflammatory molecule

Question 10

What is the difference in immunogenicity between IgA and IgG?

Answer 10

There is no difference in phagocytosis between IgA and IgG. IgA induces neutrophil migration. IgA induces more signalling compared to IgG, thus especially IgA potently activates neutrophils.

Question 11

What is the role of IgA in lineair bullus disease?

Answer 11

Auto-IgA is present in the skin of lineair IgA bullus disease. They produce auto-IgA against the skin and have a lot of neutrophils in the skin.

Question 12

Why is investigating IgA-bullus disease difficult in a mouse model?

Answer 12

Mice do not have a Fc receptor for IgA.

Question 13

What is a solution for the problem with mouse models investigating IgA-bullus disease?

Answer 13

Investigating this in a mouse model is difficult, because mice do not have a Fc receptor for IgA.
A transgenic mouse model that expresses human IgA was crossbred with mice that had green fluorescent neutrophils. Then a human-IgA knock-in mouse to produce human IgA against murine collagen XVII, the auto-antigen in lineair IgA bullus disease. Human IgA was injected into the ears (because translucent) of the mice. Neutrophil behavior is studied. Conclusion: IgA mediates Fc alfa receptor 1 dependent tissue damage in vivo.

Question 14

Do Neutrophils + FcaRI + IgA + antigen work in tumours?

Answer 14

Yes! It was tested with Cetuximab (IgG) vs. Cetuximab-IgA. In vivo: Works!

Question 15

What are the pros and cons of IgA?

Answer 15

PRO

• IgA is an extremely potent activating stimulus for neutrophils
• IgA induces phagocytosis, and release of NETs/ inflammatory mediators
• IgA induces migration of neutrophils
• The process is dependent on FcaRI

CON
-Short half life

Question 16

What is a solution for the short half-life of IgA?

Answer 16

Bispecific antibodies

Question 17

What happened when bispecific antibodies were tested with neutrophils and compared with normal IgA?

Answer 17

With cetuximab, synapsis are formed, but tumour cells are not really effected. IgA-EGFR shows rapid recruitment and killing of tumour cells. Bispecific is more effective than IgA, but the difference is in speed. The end result is the same.

Question 18

What happened when bispecific antibodies were tested with NK cells and compared with normal IgA?

Answer 18

Bispecific antibodies first tested with NK cells as effector cells: very effective! When you use an IgA-EGFR, nothing happens, because NK cells don’t have an Fc-alfa-receptor. Not much difference between bispecific Ab and cetuximab.

Question 19

How do bispecific antibodies perform in vivo?

Answer 19

Bispecific antibodies in vivo:

• Migration of neutrophils is good (like with IgA)
• Trogocytosis is very good (like IgA, although IgG does that a little too)
• ADCC NK cells is good (Like IgG, IgA doesn’t do this)
• ADCP is very good (like IgG. IgA also does that a little)
• The half life is good (the same as IgG (better than IgA))
• Bispecific performes better than IgG in reduction of tumour volume.

Question 20

How can you further enhance tumour cell killing by neutrophils?

Answer 20

Blocking SIRPa-CD47 enhances tumour cell killing by neutrophils