ANTIBODY ENGINEERING

By routinely generating full DNA sequence data for all monoclonal antibodies and antibody fragments that we generate (or are supplied with by customers), we are able to offer numerous antibody engineering services including tagging, reformatting, bi-species chimaeras, bi-specific antibodies, humanisation etc., and are also able to sequence customer supplied hybridoma cell lines.  We are also happy to discuss other customer requirements not specifically mentioned here.

Monoclonal Antibodies

There are two principal routes to generating monoclonal antibodies; either using traditional hybridoma approaches, or by using recombinant antibody technologies such as phage display.

 

Mouse Hybridoma
Hybridomas, as the name suggests, are a hybrid formed from two different cell lines.  Following immunisation, usually of a mouse or rat, spleen tissue is isolated and mechanically disrupted to release splenocytes.  Some of these are B-cells, each of which produces a single antibody, i.e. monoclonal.  The splenocytes are then fused with a suitable myeloma partner, e.g. murine SP2/0 cells, which imparts immortality to the fusion cell line.  After culturing in selective media, only fusions of a B-cell and a myeloma survive.  These are further cultured in multiple 96 well plates and supernatants screened for antigen reactivity.  Positive wells are cloned out by limiting dilution to yield wells containing single colonies each derived from a single hybridoma cell.  Further screening for antigen positive antibody allows hybridomas to be selected and expanded to yield the final hybridoma clone(s). 

Custom hybridomas are typically generated against peptides or whole proteins (recombinant or purified native), but can also be raised using crude protein preparations, whole cells, or DNA.  Whole proteins are usually customer supplied; however in-house generation can be discussed.  Scotia Biologics can assist with the design and synthesis of peptide antigens, which need to fulfil a number of specification criteria to give the best chance of success.  These include selecting surface-accessible linear epitopes that are sufficiently hydrophilic to be readily water soluble, and will likely adopt a native structure as a peptide.  These are typically 12 to 20 residues in length, and avoid sequences containing too many hydrophobic residues, glutamines, or internal/multiple cysteines, although a single N- or C-terminal cysteine can aid conjugation to carrier protein.  Non-native cysteine can be included at either terminus for conjugation purposes.

It is recommended that a monoclonal antibody program using peptide antigens include at least 2 – 3 different peptide sequences to maximise the likelihood of generating an antibody with the desired target specificity.  

A phased program with multiple checkpoints/invoicing points enables customers to control the costs of projects and balance costs with progress/success.  A Hybridoma project includes the following steps:

i)    Consultation, agreeing project objectives and strategy
ii)   Antigen (peptide) design, synthesis and preparation of conjugates (where not customer supplied immunogen)
iii)  Immunisation of multiple (4-5) mice, provision and screening of serum samples
iv)  Selection of best mouse/mice, cell fusion and initial cloning.  Screening of culture supernatants
v)   Selection of best clones, and further sub-cloning.  Supply of antibody and hybridoma (typically two clones) cryo-stocks.
vi)  Scale-up expression and purification of antibody.

Scotia will retain some vials of hybridoma for up to 6 months to provide a backup for the customer.

Although hybridomas are frequently described as immortal, they CAN over extended storage time or after large numbers of passages, show reduced yields or even stop producing antibody.  For this reason, for very important clones Scotia recommends that customers also request our hybridoma sequencing/rescue service. With the sequence of the V-genes determined, an antibody can readily be recovered by synthesising the genes and cloning them into a suitable expression vector.     

HYBRIDOMA RESCUE 

Although considered immortal, hybridoma cell lines often suffer from reduced productivity following multiples passages, and can lose viability in long-term cryo-storage.  Scotia Biologics can prevent such loses from happening by cloning and sequencing the variable region of the genes of hybridomas from mRNA.  This is also necessary where subsequent humanisation or genetic manipulation/engineering of the antibody is anticipated.  ‘Rescued’ hybridomas are tested and verified by ELISA as bacterial scFv prior to cloning as full IgG,  Whole IgG are finally verified by antigen binding ELISA of antibody expressed transiently in mammalian cells to confirm retention of antigen binding specificity.

HUMANISATION 

Non-human monoclonal antibodies are often generated via hybridoma technology (mouse, rat, rabbit), and for certain targets may have potential as therapeutic reagents in the treatment of human disease.  However, their use is often compromised by patient immune system recognition eg. Human Anti-Mouse Antibody (HAMA) responses.   In order to overcome this problem, and in so doing extend drug half-life and improve efficacy and safety, non-human antibodies can be ‘humanised’. Typically, the most immunogenic regions of foreign antibodies are the constant domains.  This can be overcome by replacing the animal-derived constant domains with the equivalent human structures.  For the variable domains, where antigen recognition is conferred, we select human framework sequences that best match those of the parental antibody.  The non-human CDRs are grafted onto these scaffolds, initially in silico, and various back-mutations designed maximise ‘human-ness’.  A small library of clones with different versions of the variable heavy and variable light chains are then screened in functional (binding) assays to identify combinations that retain as closely as possible the antigen binding characteristics of the parent antibody.  The best performing V-regions are then selected and re-engineered back into full IgG format.  The resulting antibody is expressed transiently and fully characterised.     

HYBRIDOMA

  • High yield

  • Scalable production

  • Less expensive

  • Often high affinity

  • High specificity

Advantages

Disadvantages

  • Selection process restricted

  • Selection from limited pool of clones

  • Limited opportunity to repeat selection

  • Lengthy generation time

  • Toxic antigens not possible

  • Epitope targeting not practical

  • May require humanisation

  • Sequence information not automatically available

PHAGE DISPLAY

Advantages

  • Scalable production

  • Rapid generation (naïve libraries only)

  • Large pool of clones to select from

  • Repeat selection possible

  • Greater control over selection process

  • Greater diversity of positives achievable (immune libraries)

  • High affinity (immune libraries)

  • High specificity (immune libraries)

  • Can target specific epitopes

  • Sequence information generated

  • Amenable to post-selection development or modification

Disadvantages

  • Production yield variable

  • More expensive

  • May have lower affinity (naïve libraries)

  • May require humanisation (immune/animal libraries)

ANTIBODY FRAGMENTS

Antibody fragments, most commonly scFv and Fab, have a number of advantages over whole IgG formats in many applications.  They are ideally suited to rapid, simple, cost-effective production in bacterial expression systems, and perform better than whole antibodies in many diagnostic (and some therapeutic) applications.   Scotia Biologics has developed a panel of vectors enabling whole antibody IgGs from an existing construct or expressing cell line to be converted to a variety of fragment formats (or vice versa).  Bacterial or mammalian expression system can then be employed to provide mg quantities of protein purified to customer’s specifications. 

CUSTOM MODIFICATIONS

By cloning and sequencing antibody V-region genes, Scotia Biologics is able to offer a range of additional services such as engineering antibodies or antibody fragments to introduce tags or sites for e.g. chemical conjugation of labels, purification, or for directed immobilisation.  Examples include poly-histidine, c-Myc, FLAG, and cysteine.  Customer-specified tags not listed are also available on request.

WHOLE MONCOLONAL ANTIBODIES

Scotia Biologics has developed a series of whole IgG mammalian expression vectors enabling antibodies from a wide range of isotypes to be generated.  We can provide any human IgG sub-class, together with mouse IgG2b and a variety of other host species if required to optimise reagent compatibility.  These can be generated from hybridomas, or antibody fragments originating from any species.  

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©2020 by Scotia Biologics Ltd.

Foresterhill Health Campus, Aberdeen, AB25 2ZD, UK