6BBB0333
L8 – Antibody alternatives (continuation)
DARPins
- Moving on from beta-sheet structure scaffolds which still retains antigen recognition by
loops – DARPins
- They are antibody mimetics
- DARPin: Designed Ankyrin Repeat Protein (cytoskeletal protein for stability of plasma
membrane, interactions etc – perfect protein for PP interactions)
- AR proteins are one of the most common classes of binding proteins in nature responsible
for diverse functions – signalling, structure etc
- The AR protein class mediates very high affinity protein-protein interactions in nature
- Protein designed based on analysis of sequence-structure relationship of ankyrins
- Ankyrin repeats are small units (RHS); 2 helices connected by short loop
- Modular architecture - usually used are 4 (14kD) or 5 repeats (18kD) (relatively small
molecular mass for a biologic) – the latter would provide four loops for interactions
o When you clone these darpins, you can decide how many ankyrin repeats you want
in ankyrin
LHS has 12 ankyrin repeats
Most have 4/5 repeats (have antigen-recognising loop linking 2 adjacent
repeats)
- Very stable (very important criterion), very high melting temperature due to extensive
networks of salt bridges on the surface
- If you want big interaction surface = add many ankyrin repeats in single darpin to recognise
antigen
o If antigen is big = make big darpin
o Lots of flexibility
- Combinatorial sequence libraries can be generated for the loops with huge diversity (1012
variants), binders selected using e.g. phage display or ribosome display, high affinity binding
(picomolar)
- Very high expression levels in bacteria (in fermenter up to 10g/L), stable in blood and can be
engineered to avoid T-cell epitopes (avoid immune response)
o Very efficient especially if you want to sell large quantities
,6BBB0333
Applications of DARPins
- Can improve affinity and specificity by linking different individual small (4/5 repeat) DARPins
in fusion protein
- Therefore can increase avidity/specificity by binding to multiple sites and at same time
having a functional effect:
o E.g. close down open conformations of spike protein
o Can attach a darpin that binds to serum albumin in blood, you can stabilize the
protein in blood stream
- The potential benefits of DARPins are largely due to their structural and biophysical
characteristics. Their small size (14-18 kDa) is thought to enable increased tissue penetration,
and their high potency (<5-100 pM) makes DARPins active at low concentrations.[17]
DARPins are soluble at >100 g/L, and their high stability and solubility are considered
desirable properties for drug compounds. DARPins can be produced rapidly and cost-
efficiently (i.e., from E. coli). Their pharmacokinetic (PK) properties can be adjusted by fusion
to half-life extending molecules, such as polyethylene glycol (PEG), or to DARPins binding to
human serum albumin. Because of their favorable biophysical properties,[1] DARPins are
considered highly developable using standard processes, potentially exhibiting robust class
behavior.
