Abs

Question 1

What is a biologic?

Answer 1

A medicinal product whose synthesis, extraction, or manufacture involves living sources, either human, animal, or microbiological

Question 2

Give 6 examples of biologics

Answer 2

Protein based therapeutics
Gene and cellular therapies
Stem cells and transplantation
Vaccines
Blood products for transfusion
Diagnostic reagents e.g., allergic for allergy tests

Question 3

What was the first protein biologic?

Answer 3

Insulin - released in 1923

Question 4

What are the advantages of developing insulin/protein-based therapeutics?

Answer 4

Advantages:
* Meet an unmet clinical need. The actions of insulin at its receptor have never been replicated by small molecules, so it seems a biologic was the only solution.
* Tackle targets resistant to small molecule intervention such as receptors with large complex binding sites or orphan diseases/targets with unknown binding sites
* Potential for higher affinity and selectivity e.g., selection between closely related receptor targets or mutant forms of the target contributing to disease (pharmacogenetics)
* Potential for diverse molecular mechanisms of action due to interactions with messenger molecule rather than target. Also potential for immune-directed cytotoxicity.

Disadvantages:
* Biologics tend to have a complex structure, which can complicate the complexity, reproducibility, and purity of the synthetic process.
Proteins are derived from multi-gene precursors or matured via enzymatic cleavage. For example, Insulin has 2 linked peptide chains synthesised by enzymatic cleavage from a single protein-precursor. The correct folding of the tertiary structure is necessary to avoid aggregation of the product. Also, there are process-dependent variations in proteins such as glycosylation state, amino acid modifications (e.g., oxidation), and other components within isolated preparation.
* Administration and delivery to the target tissue is via injection rather than oral administration due to reduced tissue access due to membranes e.g., intestinal lining, BBB, access to solid tumours
* There can be species variation in protein sequences which risks a lack of efficacy with non-human sequences, and potential immunogenicity.

Question 5

Where was insulin initially isolated from?

Answer 5

Bovine/porcine pancreas

Question 6

What are the 5 human antibody immunoglobulins?

Answer 6

IgA
IgD
IgE
IgG
IgM

Question 7

Describe the structure of an antibody.

Answer 7

2 heavy chains and 2 light chains. Each chain has:
* Fab region: antigen binding fragment domain. This includes a constant region and a variable region (Fv). 2 Fab domains make a immunoglobulin bivalent.
* Fc region: the constant fragment domain. Made up of 2 constant regions.

Question 8

What does the Fc region of IgE antibody’s interact with?

Answer 8

IgE, an inflammation antibody, Fc region interacts with Fcε on mast cells.

Question 9

What does the Fc region of IgG antibody’s interact with?

Answer 9

IgG Fc region interacts with FcγR on macrophages and neutrophils.

Question 10

What is the Fab region of an antibody responsible for?

Answer 10

Antigen recognition

Question 11

What is the Fc region of an antibody responsible for?

Answer 11

Directs cellular interaction and immunogenic response via interaction with an Fc receptor.
Regulate Ig transport (e.g., across the placenta)
Extends plasma half-life of Ig molecules.

Question 12

Why are different subtypes of antibody immunoglobulins (e.g., IgG1, IgG2) possible?

Answer 12

Each antibody is arranged into immunoglobulin domains (H and L). The variable regions just have VH and VL.
Within these domains are:
* 3 hypervariable segments (aka complementarity determining regions (CDR))
* 4 conserved framwork regions – one on either side of each CDR region.
The CDR regions are on the external surface of the antibody and define its binding to antigens.

Question 13

What is infliximab?

Answer 13

A monoclonal antibody which binds to TNF-alfa, an inflammatory growth factor.

Question 14

What shape is the IgG contact surface usually?

Answer 14

Flat or concave due to the 2 variable domains (VH and VL) forming a continuous surface.

Question 15

What is the difference between polyclonal antibodies and monoclonal antibodies?

Answer 15

Polyclonal antibodies – many different IgG molecules with high affinity for an antigen purified from serum after immunisation.

