1.) Antibody technologies - Biologics as current medicines Flashcards
What are the limits of small molecule (sMW) drug discovery?
- Intractable molecular targets (e.g. receptors for large hormones)
- Restricted ‘classical’ drug action at target (e.g. agonist, antagonist, inhibitors)
- Traditional drug discovery approaches by ‘big’ pharma becoming less succesful
What is a biologic?
Medicinal product whose synthesis, extraction or manufacture involves living sources (human, animal or microbiological).
Name some types of biologics.
- Protein based therapeutics
- Gene and cellular therapies, stem cells and transplantation
- Vaccines
- Blood products for transfusion
- Diagnostic reagents e.g. allergens for allergy tests
Give examples of protein based therapeutics.
- Peptide and protein hormones, growth factors
- Antibodies
- Engineered proteins (eg. receptor binding site domains)
What advances/challenges in developing insulin (1920s, the OG) can be applied to protein-based therapeutics?
Applies to all:
- Unmet clinical need, successfully addressed
(no sMW to date can imitate action of insulin at receptor)
- Due to its complex structure, single gene, enzymatic cleavage
»> PK undesirable: protein-nature means it has to be injected rather than PO (applying to all biologics)
What are the advantages of biologics vs sMW?
- Tackle targets resistant to sMW intervention e.g. receptors with large, complex binding sites, orphan diseases/targets with unknown binding sites
- Potential for higher affinity and selectivity (big surface contact between drug + receptor) e.g.
allows selection between closely related receptor targets, targeting mutant forms of the target contributing to disease (pharmacogenetics - personalised medicine) - Potential for diverse molecular mechanisms of action e.g.
interaction with messenger molecule than target, immune-directed cytotoxicity
What are the risks and disadvantages of biologics?
Lack of efficacy and PK challenges:
- Administration and delivery to target tissue: injection rather than oral, biologic is also large thus tissue access may be an issue (barriers: intestinal lining, BBB, access to solid tumours)
- Species variation in protein sequences e.g. risk of lack of efficacy with non-human sequences (porcine/bovine insulin less potent, also RISK of immunogenicity
Manufacture:
- Complexity, reproducibility and purity of synthetic process e.g.
> proteins from multi-gene precursors, or matured via enzymatic cleavage
> correct folding and tertiary structure, avoiding aggregation of product
> process dependent variations in proteins: glycosylation state, AA modifications (post-translation mods may decrease efficacy/increase aggregation) such as oxidation, other components within isolated preparation
What are the 5 types of antibodies/immunoglobulins?
5-types, distinguished by type of heavy chain:
- IgA, IgD, IgE, IgG, IgM
- Subtypes exist (e.g. IgG1, IgG2…)
What is the principle immunoglobulin class/isotype used for biologics development?
IgG:
- Main circulating antibody/Ig in serum
- Responsible for 2º adaptive response (memory B-cells from initial antigen challenge = rapid Ab production second time)
Describe the basic IgG ‘Y’ domain structure.
Fab - antigen-binding fragment domain:
- Variable regions (Fv) responsible for antigen recognition
- 2x Fab domains = IgG is bivalent
Fc - constant fragment domain:
- Directs cellular interactions and immunogenic response via interaction with an Fc receptor
- Linker domain to conjugation with drugs
- Different Ig subtypes Fc’s have different receptors
E.g. IgE (inflammation Abs) - Fc interacts with Fc-episilon R on mast cells, in IgG Fc interactions with Fc-gamma on macrophages and neutrophils
What PK properties of the Fc domain (of IgG) are beneficial in biologics development?
- Fc domain also regulates Ig transport (e.g. across placenta)
- Thus extending plasma half-life of Ig molecules: they are able to be recycled, preventing elimination via endocytosis
Describe IgG structure with a focus on protein architecture.
Human IgG - 4 polypeptide chains:
• 2 Heavy chains (H - forms Fc domain as well as inner arm of Fab domain)
• 2 Light chains (L)
- Joined by disulphide bonds (top of the Fab/Y-region)
- Separate H and L chain genes exist
How are the 4 polypeptides of IgG are arranged?
Each of the 4 polypeptide chains are arranged in respective immunoglobulin domains (circa 120 AAs):
- Fc: CH1, CH2, CH3 and CL1 domains (similar/identical domains)
- Fab: VH, VL domains (highly variable in sequence - identifying antigen)
What are hypervariable regions within IgG protein architecture?
Within VH and VL domains of Fab polypeptide chains:
- 3 hypervariable regions exist on the external surface of the antibody, defining its binding with antigens (AKA complementarity determining regions [CDR])
- separated by 4 more conserved framework regions [FR]
- CDR/hypervariable regions vary dramatically between even IgGs: 10^16 distinct IgG molecules possible
- Determining recognition and interaction with antigens
Describe what form the IgG contact surface usually takes.
AKA the hypervariable region (3 regions of the VH/VL domains of the Fab regions):
- Forms recognition site
- IgG contact surface is usually flat or concave
- Fab region is sequence of beta-pleated sheets
What are the two classes of antibodies that can be raised/generated against an antigen by immunisation?
