Recombinant antibodies for cancer therapy

Question 1

What are hybridomas? How are they created?

Answer 1

Created by injecting an antigen into a mouse. The B cells are then harvested and fused to a myeloma cell line giving a hybridoma that can infinitely produce monoclonal antibodies

Question 2

What types of antibodies are used (historically to now)

Answer 2

Mouse
Chimeric - Keep the variable regions from the mouse. Using antibody engineering, fuse this to constant regions of a human antibody
Humanised - Replace the variable region of the mouse with the closest human alternative. The only part that remains from the mouse is the CDR (responsible for binding)
Human - can be made using a transgenic humanised mouse or by building libraries using immunoglobulin genes.

Question 3

What is the benefit of using antibodies derived from cows?

Answer 3

They have a very long CDR3 heavy chains so are very good at binding to proteins that contain pockets in their 3D structure.

Question 4

What is unique to antibodies derived from llama and shark?

Answer 4

Their variable regions are composed of a single variable domain

Question 5

What are the different ways in which an antibody can inhibit cellular proliferation?

Answer 5

Prevent dimerisation of receptor by binding the ligand or receptor to block ligand binding or dimerisation

Question 6

What are the anti-tumour mechanisms mediated by Fc

Answer 6

Once a tumour cell is coated with antibodies it can:

1) Antibody dependent cell-mediated cytotoxicity (ADCC) - Antibody is recognised by immune cells (NK cells) via the FCR. This secretes grnazymes which lead to tumour lysis.
2) Phagocytosis - FC dependent recognition by macrophages which engulf the tumour cell and leads to its lysosomal degradation.
3) Complement activation - antibody-mediated recruitment of the complement cascade which leads to the formation of the membrane attack complex (MAC)

Question 7

What is the role of FcRn?

Answer 7

Extends the half life of an antibody in the blood

Question 8

How does FcRn extend the half life of an antibody in blood?

Answer 8

Antibody binds to the FcRn on endothelial cells which leads to their internalisation. As the pH becomes more acidic, the antibody remains bound to the FcRn. Unbound proteins go to the lysosome for degradation. The bound antibody is recycled to the surface where at a more neutral pH, it dissociates from FcRn.

Question 9

What kind of antibody is rituximab?

Answer 9

Chimeric antibody: Anti-CD20

Question 10

In what cells is CD20 expression high?

Answer 10

During B-cell development so is highly expressed in B cell lymphoma

Question 11

How does Rituximab work?

Answer 11

Retuximab bound CD20 on B-cells is recognised by the complimentary system. Leads to the insertion of the MAC and cell death.
May also achieve its effect through ADCC - attracting NK cells.
May also trigger apoptosis

Question 12

What is Herceptins proposed therapeutic mechanism of action?

Answer 12

Inhibition of growth signals by blocking the HER2 receptor. Also ADCC. It enhances chemotherapy and stimulates the immune system.

Question 13

What kind of antibody is Herceptin?

Answer 13

Humanised - mouse CDRs, remainder is human IgG1

Question 14

What is Avastin>

Answer 14

A humanised antibody that targets VEGF. This prevents angiogenesis.

Question 15

What can be added to an antibody to arm it against tumour cells?

Answer 15

Drugs, toxins, radioactivity

Question 16

What mode of internalisation and trafficking is normally required to get an ADC to the lysosome?

Answer 16

Clathrin dependent internalisation

Question 17

Which groove of DNA do PBDs bind to?

Answer 17

DNA minor groove - without distorting DNA

Question 18

What is the linker region required for ADC cleavage once internalised

Answer 18

Cit-Val

Question 19

What two interactions on T-cells are required for T-cell activation

Answer 19

1) TCR and antigen binding in the context of MHC

2) CD28 co-stimulation

Question 20

What happens after T-cell activation

Answer 20

T cell proliferation is inhibited by immune checkpoints such as CTLA-4 and PD-1

Question 21

What is the role of CTLA-4 and PD-1 after T cell activation?

Answer 21

Receptors on T-cells that when bound, attenuate or terminate T-cell proliferation.

Question 22

What was the result of using a super agonist of CD28?

Answer 22

Overstimulation of T-cells led to overstimulation of the immune system and cytokine release. Multiple organ failure and loss of appendages.

Question 23

What is ipilimumab?

Answer 23

A human antibody from transgenic mice that inhibits PD-1 on T-cells, preventing T cell termination.

Question 24

Why did Ipilimumab initially cause an increase in tumour burden after injection?

Answer 24

Because of the time required for T-cells to become activated and have their proper interactions with the tumour etc.

Question 25

What are scFVs?

Answer 25

Taking the variable regions of an antibody and fusing them together with a flexible linker.

Question 26

What is the advantage of using an scFv

Answer 26

Very easy to work with and can be put onto the surface of bacteriophage, producing a virus that expresses a single scFv which can be used for selction.

Question 27

What is a phage display?

Answer 27

A huge library of bacteriophages, each expressing a different surface scFv. This library can be used to challenge against a protein of interest until you have an extremely specific antibody for the protein of interest.

Question 28

How have scFvs been used in immune-oncology?

Answer 28

BiTEs - two scFvs are fused together by another flexible linker. One of the scFvs binds to a tumour antigen. The other binds to CD3 on T-cells. CD3 binding is required for T-cell activation. When both scFvs are bound to their receptors, T cell activation leads to the release of granzymes/perforin and T cell proliferation. Lysis of the tumour cell follows.

Question 29

What have BiTEs been used to treat so far?

Answer 29

Lymphoma - using CD19 targeted and CD3 targeted scFvs.

Question 30

What is a chimeric antigen receptor?

Answer 30

Uses a tumour specific scFv which is bound to CD8 which is associated to intracellular CD3 or FceRIy which leads to T cell activation.