MutaMap™ Mutational Activity Map

high throughput service for mutational activity maps of therapeutic proteins

A new key tool for developability engineering.

When it’s time to make final engineering decisions for your antibody or protein, MutaMap™ can help evaluate which individual point mutations to pursue. MutaMap™ is an in vitro assay system that helps explore the effect of substituting each amino acid in at each position in a protein sequence one by one with all 19 possible substitutions and find out the effect on protein activity. For example a sequence stretch of 100 amino acids will result in up to 2000 mutants to explore.

The approach of MutaMap™ is simple.

Each position of a protein of interest is mutated by site directed mutagenesis, expressed and tested for its affinity/activity. MutaMap™ does not use any surrogate measurement for affinity or activity. Cell free in vitro translation of proteins is combined with solution titration assays to measure affinity/activity. Both methods are optimized for high throughput processing of samples while still allowing for accurate measurements of affinity/activity. The technology is particularly suitable for investing the ligand binding interactions of high affinity monoclonal antibodies, down into the high femtomolar range where other approaches such as SPR struggle to deliver high
throughput results.

Watch our MutaMap™ Podcast

Mutations investigated in CDR1 and CDR3 of Avastin® heavy chain variable domain

 

Figure 1:
Shown above is an example of a mutagenesis heatmap generated for CDR1 and CDR3 from Avastin® heavy chain variable fragment.

 

For binding pair interactions MutaMap™ uses high throughput solution equilibrium titration (SET) immunoassays to determine the binding affinity for each construct tested.

Figure 2:
SET results for measuring the affinity of Avastin® scFv and
Lucentis® scFv. The resulting titration curve is regressed
according to the relevant mass action binding laws. The
robustness of the SET approach ensures that this high throughput
assay works well for affinities in
the single digit picomolar range, as demonstrated by the tight confidence
intervals.

What does MutaMap™ show you?

MutaMap™ delivers a heat map for your protein (see Figure 1 above) that shows you which point
mutations lead to an increase, decrease, or no change in affinity (or
other activity) or non-function of the protein when interacting with one
or more of its binding partners. Effectively you can learn which
mutations, one by one, are likely to be permissible or favourable in your
protein in terms of the key property of binding to a ligand.

MutaMap™ therefore allows you to make informed protein
engineering decisions for a range of key developability objectives which
include:

• Improving activity/affinity by cherry picking mutations, including in
  the CDRs of mAbs
• De-immunization
  while maintaining or even improving protein activity
• Altering cross reactivity, e.g. for improved cross-species reactivity
• Improved humanization or provision of other engineered features
• Stability/Manufacturability
  and other key developability improvements, achievable through deliberate
  point mutations
• Prolonging half-life
• Developing unique new composition of matter IP

 

Focus on position S105 in CDR-H3 of Avastin®
heavy chain variable domain

Figure 3: What is clear is that the MutaMap™ heatmap shows permissible
mutations, especially in CDR-H3 in cases that are not normally
considered conservative, e.g. in position S105. This opens up choices for re-designing the molecule
that would not normally be available based on computational assessments.

 

Figure 4: Position S105 in Avastin® CDR-H3 is mutated to T in
in Lucentis® MutaMap™ reconfirms that this mutation is indeed beneficial for
improving binding. It also shows that a number of other mutations
are available to match or improve the affinity of the construct
over the Avastin® wild type.

 

How does MutaMap™ compare to molecular evolution technologies?

Molecular evolution techniques such as phage display and other phenotype-genotype
coupled randomization techniques are most commonly used in the affinity
maturation process for monoclonal antibodies and other binding
scaffolds. The advantage of these technologies is that they help explore
a very large sequence space of combined mutations.

There comes a point
however when final decisions have to be made on the implementation of a
protein sequence where individual point mutations may be considered in
an antibody or therapeutic protein to meet a variety of design
objectives. Randomized molecular evolution is not appropriate for this
step. Exploring individual point mutations is nothing new, but it has
been difficult to carry this step out in very high throughput way,
especially where the objective is to clone and express every mutant and
then measure its affinity/activity with reasonable accuracy. This
is what MutaMap™ can achieve.

Where does MutaMap™ fit in as part a project for e.g. generating a candidate
monoclonal antibody for clinical development?

Figure 5:Example work flow for pre-clinial protein engineering of therapeutic
monoclonal antibody; individual projects may differ.

How long does a MutaMap™ project take?

Our objective is to complete medium size projects of exploring 500-2000 mutations in
approximately 8-12 weeks from receiving the customer’s protein sequence.
Larger projects will take slightly longer, depending on complexity.

What is the stepwise process for carrying out MutaMap™?

• First
  we will discuss with you the sequence space you want to explore. This
  may be the known paratope of a protein or the CDR and flanking regions
  of a monoclonal antibody.

• To save time and cost you may not want to explore substituting amino
  acids into the sequence that are considered highly non-conservative or
  prone to degradation when exposed. We will also discuss what you know
  about the binding interaction of your protein with its target, whether
  you have working immunoassays for this interaction and whether the wild
  type protein and isolated ligand is available to work with in an
  immunoassay system. Depending on the nature of the interaction and the
  binding partners we will ensure that the base assay of binding wild type
  protein to target works well.

• Once these
  details are agreed and the project is initiated we will run the
  mutagenesis agreed for each position, express the protein at small scale
  and carry out the equilibrium binding titration affinity measurement in
  high throughput.

What will you get?

A final technical
report delivered via our secure webserver showing you the affinity or
activity determination for the wild type and each mutant with confidence
interval. This will be presented in various formats for ease of
interpretation, including a standard heatmap.

For customers that
want to carry out protein antigenicity studies in parallel, these can be
carried out in approximately the same timeframe as the MutaMap™. This
means that within a period of approximately 8-12 weeks we will have
determined experimentally both the putative T cell epitopes and the
MutaMap™ of permitted mutations in your protein sequence. This
information can allow you to proceed with much better informed decisions
on how to address immunogenicity related issues for your program while
addressing simultaneously other developability related design decisions
for your sequence.