Antibody Technologies 2

Question 1

Why you might adopt an antibody vs small molecule approach?

Answer 1

IgG target binding has high affinitity and selectivity, can be raised without knowledge of molecular binding sites, can tackle intractable targets.

Broad range of pharmcological mechnanisms such as recruitment if immune response (ADCC)

Question 2

What are the potential problems using this strategy?

Answer 2

• IgG are larger molecules, H+L chains. post-trans mod and require in vivo immunistaion in mammalian species to generate.
• Humanisation to avoid immunogenicity
• All adds to cost and complexity of synthesis of an appropriately regulated therapeutic- of small molecules
• Oral not possible, IgG distribution may be excluded from targte site.

Question 3

Antobody-ab

If you can make one change to the design of Ab based therapeutics to address some challenges, what would it be?

Answer 3

Keep the target binding variable regions (responisble for the adv in affinity/ selectivity)

Thus just make it smaller to achieve this.

Question 4

Smaller antibody templates include:

Answer 4

• Humanise amino acid seq
• Increase stability of therapeutic
• Improve access to target tissues such as the CNS or solid tumours.

Question 5

Tell me about IgG fragment cleavge that can retain antigen binding?

Answer 5

Enzymatic digestion like pepsin which retains the disulphide linjed bivalent antigen binding domains F(ab)2 which is what recognises the antigens.

Pepsin Cleaves FC domain which stands for crystalisable part of the AB.

Or have **papain **which cleaves at different site to release monomeric F(ab) domains

Question 6

look on slide

Explain about the use of recombinant technology. (scFv)

Answer 6

You can go even smaller than this which would be making a single polypeptide chain by linking the entrance to the C terminus on the DNA.

Then express it in E.coli/yeast or mamalian cells to purify ir

Question 7

What are the advantages of using scFv or F(ab)

Answer 7

Reduction in size (160kD) to scFv (30kD)
Simplified manufacture like single gene encoding, lack of glycosylation.

BUT…….
* Fc domain could be important to the ADCC MOA.
* Engineerd fragments may have reduced structural stability and be prone to aggregration.
* Shorter plasma half life.

With AB there is no renal elimination due to their size and reduced proteolytic degradation after phagocytosis, due to unbound IgG being recylced to the plasma via cellular FCgammaR receptors.

Having lack of Fc domain and smaller domian may lead to greater ELIMINATION.

Question 8

Examples such as Abciximab

Answer 8

This drug blocks blood clotting by binding to a platlet glycoprotein prevnting fibrinogen cross-linking and aggregration.
Short half life which is suitable for the indication
Higher affinity to glycoprotein means binding to target is long lasting which is also ideal.

Question 9

What is the most saturated protein in out blood?

Answer 9

Albumin which transports fat molecules, large protein 66kD in size.

Question 10

Nanobodies?

Answer 10

Only two chains which are slightly shorter but heavier.

The antigen binding domain is fully encoded by single heavy chain, allowing its cloning as a VH domain nanobody.

Sequences require humanisation.
But because they are so small they have less risk of aggregation.

Question 11

GPCR example with nanobodies

Answer 11

They have a ligand binding site and GPCR recognition site.

There is only one domain so will be smaller and these variable loops are a bit longer.

They are also better at recognising 3D shapes.

The sites on the slide are concave or flat thus are poorly immunogeneic for standard IgG molecules.

Question 12

Nanobodies with lysozyme

Answer 12

The hypervariable regions of nanobodies have longer CDR loops- producing a convex binding surface able to recognise internal binding sites.

Question 13

Nanobodies: therapeutics?

Answer 13

Caplacizuman was approved for thrombotic thrombocytopenic purpura
Causes build up of vWF proteins thus aggregating platlets.

Question 14

Multivalency?

Stringing nanobodies together?

Answer 14

Causes agonism through receptor cross linking, higher affinity of the nanobody therapeutic for a single target.

Dual specificity- nanobodies against different targets to improve efficacy.

Question 15

Removing the need for injection.

Optimising the pk properites via conjugation.

Answer 15

Injecting everyday becomes expensive so in order to remove this you need more half life.

Add albumin, PEG, Fc domains in order to optimise rate of elimination and extend plasma half life.

Question 16

Can CNS penetration be achieved?

Answer 16

Excluded from crossing BBB.

Alternative strat under investigation is by hijacking transcytosis across the BBB by AB conjugation to an appropriate ligand

E.g. IgG against amyloid beta protein, conjugated to Fab domain binding transferrin receptor, then the T recptor carrier the cojugatee dantibody across the BBB.

This is theoretical, drugs havent been approved yet.
On rodent models, fluorecent labels shown they crossed BBB.

Question 17

Antibodies as delivery vehicles.

Answer 17

1. Recruitment of immune system such as Fc domains to restore ADCC
2. Anti -cancer=selective delivery of toxic payloads to target cells e.g. trastuzumab.
3. Biomedical imaging Nucleotide labelling for PET scanning=monitoring tumour metasases.