Monoclonal antibodies – IgG producing B cells are isolated from an immunised mouse to produce identical IgG antibodies.

Question 16

How are monoclonal antibodies usually produced?

Answer 16

1. A mouse is immunised with the target antigen and its immune system makes antibodies against it.
2. The immune cells are isolated to find the antibody-forming vells which are fused with tumour cells to produce hybridomas.
3. These hybridomas are screened for production of the desired antibody, and those which do are cloned.
4. Clonal expansion is then done to isolate the target monoclonal antibodies.

Question 17

What are some issues with monoclonal IgG antibodies being produced in mice?

Answer 17

There is a risk of generating human anti-mouse antibodies (HAMAs) which will cause rapid degradation of mouse IgGs resulting in a short plasma half-life. It also could induce an inflammatory response.

Mouse Fc domains may contribute to lack of efficacy.

Question 18

What are chimeric and humanised antibodies?

Answer 18

Chimeric antibodies - Human constant domain, Mouse variable domains

Humanised antibodies - Human constant domains, Mouse CDR regions of variable domains

Question 19

Name 1 chimeric, humanised, and human antibody.

Answer 19

Chimeric antibodies (-xi-) e.g., Infliximab

Humanised antibodies (-zu-) e.g., trastuzuma

Human (-u-) e.g., adalimumab

Question 20

What are 3 ways of producing human antibodies?

Answer 20

In vivo immunization of transgenic mice, engineered to produce human IgG. This is used for the majority of fully “human” IgGs currently marketed derived in this manner.

Phage display – a process where a virus coat is engineered to express human IgG domains which allows screening and selection for target affinity without immunization. Associated IgGs lack key post translational modification (e.g. glycosylation)

Mammalian cell antibody display systems in development

Question 21

Name a IgG antibody which acts as a receptor antagonist, and its mechanism.

Answer 21

Cetuximab (Erbitux®) is a Mab licensed for head, neck, colon, and lung cancers. It acts extracellularly as an antagonist for ligand binding at EGFRs therefore limiting EGFR dependant proliferation of tumour cells.

Question 22

Name a mAb which acts as an antagonist of a growth factor or messenger. and its mechanism.

Answer 22

Anti-VEGF therapies such as bevacizumab (Avastin®) work by binding to VEGF and preventing it from binding to VEGFR (a tyrosine kinase) and promoting tumour angiogenesis.

Question 23

Name a mAb which acts as an agonist, and its mechanism.

Answer 23

Antibodies have been trialled as agonists for death receptors linked to tumour apoptosis. Normally, a chemokine ligand (TRAIL) binds to death receptors (e.g., DR4/5) on tumour cells, activating pro-apoptotic signalling. Bivalent antibodies such as conatumumab can mimic this activation by cross-linking the DR receptors. However, current IgGs have not passed phase I/II trials.

Question 24

Name a mAb which blocks interleukins and its mechanism.

Answer 24

Severe COVID infection can lead to a cytokine storm, a fatal event caused by infiltration of T cells and neutrophils, driven by IL6 release and binding of IL6 receptors to gp130. Tocilizumab blocks IL6 receptor signalling to reduce the likelihood of mechanical ventilation and death (NEJM). Currently, not all trials agree on the benefits of this.

Question 25

How might a mAb be used to stimulate antibody directed cell cytotoxicity?

Answer 25

The Fc domains of IgG subtypes (most notably IgG1 and IgG3) recruit macrophages to IgG bound antigens or tumour cells. This leads to destruction by complement/cell mediated mechanisms. Long term, this response may be reinforced by adaptive immunity, to the antigens presented by the macrophages following phagocytosis.

Question 26

How does Trastuzumab work?

Answer 26

Trastuzumab (Herceptin®), targets the EGF-related receptor HER2, for treatment of HER2 positive breast cancer via a number of actions:
o Direct binding and inhibition of HER2 dimers and signalling
o Prevention of HER2 cleavage to an constitutively active form
o Endocytosis and degradation of HER2 receptors in cancer cells
o Antibody dependent cytotoxicity by recruiting natural killer cells to destroy tumour cells

Question 27

Benefits of smaller antibody templates such as nanobodies:

Answer 27

Simplify genetic engineering e.g. to humanise amino acid sequence.