- DARPins can be manipulated in many ways
o Add GFP – folloe fluorescence
o Can attach enzyme
o Can put binding site for metals and e.g. add radioactive metals binding sites like
uranium to destroy the target cell with the radiation
o Cannot do all of this with classical Abs
, 6BBB0333
Affibodies
- DARPins do away with beta-sandwich scaffold but still have loops
- Affibodies do not have either
- Scaffold is derived from the IgG binding Zdomain of protein A (immune system protein that
binds to antibodies)
- Binds side-on with the helices (not head on with loops)
- Very small domain: 58 - AA 6kD
- Very stable (Tm > 90oC) and well expressed in bacteria
- Binding sites on surface of helices H1 and H2 by randomisation of 13 amino acids
o With affibodies there are no binding loops but instead binding of two of the three
helices in the structure
- Sub-nanomolar affinities from library selections progressed to picomolar affinities after
maturation
-
- Original scaffold
- The green bits in helix 1 and 2 have been manipulated
- Ensured that residues important in packing of structure have not been changed and only the
exposed residues (green) are
- This is more risky than the other folds where the scaffold and antigen-binding portions are
clearly separate
L8 – Antibody alternatives (continuation)
DARPins
- Moving on from beta-sheet structure scaffolds which still retains antigen recognition by
loops – DARPins
- They are antibody mimetics
- DARPin: Designed Ankyrin Repeat Protein (cytoskeletal protein for stability of plasma
membrane, interactions etc – perfect protein for PP interactions)
- AR proteins are one of the most common classes of binding proteins in nature responsible
for diverse functions – signalling, structure etc
- The AR protein class mediates very high affinity protein-protein interactions in nature
- Protein designed based on analysis of sequence-structure relationship of ankyrins
- Ankyrin repeats are small units (RHS); 2 helices connected by short loop
- Modular architecture - usually used are 4 (14kD) or 5 repeats (18kD) (relatively small
molecular mass for a biologic) – the latter would provide four loops for interactions
o When you clone these darpins, you can decide how many ankyrin repeats you want
in ankyrin
LHS has 12 ankyrin repeats
Most have 4/5 repeats (have antigen-recognising loop linking 2 adjacent
repeats)
- Very stable (very important criterion), very high melting temperature due to extensive
networks of salt bridges on the surface
- If you want big interaction surface = add many ankyrin repeats in single darpin to recognise
antigen
o If antigen is big = make big darpin
o Lots of flexibility
- Combinatorial sequence libraries can be generated for the loops with huge diversity (1012
variants), binders selected using e.g. phage display or ribosome display, high affinity binding
(picomolar)
- Very high expression levels in bacteria (in fermenter up to 10g/L), stable in blood and can be
engineered to avoid T-cell epitopes (avoid immune response)
o Very efficient especially if you want to sell large quantities
,6BBB0333
Applications of DARPins
- Can improve affinity and specificity by linking different individual small (4/5 repeat) DARPins
in fusion protein
- Therefore can increase avidity/specificity by binding to multiple sites and at same time
having a functional effect:
o E.g. close down open conformations of spike protein
o Can attach a darpin that binds to serum albumin in blood, you can stabilize the
protein in blood stream
- The potential benefits of DARPins are largely due to their structural and biophysical
characteristics. Their small size (14-18 kDa) is thought to enable increased tissue penetration,
and their high potency (<5-100 pM) makes DARPins active at low concentrations.[17]
DARPins are soluble at >100 g/L, and their high stability and solubility are considered
desirable properties for drug compounds. DARPins can be produced rapidly and cost-
efficiently (i.e., from E. coli). Their pharmacokinetic (PK) properties can be adjusted by fusion
to half-life extending molecules, such as polyethylene glycol (PEG), or to DARPins binding to
human serum albumin. Because of their favorable biophysical properties,[1] DARPins are
considered highly developable using standard processes, potentially exhibiting robust class
behavior.
- DARPins can be manipulated in many ways
o Add GFP – folloe fluorescence
o Can attach enzyme
o Can put binding site for metals and e.g. add radioactive metals binding sites like
uranium to destroy the target cell with the radiation
o Cannot do all of this with classical Abs
, 6BBB0333
Affibodies
- DARPins do away with beta-sandwich scaffold but still have loops
- Affibodies do not have either
- Scaffold is derived from the IgG binding Zdomain of protein A (immune system protein that
binds to antibodies)
- Binds side-on with the helices (not head on with loops)
- Very small domain: 58 - AA 6kD
- Very stable (Tm > 90oC) and well expressed in bacteria
- Binding sites on surface of helices H1 and H2 by randomisation of 13 amino acids
o With affibodies there are no binding loops but instead binding of two of the three
helices in the structure
- Sub-nanomolar affinities from library selections progressed to picomolar affinities after
maturation
-
- Original scaffold
- The green bits in helix 1 and 2 have been manipulated
- Ensured that residues important in packing of structure have not been changed and only the
exposed residues (green) are
- This is more risky than the other folds where the scaffold and antigen-binding portions are
clearly separate
