Affinity maturation

Introduction

In immunology, affinity maturation is the process by which TFH cell-activated B cells produce antibodies with increased affinity for antigen during the course of an immune response. With repeated exposures to the same antigen, a host will produce antibodies of successively greater affinities. A secondary response can elicit antibodies with several fold greater affinity than in a primary response. Affinity maturation primarily occurs on surface immunoglobulin of germinal center B cells and as a direct result of somatic hypermutation (SHM) and selection by TFH cells.In vivo, natural affinity maturation by the immune system takes place by somatic hypermutation and clonal selection. In vitro, in the laboratory affinity maturation, can be obtained by mutation and selection.

Affinity maturation occurs within the GC, where somatically mutated BCRs undergo selection on antigen retained on FDCs. Antigen is retained in the form of ICs and involves the interaction of both complement receptors and FcγRIIB with these ICs on FDCs. B cells also express both complement and FcγRIIB. An analysis of the role of RIIB in affinity maturation thus represents the contribution of both FDCs and RIIB to this process. The role of FDC-expressed FcγRIIB has been clarified by the development of chimeric mice in which only the FDC FcγRIIB is deficient and the B-cell FcγRIIB is retained. In these mice, potentiation of affinity maturation is seen. This potentiation likely results from the increased stringency of selection that occurs in the GC that, in turn, results from unopposed access of FDC ICs to B-cell FcγRIIB, as has been proposed previously. Coligation of the BCR and FcγRIIB attenuates the BCR signal by the negative signaling role of SHIP This negative signal would impose a requirement for a higher threshold for effective BCR stimulation and thus favor higher affinity BCRs. In addition, under those conditions where coligation to BCR is ineffective, ligation of FcγRIIB alone results in an apoptotic signal, resulting in the elimination of those somatically mutated B cells with low affinity for antigen. The situation is reversed when B cells that lack FcγRIIB expression are transferred. In that case decreased apoptosis is observed, and the resulting B cells display a reduced affinity for antigen (Kalergis and Ravetch, unpublished observations). This is perhaps due to the persistence of low-affinity B cells that arise as a consequence of somatic mutation and are normally eliminated by a negative selection mechanism involving FcγRIIB crosslinking and induce apoptosis. Because FcγRIIB is regulated on both FDCs and B cells, the potential is provided for fine-tuning the survival and selection of B cells in the GC by the interaction with ICs on FDCs.

Untargeted Mutagenesis

We use an error-prone PCR integrated DNA-shuffling approach to mutate mainly CDR regions during sub-library construction. If the potential of introducing immunogenic mutations to framework positions is not a concern, we usually use this approach to create mutations at completely random positions across the entire VH and VL fragments. In these cases, the genetic diversity of the sub-library is further increased via passage through our proprietary bacterial mutator strain, CD-affi™.

Oligonucleotide-directed Mutagenesis

We are able to create any sub-libraries to incorporate the defined mutations using trimer codon technology. Most of the time, we need study the AA sequences of the antibody to find out the conserved sequences (in comparison with the germ-line and antibody subfamily sequences). We may then introduce mutations to the positions in the frame work regions that are not conserved. Supposedly, these regions will be antigen-specific and change in these regions may not increase immunogenicity.

Antibody Affinity Measurement

We offer Biacore Analysis services for binding kinetic analyses of antibodies. We typically capture the antibody on the chip and run antigen on top of the captured antibody. The antigen will be ran at 6 different concentrations for each antibody and chi-square analysis will be performed on the binding constants we obtain from each antigen concentration. The documentation package will include a real time on-rate (Ka), off rate (Kd), an affinity constant (KD), chi square value and a graph of real-time binding kinetics. We would like to obtain ~50 uL of 1 mg/mL antigen and antibody solutions. We will need ~100 ug of antigen and ~50ug for each antibody. We would need MW information for the antigen as well. It may require special considerations for antigens with repeated or multiple epitopes for affinity determination.

Peptide Affinity Maturation

Alanine scanning mutagenesis is our favorite method in affinity maturation of peptide binders. In this method, each single AA of a selected binding peptide will be replaced with an alanine, and then the binding of the modified peptides to the target protein will be assayed using Biacore technology. The non-essential AAs will be specifically identified. After that, we will create a directed/constrained peptide sub-library that contains random sequences on the non-essential AA positions. Here, again, we frequently randomize the non-essential residues using "NNK" or "trimer codon" strategy. Mutants with increased binding affinity are identified by enhancing the screening stringency, followed by phage ELISA.

Affinity maturation is the process by which antibodies increased its affinity, avidity and activity through multiple rounds of somatic hyper mutation (SHM) in germinal centers. Successive generations of B cells mutate and present to the antigen, that recognize the antigen with high affinity will survive and the low affinity ones are eliminated. Affinity maturation process more often applied to antibody leads that are selected from library approaches using a phage display technique and the affinity of the leads have 10 –100 nM affinity levels to the target. In antibody drug discovery, affinity maturation is applied to antibody leads to increase the affinity at least 10 to 50 folds.

Our antibody affinity maturation process

• Binding test in relevant application

• Construction of a phage display screening protocol

• Definition of a strategy

• Assessment report including evaluation of your project’s feasibility and chance of success

• Library design using the mutagenesis strategy previously established

• Creation of an antibody DNA variant library

• Creation of the phage display library

• Library screening using the custom protocol established in first phase (4 to 6 panning rounds)

• Validation of the best phage variants in the relevant application

• Affinity comparison of the best single phages

• Antibody sequencing

• Gene synthesis and subcloning in an adapted expression vector

• Transient expression test in mammalian cells

No.1Preliminary study by our affinity maturation expert

No.2Design and construction of a mutated phage display library

No.3Library screening and variants selection

No.4Sequencing and recombinant expression of the best variants

No.5Antibody tests

Antibody characterization depending on customer’s request:

.        – KD determination: Biacore (SPR), Xelplex (SPRi), OctetRed

.        – Binding analysis: ELISA

.        – Stability analysis: DSC

.        – Aggregate analysis: SEC-HPLC

.        – Other options available

Affinity maturation is important in diagnostics and biotherapeutics as well as many other applications requiring strong antibody-antigen interactions.

When developing antibodies, many features can be required such as functionality (inhibiting activities for instance), specificity, use in particular applications and of course affinity. But it can be tricky to select an antibody with all these features from the start. It is therefore relevant to first focus on the functionality and application before working on improving the affinity using our high-class phage display platform.

For more information or to get a quote for our phage display services with the use of Affinity Maturation, don’t hesitate to contact us.