4. Antibodies

Antibody

An antibody unit consists of four polypeptide chains: two identical heavy chains and two identical light chains connected by disulfide bonds. Different B cells produce different antibodies.

Fragment antigen-binding region (Fab region)

The part of an antibody that binds to antigens is called the Fab region. It is composed of one constant and one variable domain of each of the heavy and the light chain (carries the most diversity).

The fragment crystallizable region (Fc region)

The tail region of an antibody that allows other components to bind (like protein). Defines antibody isotype (IgA, IgD, IgE, IgG, IgM)

Epitope

An epitope is the part of an antigen recognised by antibodies (or T cells). Different antibodies may recognise very similar epitopes or very different epitopes.

Overlapping epitopes

Two or more epitopes on an antigen that share some portion of their molecular structure. Antibodies that recognize overlapping epitopes may compete for binding (the antibodies block each other’s binding sites).

Non-overlapping epitopes

May be simultaneously recognised by antibodies without interfering with each other’s binding

Polyclonal antibodies

A mixture of antibodies found e.g. in serum that origins from different antibody-producing B cells. High batch variability (problematic for production) and impossible to standardize completely. They bind many different epitopes with different functionalities. They can bind epitopes with different affinities and reaction rate kinetics.

Monoclonal antibodies

A single antibody that origins in a single antibody-producing B cells. It – recognizes one part (epitope) of the target (the antigen) with a single, defined binding strength (affinity). Different preparations have the potential to be very similar to each other.

Primary screening phase

Evaluate the binding affinity and specificity of antibodies to the target antigen. This step helps identify lead candidates for further development.

• Immunochemical assays (e.g. ELISA)

• High throughput flow cytometry screening assay (can my antibody bind to these cells that carry my target or not)

• Next Generation Sequencing (look for antibodies that are enriched suggesting that they have some binding ability to the target) 

 What techniques can be used for the target-based approach (to decide on a potential target)?

Gene expression profiling:

• Microarrays

• Next generation sequencing (NGS)

Proteomics:

• to define possible targets

Function-based approach

If you don’t have detailed knowledge about the target of interest. Look at different antibodies and see which would functioning.

Pre-existing functional knowledge approach

Build on the knowledge on receptor binding, function etc.

Sequencing

Determine the amino acid sequence of the selected antibodies. This information is crucial for downstream development and manufacturing.

Affinity determination

Measure the strength of binding between the antibodies and the target antigen. Want to identify variants that stay bound very well to the target.

Specificity screening assays

Assess the specificity of antibodies by testing their binding to related or structurally similar antigens to ensure selection for the intended target. The goal is to identify antibodies that specifically recognize the target of interest, thus minimizing the risk of off-target effects.

Orthogonal screening assay

Further validation of the binding specificity and characteristics of the antibodies. Performed on antigen on cells by flow cytometry.

Cross-reactivity

The ability of an antibody or a receptor to bind to different antigens or ligands that share similar structures. Can be beneficial (can respond to a more wide range of pathogens) but may also lead to unwanted immune responses such as autoimmune reactions.

Improving half-life of antibody

• Binding to FcRn: recycling of antibodies → extended half-life in circulation.

• PEGylation of smaller antibody fragments: (increases size → more difficult to secrete)

Antibody that does not bind cellular Fc receptors or complement

It helps minimize unintended immune responses.

Functional assay

Evaluate the ability of the antibodies to elicit the desired biological response or function

Examples:

• Cell killing 

• Apoptosis 

• Complement fixation 

• ADCC (Antibody-dependent cellular cytotoxicity)

• Inhibition of migration 

• Checkpoint inhibition

Epitope mapping

Define where on the molecule that antibody binds. Understanding epitope specificity is important for understanding antibody mechanism

– Immunoassay (ELISA, FRET assays, blocking of function etc.)

– Label-free assays e.g. surface plasmon resonance (SPR)

– HDX-MS (hydrogen-deuterium exchange mass spectrometry)

– X-ray crystallography

Developability

Evaulate if the product will have well behaving properties. Identifying problematic features for drug development like aggregation. Evaluation of factors such as stability, solubility and potential immunogenicity.

Animal model testing

Asses the therapeutic potential and safety of the antibodies in relevant animal models.

Antibody mechanisms of action

e.g.

•  Blocking

•  Signalling

•  Payload delivery (e.g. radioactivity, drugs)

•  Inhibition of migration

•  Induction of apoptosis (a cellular suicide process)

•  Recruitment of cell killing immune mediators (NK cells, complement etc.)

•  Cell crosslinking by bispecific antibodies (Bispecific antibodies = binds to different targets)

•  Cell killing through CAR T cells (will bind to tumor cells)

•  Checkpoint inhibitors – allowing immune control to be active

Check-point inhibitor

Immune checkpoints are key regulators of the immune system that when stimulated can weaken the immune response to an immunologic stimulus. Tumour cells can stimulate immune checkpoints → weaken immune response to protect themselves from attack

How is severity of anti-drug antibodies (ADA) impact correlated to frequency of occurence?

In general, the severity of anti-drug antibodies (ADA) impact and the frequency of occurrence have been inversely correlated to impact 

Humanization

Redesign of animal (mouse) antibody into protein with a human-like sequence

Chimerization

Redesign of animal (mouse) antibody into a molecule with partial human sequence

Immunization

To invoke production of specific antibodies (typically mice, now also e.g. rabbits)

Immortalization

Immortalization of antibody-producing capability through a fusion of antibody-producing B cells with immortal cell lines (survival and antibody-producing properties)

Hybridoma technology

Hybridoma technology is a method to produce human monoclonal antibodies by fusing B cells with myeloma cells, creating immortal cell lines that secrete specific antibodies.

Antibody phage display library

An antibody phage display library consists of bacteriophages presenting diverse antibody fragments on their surface. These libraries facilitate the selection and isolation of antibodies that bind to specific targets,