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