2.) Nanobody technologies and conjugates Flashcards
What IgG fragments exist?
IgG fragments that retain antigen binding:
- F(ab)2
- F(ab)’
- scFv
Why is there a need to develop antibody fragments?
Smaller antibody templates streamline synthesis and manufacture = reduced cost:
- SImply genetic engineering e.g. to humanise AA sequence
- Increases stability of therapeutic e.g. gut acidic compartments
- Improves access to target tissues such as the CNS (crossing BBB) or solid tumours
What strategies exist to generate IgG fragments and what do they entail?
Enzymatic digestion:
- Pepsin: cleaves Fc domain, retaining disulfide linked bivalent antigen binding domains F(ab)2 (leaving bivalent top of Y antigen-binding variable domain)
- Papain: cleaves at different site to yield individual monomeric F(ab)’ (prime) domains (one side of the antigen-binding variable Y domain)
Recombinant technology:
- Single chain (sc)Fv: variable domains (Fv) are joined by peptide linker in one polypeptide encoded by one gene
- Artificially join Fv with peptide linker
- H & L domains in single polypeptide encoded in 1 gene
- Genetic engineering approach
What advantages are there associated with scFv or F(ab)’ domains?
- Reduction in size from IgG (160 kD) to scFv (30 kD; 1/5 of size)
- Simplified manufacture (complexity): more reproducible, single gene encoding, lack of glycosylation allowing antibody production in bacterial cell systems over mammalian (bacteria can’t conjugate)
- Increased tissue perfusion
What are the potential disadvantages to using scFv/F(ab)’ domains?
- Fc domain (constant) may be important (e.g. ADCC mechanism of action)
- Engineered fragments may have reduced structural stability, prone to aggregation (fold/misfold/unfold due, exposed hydrophobic parts = aggregation)
- Potential issues with yield in manufacture, risk of immunogenicity (due to aggregation?)
- Engineered fragments/domains may have short plasma half-life (PK issue - elimination vulnerability)
What is an advantage of full-length IgGs over IgG fragments?
Predictable PK profile:
Long plasma t1/2 (15-30 days depending on IgG subclass) due to:
- No renal elimination (too big for glomerular filtration)
- Reduced proteolytic degradation after phagocytosis of IgG-antigen complexes: unbound IgG recycled to plasma via cellular FcγR receptors
What IgG fragment technology is Abciximab (ReoPro) an example of? Indication?
Abciximab is a chimeric F(ab)’:
- Binds surface platelet glycoproteins IIb/III (imitates coagulation cascade)
- Thus preventing fibrinogen cross-linking and aggregation
»> Acute anti-thrombotic therapy during surgical procedures e.g. CVS angioplasty
> Future indication for stroke?
Why is the short plasma half-life of Abciximab (chimeric Fab’) not an issue/advantageous?
Abciximab has a half-life of 10-30 minutes:
- Thus suitable for indication of preventing thrombosis during surgery (not an acute indication)
- Abciximab also has a high affinity for GP IIb/III: binding to target is long-lasting (covalent character, slow dissociation rate with effects taking > 24 hours to wear) even after plasma clearance
What IgG fragment technology is Certolizumab pegol an example of? Indication?
Certolizumab pegol is a humanised F(ab)’, which is also PEGylated (conjugated to increase size, reducing renal elimination):
- TNF-alpha biologics (e.g infliximab, adalimumab)
- Anti-inflammatory, licensed for RA/Crohn’s
- PEG (polyethylene glycol) adds 70 kD, which increases solubility as well as the above to improve PK
»> Also increases cost and complexity of manufacture
What is the significance associated with select camelid and shark antibodies? Caveat?
They only have heavy chains:
- Antigen-binding domain is encoded fully by a single heavy chain, allowing its cloning as a VH domain ‘nanobody’
- Naturally occurring single chain antibodies
»> Sequences still require humanisation (reduce immunogenicity risk)
How are nanobodies defined?
- Small (15 - 18 kD)
- Monomeric: less risk of aggregation
- pH stable (potential for oral delivery to gut lumen e.g. Crohn’s)
What advantage to nanobodies convey over IgGs with accessing target sites?
Ability to access internal target sites:
- Most protein drug targets have binding sites recessed in their structure (cavities/pores) E.g. active sites of enzymes, ion channel pores, NT binding sites in receptors
- Antigen binding site of IgG is flat/concave
- Nanobodies have longer CDR loops within their hypervariable regions; producing a CONVEX binding surface able to recognise and explore internal binding sites
Do any licensed therapeutics employ nanobody technology currently?
No, but some in PIII trials:
Caplacizumab (TTP - Thrombotic Thrombocytopenic Purpura):
- Congenital/acquired autoimmune disease
- Deficient processing of blood glycoprotein, von Willebrand factor (vWF)
- Build-up of ultra large vWF proteins, constitutively aggregating platelets (unregulated)
- Orphan disease without effective therapeutics: streamlines licensing for FDA/EU, easier for approval of common indications after
»> Caplacizumab is an anti-vWF nanobody, preventing platelet aggregating (inhibiting platelet recruitment associated w/ultra large vWF)
Why may multivalent binding properties of OG IgG be desirable WRT to nanobody technology?
Ease of implementing?
Stringing nanobodies together to yield multivalency can give:
- Higher affinity of nanobody therapeutic for single target
- Altered pharmacological properties e.g. agonism through receptor cross-linking
- Dual specificity therapeutics: different nanobodies strung together to be active against different targets (improving efficacy)
»> Straightforward to engineer in nanobodies due to their domain and gene organisation?
What is an example of multivalent nanobody therapeutics?
Ablynx Pharma’s BI 836 880:
- Dual specificity therapeutics
- Anti-VEGF nanobody AND Anti-Ang II nanobody
- To inhibit tumour angiogenesis, targeting two different messengers
- Proprietary domain also tagged on to extend plasma half-life
