introduction to in silico engineering for biologics
TRANSCRIPT
Lecture Aim
This lecture aims to provide a basic understanding of the concept of in silico engineering/design as part of the antibody therapeutic/biologic drug development process:-
Introducing theory and approaches, drivers, databases and software – and with a focus on safety and efficacy.
This Lecture Covers
• Biologics – antibody therapeutics•Engineering biologics for safety – reducing immunogenicity•Considering efficacy of biologics
What are Biologics?
Typically biologics are thought of as being either antibody therapeutics or components of vaccine products.
However... (from FDA CBER)
Biological products include a wide range of products such as vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins. Biologics can be composed of sugars, proteins, or nucleic acids or complex combinations of these substances, or may be living entities such as cells and tissues. Biologics are isolated from a variety of natural sources - human, animal, or microorganism - and may be produced by biotechnology methods and other cutting-edge technologies. Gene-based and cellular biologics, for example, often are at the forefront of biomedical research, and may be used to treat a variety of medical conditions for which no other treatments are available.
Center for biologics evaluation and research
We will just consider antibodies here...
Safety and Efficacy of Biologics
• Safety: safety issues primarily focus on the potential of the protein biologic to raise an immune response in the subject. This could be mild or severe.
• Efficacy: efficacy issues focus on either the raising of anti-drug antibody responses, or the in vivo half life of the protein
Making suitable Abs for therapy
Monoclonal antibodies are traditionally made using Mice* – these are fine for R&D use, but bring problems for use in Humans
When developing Abs for therapeutic use there are very few requirements for modelling or in silico engineering as most of the work can be simple molecular biology (gene editing/expression systems)
However, the use of in silico engineering provides further options for improving or modifying function – particularly considering safety and efficacy.
*also phage or ribosome display – or now, humanised mice, which can avoid these problems – but are beyond the scope here
Immune response: B-cell activation
a) "B cell activation" by Fred the Oysteri. Licensed under Public domain via Wikimedia Commonsb) "T-dependent B cell activation" by Altaileopard - Own work. Licensed under Public domain via Wikimedia Commons
(a)
(b)
Antibody structure
By Dan1gia2 (Own work) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons
Size relationship
antibody
rhinovirus
DNA and DNA polymerase
ribosome
rhodopsin
membrane
cyclooxygenase
http://www.rcsb.org/
Chimeric Ab:
Retain the murine variable domains – splice to Human constant domain.
75% Human*
Humanised Ab:
Retain the murine CDRs – splice to Human variable framework & constant domain.
95% Human*
Best to try and ‘humanise’ them as a first step – helps both:
Safety and Efficacy
Engineering:* refers to percentage Human origin. Of course, being both mammals the mouse and Human have fairly high antibody sequence similarity
Targets for engineering
By Dan1gia2 (Own work) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons
CDR – tweak to remove unwanted PTM sites – mitigate immunogenicity (more later) at human/mouse interface
VL/H – remove unwanted PTMs. If Chimeric, reduce immunogenicity at C/V interface
Fc – Select effector functions, remove unwanted PTMs, enhance function?
Other – Add drug conjugates?(Beyond the scope of this talk)
What about Fc selections?
Salfeld, J.G., 2007. Isotype selection in antibody engineering. Nature Biotechnology, 25(12), pp.1369-1372.
Half life
• Proteins & Biologics will be slowly cleared by the system (either immunologic response or cellular uptake/destruction)
• Two main strategies to increase serum halflife: increase the size (pegylation) or exploit (enhance?) natural protein recycling (via FcRn)
FcRn – neonatal Fc Receptor
Roopenian, D.C. & Akilesh, S., 2007. FcRn : the neonatal Fc receptor comes of age. Nature Reviews, Immunology, 7, pp.715-725.
FcRn in the adult
Roopenian, D.C. & Akilesh, S., 2007. FcRn : the neonatal Fc receptor comes of age. Nature Reviews, Immunology, 7, pp.715-725.
IgG : FcRn binding
Roopenian, D.C. & Akilesh, S., 2007. FcRn : the neonatal Fc receptor comes of age. Nature Reviews, Immunology, 7, pp.715-725.
Deimmunisation & ADA• If part of the Ab is recognised as foreign – it can stimulate
a T-cell response when the fragment is presented on MHCII, and...
• If the Ab contains a B-cell epitope (it will), then...
• The immune system will raise antibodies to the biologic which may be harmful to the patient or at least reduce the usefulness of the drug
• Engineer to remove the T-cell epitopes (Humanisation + deimmunisation strategy)
Safety: reducing immunogenicity
a) "T-dependent B cell activation" by Altaileopard - Own work. Licensed under Public domain via Wikimedia Commons
(a)
If the Antibody (antigen) doesn’t have any epitopes that will (a) bind MHC II or (b) be recognised by a TCR – the B-cell will not be activated, and no ADA
We can deal with (a) though engineering - deimmunisation
Predicting T-cell epitopes http://www.iedb.org/
Sequence-level engineering
PGLVRPSQTLSLTCT = T-cell epitope
PGLVRPSATLSLTCT = weak or non-epitope?
Remove or mitigate the risk – taking into account the promiscuity of the epitope for HLA types, and population variation.
MHCII varies by population, but so does IgG...
Jefferis, R. & Lefranc, M.-paule, 2009. Human immunoglobulin allotypes. Possible implications for immunogenicity. mAbs, 1(4), pp.1-7.
Aggregation & ADAT
-cel
l epi
tope
sA
ggregatio n
a) "B cell activation" by Fred the Oysteri. Licensed under Public domain via Wikimedia Commons
(a)If antigen can cross-link the B-cell receptor, the cell will become activated without the presence of a T-cell
The result is mainly IgM, but can still be a problematic response
Aggregated antigen can cause the cross-linking – even when as “Human-like” as possible
This is T-cell Independent B-cell Activation
Aggregation & ADA
Engineer to remove potential aggregation hotspots (disorder/hydrophobicity, PTMs and pI shift potential, hydrophobic patches)
Predicting aggregation is really hard!
Problem – sometimes this is due to formulation!