Background: Affinity maturation is one of the most applied strategies for monoclonal antibodies to enhance efficacy, reduce immunogenicity and improve potency. Sanyou Bio offers antibody affinity maturation services, which can improve affinity, blocking activity, species cross reactivity and developability.
Extreme enhancement of affinity activity
By applying advanced structure simulation and multiple mutagenesis strategies, the affinity of antibodies can be enhanced, and the average enhancement ranges from 5 to 100 folds.
Comprehensive verification with eukaryotic proteins
With mammalian cell expression system, our platform can provide natural conformation-like recombinant proteins. Moreover, comprehensive QC will be conducted to ensure the quality of the produced protein.
Suitable for various types of targets
Our SY-FLAM platform can serve for all kinds of targets, and can be customized for projects with high difficulty or special requirement. Representative targets: LAG3, IL- 23, CD47, Claudin18.2, etc.
Extensive experience project development
Our platform has performed more than 180 affinity maturation.The core R&D personnel have more than ten years of experience, and can carry out diversified affinity maturation according to project needs.
Comprehensive upgrade of technical processes
Develop various personalized screening strategies for different modified molecules, match with a series of high-throughput and automated screening equipment, obtain eukaryotic antibody proteins rapidly.
Rapid and qualified delivery within 4 weeks
Our SY-FLAM platform can achieve a single round affinity maturation within 4 weeks. In coordination with comprehensive QC system, we are able to accomplish delivery with high efficiency and quality.
1. Multiple strategies for library construction
SY-FLAM platform integrates a variety of library construction strategies, including combined single site mutagenesis covering all CDRs, serial mutagenesis of key CDR and chain shuffling based on high-capacity natural antibody libraries. Multiple strategies endow the constructed library with capacity over 1*109.
Strategy 1: A point-mutation antibody library covering each amino acid site of 6 CDRs
Strategy 2: Antibody library with sequential mutation of multiple amino acid sites against key CDRs
2. Diversified high-throughput screening system
2.1 High-throughput screening syste
The platform is equipped with automation solutions for the workflow of high-throughput phage display, including integrated high-throughput screening systems such as high-throughput screening, high-throughput library construction, and high-throughput expression. The high-throughput system ensures fast and effective screening and supports the subsequent delivery with efficiency.
2.2 Multiple screening approaches
The platform integrates multiple screening methods such as solid-phase screening, liquid-phase screening, solid-liquid cross screening, cell screening, and protein-cell cross screening. We also offer customized plan for screening based on the project objectives.
1. Antibody Functional Validation
1.1 Simultaneous improvement of affinity and functional activity
Background: The Parental Ab is superior to the Benchmark (reference antibody) in functional activity, but inferior in affinity activity.
Method: Sanyou adopted the single- and double-site saturation mutation library construction strategies, combining with targeted panning strategy, to improve affinity, to obtain optimal antibodies with affinity activity comparable to that of the Benchmark.
Results: As shown in Fig. 1A, Parental Ab (before modification) showed a 5-fold increase in affinity activity after modification, which was comparable to that of the Benchmark; as shown in Fig. 1B, the functional activity of the Engineered Ab (after modification) was improved 350 times when compared with Benchmark, indicating a simultaneous improvement in affinity and functional activity and a successful modification of affinity maturation.
Fig. 1A Binding affinity
Fig. 1B Functional activity
1.2 Dual enhancements of binding and blocking
Background: Since the binding and blocking activities of the Parental Ab were significantly weaker than the benchmark, optimized binding and blocking activities are desired.
Method: Affinity maturation based on multiple mutation libraries were performed on the parental Ab, and three rounds of solid-phase and liquid-phase screening were carried out to obtain the Engineered Ab.
Results: As shown in Fig. 2, the Engineered Ab showed comparable binding and blocking activity to the benchmark, indicating a significant improvement on the Parental Ab.
Fig. 2A Affinity activity determination
Fig. 2B Blocking activity determination
1.3 Improved species cross-reactivity
Background: Parental Ab demonstrate good binding ability against human antigen, but little against mouse antigen, indicating that its cross reactivity between human and mice is weak.
Method: With our SY-FLAM platform, single site and double site mutagenesis based libraries of Parental Ab were constructed, and three rounds of panning for mouse antigens were carried out to obtain antibodies with human-mouse cross-reactivity.
Results: As shown in Fig. 3, the Engineered Ab demonstrates human-mouse cross-reactivity. While maintaining human affinity activity,the EC50 value of the Engineered Ab for mouse antigen increased from 695.921 nM to 5.823 nM (120-fold increase).
Fig. 3A Human antigen binding activity
Fig. 3B Mouse antigen binding activity
1.4 Significant improvement of pH-dependent activity
Background: The Parental Ab shows no significant differences in binding properties at pH 6.0 or pH 7.2.
Method: To obtain antibodies with good binding activity under different pH conditions, Sanyou applied a single- and double-site combination method to construct the library, performed three-round panning at pH 6.0 condition and ensured that the affinity activity remained unchanged at pH 7.2 simultaneously.
Results: As shown in Fig. 4, the affinity activity of the Engineered Ab was comparable to that of the Parental Ab at pH 7.2 and was significantly lower than that of the Parental Ab at pH 6.0, indicating a successful modification of pH-dependent antibody.
Fig. 4 pH-dependent activity assay
2. Full-length antibody kinetic assay
2.1 Fortebio-based assay
Background: The fast dissociation rate (KD) of the Parental Ab results in unsatisfying in vivo pharmacokinetics.
Method: With our SY-FLAM platform, multi-site mutagenesis based libraries of Parental Ab were constructed, and after 4 rounds panning based on KD (determined by Fortebio), antibodies with low KD were obtained.
Results: As shown in Fig. 5, the KD value of Engineered Ab increased from 6.53E-8 M to 2.37E-10 M (276 fold increase).
Fig. 5A Parental Ab
Fig. 5B Engineered Ab
2.2 Biacore-based assay
Background: While benchmark-comparable antibody had been obtained, a 10-fold increase in KD was desired.
Method: With our SY-FLAM platform, single-site and continuous double-site mutagenesis based libraries of Parental Ab were constructs. The affinity kinetics of the Engineered Ab acquired by 3 rounds of screening was determined by Biacore.
Results: As shown in Fig. 6, while the activity of the Parental Ab was comparable to that of the Benchmark antibody, the KD value of Engineered Ab increased from 1.98E-9 M to 1.01E-10 M (20-fold increase).