Increase stability of the therapeutic (e.g. gut acidic compartments)

Improve access to target tissues such as the CNS or solid tumours.

Question 28

What are the different types of antibody fragments?

Answer 28

F(ab)2 - disulphide linked bivalent antigen-binding domains (top 2 arms, no base).
F(ab)’ - individual monomeric antigen-binding domains (1 arm).
scFv - 2 Fv domains joined by peptide linker in one polypeptide encoded by one gene.

Question 29

How can antibody fragments be produced?

Answer 29

Enzymatic digestion:
o Pepsin – cleaves Fc (constant region), retaining F(ab)2.
o Papain – cleaves at a different site to release individual F(ab)’ domains.

Recombinant technology:
o Single chain (sc) Fv – variable domains (Fv) are joined by peptide linker in one polypeptide encoded by one gene.

Question 30

What are the pros and cons of small antibody fragments such as scGv and F(ab)’ domains?

Answer 30

Pros:
* Reduction in size from IgG (160kD) to scFv (30kD).
* Simplified manufacture due to single gene encoding and lack of glycosylation. Can allow antibody production in bacterial, rather than mammalian cell systems.

Cons:
* The Fc domain may be important for function, for example in the antibody-dependent cellular cytotoxicity (ADCC) mechanism of action or in recycling of the antibodies.
* Engineered fragments may have reduced structural stability and be prone to aggregation. This can cause issues with problems with yield during manufacture and risk of immunogenicity.
* Engineered fragments/domains may have a short plasma life as a lack of Fc domain and smaller domain size can contribute to greater elimination. Normal, full length IgGs have a predictable pharmacokinetic profile. They have a long plasma half-life (15-30 days depending on subclass) as it is not renally eliminated (too big). The fragments, however, are small enough to be excreted by the kidneys so have shorter half-life. This is not always a disadvantage, as depends on the indication.
Also, full size IgGs have reduced proteolytic degradation after phagocytosis as unbound IgG is recycled to the plasma via cellular FcγR receptors.

Question 31

Whatis Abciximab?

Answer 31

A chimeric F(ab)’ which binds to platelet glycoprotein IIb/IIIa to prevent fibrinogen cross-linking between platelets leading to aggregation and clot formation. It is used as an acute ant-thrombotic therapy during surgical procedures (e.g., cardiovascular angioplasty) and has the potential to be used as a stroke therapy.

Question 32

How does the size of Abciximab contribute to its function?

Answer 32

It has a short plasma half-life of 10-30 minutes as it is small enough to be renally excreted. However, this is suitable for its current licensed indication of preventing thrombosis during surgery.
Also, the high affinity of abciximab for GP IIb/IIIa means its binding to the target is long-lasting (effects on platelets take >24h to wear off), even after plasma clearance.

Question 33

What is the similarities and difference between Certolizumab and other anti-TNF alpha biologics?

Answer 33

Like Infliximab and Adalimumab, Certolizumab has an anti-inflammatory effect for use in the treatment of RA and Chron’s disease.
However, unlike Infliximab and Adalimumab, Certolizumab pegol is conjugated to PEG (polyethylene glycol). PEG has a size of 70kD, which increases the size of the molecule, thus reducing renal elimination, as well as increasing the F(ab)’ solubility, thus improving its pharmacokinetic characteristics.

Question 34

Name 2 disadvantages to PEGylated antibodies/fragments.

Answer 34

Increases the cost and complexity of the manufacture.
Since use of PEG has become more mainstream and is used in food items such as ice cream (prevents crystal formation during freezing), around 30% of individuals have developed antibodies against PEG, which means antibodies will be attacked and won’t work.

Question 34

What are nanobodies?

