Antibody is an indispensable tool for basic research, clinical diagnosis, and therapeutics. However, sometimes an antibody with the most favorable specificity lacks sufficient affinity for a desired application. Affinity maturation is a process in which antibodies gain improved affinity, avidity, and antipathogenic activity. It is a result of somatic hypermutation (SHM) of immunoglobulin genes in Tfh cell (T follicular helper cell)-activated B cells, which combines with the selection for antigen binding. This iterative process occurs in germinal centers and proceeds for weeks after infection or vaccination. The resulting antibodies can be highly mutated with increased affinity to antigens by an order of magnitude.
Fig.1 Overview of affinity maturation. (Doria-Rose, 2015)
In Creative Biolabs, we conduct antibody optimization and antibody affinity maturation service that combines with molecular evolution and high-throughput phage library construction and screening to improve antibody potency and to save the cost of antibody production.
The in vitro affinity maturation has been used to optimize antibodies, antibody fragments or peptide molecules. Here, Creative Biolabs hopes to introduce Antibody Affinity Maturation Service to improve the target binding affinity of antibodies to antigens, based on principles of mutation and selection. In addition, we also offer HIAF-ProNano™ Platform to increase the affinity of single domain antibodies by engagement of protein nanocages without changing physicochemical properties of single domain antibodies. A standard maturation process is performed through the following steps.
Fig.2 An optimized process of affinity maturation in Creative Biolabs.
Phage display is the most commonly applied molecular technology in display and affinity maturation for a wide range of proteins. This method enhances the affinity of recombinant antibody fragments by virtue of random mutagenesis and phage display under stringent conditions. Random mutations are inserted by performing radiation, chemical mutagens, or several rounds of error-prone PCR. After construction of a mutated antibody gene library, affinity selection is started by panning with washing solutions optimized for off-rate-dependent selection. As a result, antibody affinity can be improved by at least 10 folds after each round of antibody affinity maturation through phage display.
Except that, Creative Biolabs can take advantage of computer hardware and modeling algorithms to complete a rational design of antibody structures and functions. With the help of computational methods, sampling antibody conformations and scoring designed antibody variants are convenient in antibody affinity maturation process.
➢ Bacterial display – The bacterial display presents thousands of copies of displayed protein on the cell surface. The most important advance made by cell-based bacterial display is quantitative screening and high enrichment ratios of proteins bound to fluorescently labeled ligands using fluorescence-activated cell sorting. In Creative Biolabs, our bacterial display retains many helpful features of phage display, including the ease of manipulation, high transformation efficiency, and familiarity of handling E. coli. Aided by this system, clients can obtain affinity maturation (at least 5 folds, even up to 100 folds), expression level and thermostable candidates.
➢ Yeast display – Yeast display can overcome limitations of bacterial hosts requiring of phage and bacterial display, since these methods potentially contain variants due to mismatched human/bacterial codon bias. In Creative Biolabs, yeast display benefits from the advantage of FACS-based selections, yet has the characteristic of eukaryotic protein folding pathways and codon usage greatly similar to mammalian cells. Thus, it may be the most ideal system for the surface display of mammalian cell-surface and secreted proteins, particularly antibodies.
➢ Ribosome and mRNA display – In Creative Biolabs, we conduct ribosome and mRNA display that both apply an in vitro transcription/translation (IVTT) system circumventing growth and transformation of cells. IVTT-based techniques promisingly create much larger libraries (>1012) than cell-based systems and potentially avoid expression bias. Notably, IVTT methods have a potential for affinity maturation via recursive mutation, in which candidates are further mutated after each round of selection.
i. Design phage library and perform library construction: 2 -3 weeks;
ii. High-throughput phage library screening: 1.5-2 months;
iii. Construction and expression of recombinant antibodies: 2-3 weeks.
iv. Antibody affinity measurement: 2-3 weeks
v. Deliverables: Top 3 clones, 100 μg purified antibody for each clone, full report.
✔ 10-100 folds affinity improvement;
✔ Remarkable homogeneity and biocompatibility;
✔ Making further affinity maturation by directed evolution or computational design;
✔ Multiple tools (ELISA, DSC, SEC-HPLC, etc.) for antibody characterization;
✔ Fast cycle and high efficiency: about 3-5 months for a project;
✔ Extensive experience and guaranteed services.
Antibodies are widely used as diagnostics and therapeutic reagents. High-affinity binding is essential for extending dissociation half-times, expanding detection limits, reducing drug dosages and improving drug efficacy. Our antibody maturation services are tailored for each project to ensure that affinity or other objectives from clients are met or exceeded. Experienced teams are assigned to focus on progressing projects and deliver high-quality results in a timely manner, so that we are considered as professional provider and attentive service partners.
For more information or quotes on our antibody engineering services, please contact us by e-mail.
For Research Use Only | Not For Clinical Use
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