Antibody Technologies 1 Flashcards
Small molecules drug discovery has limits?
Receptors for hormones are large so small molecule drugs will find it difficult to block it.
Restricted drug action at target(agonist, antagonist, inhibitor)
What is biologic?
Medicinal producy whose synthesis, extraction, or manufacture involves living sources
Includes:
Protein based therapeutics
Gene and cellualr therapies, stem cells and transplantation
Vaccines
Blood products for transfusion
Diagnostic reagents (allergen tests for allergies
Injection rather than oral, cant be absorbed through the gut,risk of imm
Protein based therapeutics (insulin)
immunogeneicty
Peptide and protein hormones and growth factors
Antibodies
engineered proteins (receptor binding domains)
First protein biologic was insulin, which was from pigs pancreas.
Complex structure, two linked peptide chains synthesised by enzymatic cleavage from a single protein cleaver.
What is immunogenicity?
The ability of a foreign substance such as an antigen to provoke an immune respone.
Why adopt a biopharmaceutical versus small molevule approach?
Advantages:
Tackle targets to small mole intervention
Potential for higher affinity and selectivity
Potential for diverse molecular MOA (intercation with messenger molecule rather than target immune directed cytotoxicty)
What are the risks of protein therapeutics?
Lack of efficacy: delivery to the target tissue rather than oral/ also tissue access (BBB or solid tumours)
Specicies variations in protein sequences potential immunogenicity.
Complexicity, reproducibility and purity of synthetic process (antibodies: have to make a much higher MW amount, multiply by a factor of 300 which becomes quite challenging)
Also there are post translational modifications in animals which are different to humans.
Look at the slide of the diagram on the lecture to get better understand
Antibody structure
5 human antobody immunoglobulins (IgA, D,E, G, M)
IgG is the main circulating antibody in plasma (2ndry adaptive response)
Fab: The antigen binding fragment domain which has variable region Fv responsible for antigen recognition. It is bivalent
IgE (inflamation) Fc interacts with R on mast cells
IgG- Fc interacts with Fc gamma on macrophages and neutrophils
Fc regultes Ig transport and extends plasam1/2 life
IgG structure
Two heavy chains linked via disulphie bonds, two light chains alsovia DSB.
Each chain contains multiple domains which is a stable protein fold, linked like on a string together. Also have variable domains which change loops and due to the response of a new antigen.
3 hypervariable regions (CDR loops), dont form a structural backbone, the HV segments are o the external surface and define its binding with antigens.
Example of infliximab binding with TNFalpha
IgG contact surface is usually flat or concave.
They recognise the loops of the hormone target.
Poly and mono
Monoclonal antibody generation.
Polyclonal-many different IgG molecules with high affinity for antigens after immunization
Mono- IgG producing B cells isolated forom the mouse producing identical IgG molecules.
- Antigen is injected in to the mouse (immunization)
- Isolation of immune cells to form antibody forming cells.
- Tumour cells fuse causing hybridomas
- These hybridomas are then screened for procution of desired antibody.
- Antibody producing hybridomas are cloned
- Clonal expansion leads to monoclonal antibodies being formed.
PK challenges
Humans will develop an immune response, take antibodies out of the blood and degrade them, thus they are no longer active
Worst scenario is inflammation and non-desired also could be quite dangerous
Mouse Fc domains may also contribute to lack of efficacy
Fullly humanised antibodies
Replacing the complement CDR regions from mouse antibodies and splice them onto the human antibodies, so only the CDR regions are from the mouse and is nearly completely humanised
Not all perfect, splicing can go wrong and lose efficacy by not recognising the target.
Humanised antibodies
In vivo immunization of transgenic mice which are egineered to prodcue human IgG.
Via phage display
* Virus coats engineered to express human IgG domains
* Allows screening and selectrion for target affinity without immunization
* Asscociated IgGs lack post-trans mod
* Mammalian cell antibody display systems in development
MOA of IgG
Receptor antagonists and inhibitors
Cetuximsb
Antagonist of EGF receptor
Limits EGF R dependent proliferation of tumour cells.
Thus preventing activation of the receptor.
ANti-VEGF therapies like bevacuziman, acts as an antagonist of the stimulating VEGF which could cause tumour angiogenesis.
Agonists of chemokine ligand TRAIL binds to tumour cells activating pro-apoptotic signalling.
(Antibody mimics hormone and induces apoptosis)
Not past phase 1 yet.
COVID-19:Tocilizumab
Causes infliltration of T cells/neutrophils driven by interleukin 6 release
Tocilizumab blocks IL6 receptor signalling, therefore reducing inflammatory storm.
Antibodies bind to ACE2 (USED BY COVID TO ENTER CELLS)
Thus blocks infection and cell entry.
Trastuzmab
Targets herceptin
Targets EGF related HER2(HER2+ve breast cancer)
* Direct binding and inhibition of HER2 dimers
* Prevention of HER2 cleavage
* Endocytosis and degration of HER2
* Antibody dependent cytotoxicity to destroy tumour cells.
Doesnt prevent cancer from devloping, but slows it down.