- Polyclonal antibodies (pAb)
- Monoclonal antibodies (mAb)
How do polyclonal and monoclonal antibodies differ?
Polyclonal (pAb):
- Many different IgG molecules (many subtypes) with high affinity for antigen which are purified from serum after immunisation
- Taken from rabbit, IgGs are not identical, may recognised different parts of the antigen
»> Not heavily used in medicine
Monoclonal (mAb):
- IgG producing plasma B cells isolated from immunised mouse, producing identical IgG molecules
- Liver B cells are cultured, with each cell producing the single antibody desired
- Current Ab therapeutics are monoclonal, hence -mab suffix (e.g. trastuzumab, infliximab)
How do monoclonal antibody therapeutics compare with sMW drug discovery?
Monoclonal antibodies technically:
- Are applicable to any antigen (e.g. microbe, protein, chemical target - inject anything/desired antigen into mouse hypothetically)
- Do not need structural knowledge of ‘binding site’ - mouse B-cells do the work/recognise antigen, naturally developing high affinity Abs
- IgGs can be identified by high affinity binding, then molecular properties elucidated.
What issues do solely mouse-derived monoclonal IgGs present?
PK issues:
- Risk of generating human anti-mouse antibodies (HAMAs); recognise mouse IgGs, leading to rapid degradation of human IgG and short plasma t1/2
»> Also potential immunogenicity (inflammatory response)
- Mouse Fc domains may also contribute to lack of efficacy
How are issues with sole mouse-derived monoclonal IgGs combatted? What are they?
Chimeric/humanised Abs are required for clinical IgGs:
Chimeric ( xi )
- Fc domains: human (constant)
- Fab domain: mouse (variable)
E.g. infliXImab
Humanised (zu)
- Fc domains: human (constant)
- Fab domain: human, aside from CDR/hypervariable regions which are mouse
E.g. trastuZUmab (Herceptin)
Human (u)
- Fc domains: human
- Fab domain: human (whole, inc. CDR regions)
E.g. AdalimUmab
What new technologies exist to improve cloning and production of human IgG therapeutics?
- In vivo immunisation of transgenic mice, engineered to produce human IgG (majority of fully ‘hUman’ IgGs marketed derived in this manner)
- Phage display: virus coats engineered to express human IgG domains, allowing screening and selection for target affinity without immunisation. Associated IgGs lack key post-translational modification (e.g. glycosylation - can be a pro or a con)
- Mammalian cell antibody display systems
What different mechanisms of action do IgGs exhibit?
- Receptor antagonism/inhibitors
- Antagonism of stimulating growth factor/messenger
- Agonists
- Ab-directed cell cytotoxicity (ADCC)
Describe the mechanism of action of cetuximab (licensed for head & beck, colon and lung cancers).
Antagonist for ligand (EGF) binding at the EGF receptor:
- Thus limiting EGFR dependent proliferation of tumour cells
- Classical antagonist (blocking EGF reaching receptor site)
Describe the mechanism of action of IgGs such as bevacizumab and infliximab.
Antagonism of stimulating growth factor/messenger:
- Bevacizumab: anti-VEGF therapy (growth factor for tumour angiogenesis; coats VEGF growth factor to prevent binding to tyrosine kinase VEGF receptor, turning off the signal)
- Infliximab: anti-TNFα therapy
> > > Targets messenger, NOT the receptor
How are IgGs used as agonists therapeutically?
Trial as agonists for death receptors linked to tumour apoptosis:
- Chemokine ligands (TRAIL) normally bind to ‘death’ receptors (e.g. DR4, 5) on tumour cells, activating pro-apoptotic signalling
- Bivalent antibodies (e.g. conatumumab) can mimic this activation by cross-linking DR4, 5 receptors - forced dimerisation results in downstream signalling for apoptosis of tumour cells (hijack death receptors)
> > > Not in clinic: not passed PI/II trials yet
Describe the IgG mechanism of action for ADCC (antibody directed cell cytotoxicity).
‘Immunotherapy’:
- Fc domains of IgG subtypes (particularly IgG1 and IgG3) recruit macrophages to IgG bound antigens or tumours cells)
- Leads to destruction by complement/cell mediated mechanisms (T cells)
- In longer term, response can be reinforced by adaptive immunity to the antigens presented by the macrophages following phagocytosis
What is adaptive immunity?
- Immune system/phagocytes ingest tumour cells and then present tumour antigens on their surface
- Allows targeting from T cells
Describe how trastuzumab (herceptin) combines different molecular mechanisms.
Trastuzumab targets the EGF-related receptor HER2 in HER2+VE breast cancer:
- Direct binding and inhibition of HER2 dimers and signalling
- Prevention of HER2 cleavage to a constitutively active form
- Endocytosis and degradation of HER2 receptors in cancer cells
- Antibody dependent cytotoxicity to destroy tumour cells (Fc domains recruiting macrophages etc)
