Recombinant antibodies for cancer therapy Flashcards
What are hybridomas? How are they created?
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
What types of antibodies are used (historically to now)
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.
What is the benefit of using antibodies derived from cows?
They have a very long CDR3 heavy chains so are very good at binding to proteins that contain pockets in their 3D structure.
What is unique to antibodies derived from llama and shark?
Their variable regions are composed of a single variable domain
What are the different ways in which an antibody can inhibit cellular proliferation?
Prevent dimerisation of receptor by binding the ligand or receptor to block ligand binding or dimerisation
What are the anti-tumour mechanisms mediated by Fc
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)
What is the role of FcRn?
Extends the half life of an antibody in the blood
How does FcRn extend the half life of an antibody in blood?
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.
What kind of antibody is rituximab?
Chimeric antibody: Anti-CD20
In what cells is CD20 expression high?
During B-cell development so is highly expressed in B cell lymphoma
How does Rituximab work?
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
What is Herceptins proposed therapeutic mechanism of action?
Inhibition of growth signals by blocking the HER2 receptor. Also ADCC. It enhances chemotherapy and stimulates the immune system.
What kind of antibody is Herceptin?
Humanised - mouse CDRs, remainder is human IgG1
What is Avastin>
A humanised antibody that targets VEGF. This prevents angiogenesis.
What can be added to an antibody to arm it against tumour cells?
Drugs, toxins, radioactivity
What mode of internalisation and trafficking is normally required to get an ADC to the lysosome?
Clathrin dependent internalisation
Which groove of DNA do PBDs bind to?
DNA minor groove - without distorting DNA
What is the linker region required for ADC cleavage once internalised
Cit-Val
What two interactions on T-cells are required for T-cell activation
1) TCR and antigen binding in the context of MHC
2) CD28 co-stimulation
What happens after T-cell activation
T cell proliferation is inhibited by immune checkpoints such as CTLA-4 and PD-1
What is the role of CTLA-4 and PD-1 after T cell activation?
Receptors on T-cells that when bound, attenuate or terminate T-cell proliferation.
What was the result of using a super agonist of CD28?
Overstimulation of T-cells led to overstimulation of the immune system and cytokine release. Multiple organ failure and loss of appendages.
What is ipilimumab?
A human antibody from transgenic mice that inhibits PD-1 on T-cells, preventing T cell termination.
Why did Ipilimumab initially cause an increase in tumour burden after injection?
Because of the time required for T-cells to become activated and have their proper interactions with the tumour etc.
What are scFVs?
Taking the variable regions of an antibody and fusing them together with a flexible linker.
What is the advantage of using an scFv
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.
What is a phage display?
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.
How have scFvs been used in immune-oncology?
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.
What have BiTEs been used to treat so far?
Lymphoma - using CD19 targeted and CD3 targeted scFvs.
What is a chimeric antigen receptor?
Uses a tumour specific scFv which is bound to CD8 which is associated to intracellular CD3 or FceRIy which leads to T cell activation.