Answer 34

An antigen-binding domain fully encoded by a single heavy chain, therefore existing as a small monomeric VH domain.

Question 35

What species are nanobodies native to?

Answer 35

Camelids - llamas, alpacas, camels
Sharks

Question 36

How much smaller are nanobodies than antibodies?

Answer 36

Around 10x,
15-18kd, compared to 160kD antibodies

Question 37

What are some advantages of nanbodies?

Answer 37

Monomeric - less risk of aggregation
pH stable - allows oral delivery to the gut lumen (e.g., for Crohn’s treatment).
Recognise internal binding sites
Can be strung together to have multiple functions.
Can penetrate deeper into tissues to reach poorly vascularised areas.

Question 38

How do nanobodies recognise internal binding sites?

Answer 38

Most protein drug targets have binding sites recessed in their structure which are poorly immunogenic for standard IgG molecules in which the antigen binding surface is flat or concave. However, the hypervariable regions of nanobodies have longer CDR loops which produce a convex binding surface which is able to recognise internal binding sites.

Question 39

How does Caplacizumab work?

Answer 39

Caplacizumab is a bivalent nanobody (2 of same nanobidy linked together) for treatment of the congenital/acquired auto immune disease, Thrombotic Thrombocytopenic Purpura (TTP). This condition causes a lack of ADAMTS13 activity (normally cleaves vWF proteins into multiple smaller ones) resulting in deficient processing of the blood glycoprotein von Willebran factor (vWF) which leads to a build-up of ultra large vWF proteins which constitutively aggregate platelets.
Caplacizumab binds to the vWF and blocks the site which recruits platelets, thus reducing their aggregation.

Question 40

What are multivalent nanobodies?

Answer 40

Nanobodies which are linked to other nanobodies as well as other domains to improve efficacy and PK properties.

Question 41

Name a multivalent nanobody

Answer 41

BI836880 - tumour angiogenesis inhibitor which contains:
* Anti-vEGF nanobody – target vEGF
* Anti-AngII nanobody – target angiopoietin II messengers
* Proprietary domain to extend plasma half-life.

Question 42

What is a bispecific antibody?

Answer 42

The two arms of the antibody are different, allowing simultaneous binding to two different targets.

Question 43

Name a bispecific antibody

Answer 43

Normally, during the clotting cascade, factor VIII acts as a cofactor in the proteolytic activation of factor X by factor IXa.
In haemophilia A there is deficiency of factor VIII leading to reduced clot formation and increased bleeding. Emicizumab (HemLibra) is bispecific antibody which mimics factor VIII by crosslinking Factor IXa and X.

Question 44

Name some some methods of extending fragment plasma half-life.

Answer 44

Conjugate albumin (67kDa), PEG (70kDa), or Fc domains (from different types of antibodies to produce a different function, or to silence it from the immune system).

Question 45

How has conjugation been used to increase IgG CNS penetration?

Answer 45

sFab, an IgG against amyloid beta protein for treatment of Alzheimer’s plaques, is conjugated to a Fab’ domain-binding transferrin receptor. This transferrin receptor “carries” the conjugated antibody across the BBB via the endosomal transcytosis pathway.
After IV administration in rodent models, sFab binding was detected in CNS plaques, but not in negative controls.

Question 46

Name different ways in which conjugated antibodies can act as delivery vehicles.

Answer 46

Recruitment of immune system (fragment/domain therapeutics)
o Fc domains to restore antibody dependent cytotoxicity (ADCC)
o Cytokine peptides (e.g., TRAIL death receptor agonists).

Anti-cancer therapeutics – selective delivery of toxic payloads to target cells. These include:
o Radionuclides (e.g., I131 conjugates) – attached to antibody which targets certain cancer cells to allow targeted radiation therapy to cancer cells.
o Cytotoxins - trastuzumab emtansine is an FDA approved antibody delivers microtubule inhibitor to target breast cancer cells.

Biomedical imaging
o Nucleotide Labelling for PET scanning e.g., monitoring tumour metasases
o Tumour imaging with 89Zr-trastuzumab