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Service Overviews

Background: Affinity maturation is one of the most applied strategies for monoclonal antibodies to enhance efficacy, reduce immunogenicity and improve potency. SanyouBio offers antibody affinity maturation services, which can improve affinity, blocking activity, species cross reactivity and developability.


Methods: According to the principle of in vivo affinity maturation, we adopt a variety of mutation strategies, and used phage display technology to construct a large-capacity mutation library to obtain antibodies with significantly improved affinity.


Advantages: Antibodies with an affinity increase of several times to nearly a hundred times are obtained in 5-6 weeks.

Cases: By March 2022, our SY-FLAM (established in 2017) has completed more than 180 affinity maturation projects, and the antibody affinity can be improved up to 100 times.

Service Overviews Background: Affinity maturation is one of the most applied strategies for monoclonal antibodies to enhance efficacy, reduce immunogenicity and improve potency. SanyouBio offers antibody affinity maturation services, which can improve affinity, blocking activity, species cross reactivity and developability. Methods: According to the principle of in vivo affinity maturation, we adopt a variety of mutation strategies, and used phage display technology to construct a large-capacity mutation library to obtain antibodies with significantly improved affinity. Advantages: Antibodies with an affinity increase of several times to nearly a hundred times are obtained in 5-6 weeks. Cases: By March 2022, our SY-FLAM (established in 2017) has completed more than 180 affinity maturation projects, and the antibody affinity can be improved up to 100 times. Service Contents Service Service Details Client Provides Deliverables and Standards Time Affinity improvement 1. Quality control for antigen 2. Quality control for parental antibody 3. Library construction based on multiple mutagenesis strategies 4. Customized panning and screening plans 5. Eukaryotic expression of antibodies 6. Quality control of engineered antibody 7. Project-specified assays 1. Antibody sequence 2. Benchmark 3. Antigen 2-5antibodies (≥5-fold higher affinity than the Parental Ab ) 6 weeks Developability improvement 1. Antibody sequence 2. Benchmark 3. Antigen 2-5 optimized antibodies 5-6 weeks Block activity improvement 1. Antibody sequence 2. Benchmark 3. Antigen 4.Acceptor/ligand 2-5 antibodies (≥5-fold greater blocking activity than the Parental Ab ) 6 weeks Species cross reactivity improvement 1. Antibody sequence 2. Benchmark 3. Human antigen 4. Rat/monkey antigen 2-5 antibodies ( ≥5-fold enhanced species cross reactivity compared to the Parental Ab) 6 weeks Service Highlights 1. 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 to100 folds. 2. Comprehensive verification of eukaryotic expression With mammalian cell expression system (CHO/HEK293), our platform can provide natural conformation-like and highly bioactive recombinant proteins. Moreover, comprehensive QC will be conducted to ensure the quality of the produced protein. 3. Unlimited competencies for various target types 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. 4. Extensive experience project development Our SY-FLAM platform has performed more than 180 affinity maturation projects. Our core R&D experts have more than 10 years of experience, and will offers the best service based on the requirements of the project. 5. Rapid and qualified delivery in 6 weeks Our SY-FLAM platform can achieve a single round affinity maturation within 6 weeks. In coordination with comprehensive QC system, we are able to accomplish delivery with high efficiency and quality. Case Stastics 1. Validation of full-length antibody based on ELISA 1.1. 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. Methods: 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. 1, the Engineered Ab showed comparable binding and blocking activity to the benchmark, indicating a significant improvement on the Parental Ab. Fig. 1A Affinity Fig. 1B Blocking 1.2. 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. Methods: 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. 2, 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 695.900 nM to 5.810 nM (120-fold increase). Fig. 2A Human antigen binding activity Fig. 2B Mouse antigen binding activity 2. Full-length antibody kinetics assay 2.1. Fortebio-based assay Background: The fast dissociation rate (KD) of the Parental Ab results in unsatisfying in vivo pharmacokinetics . Methods: 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. 3, the KD value of Engineered Ab increased from 6.53 x 10-8 M to 2.37 x 10-10 M (276 fold increase). Fig. 3A Parental Ab Fig. 3B Engineered Ab Samples ID KD (M) ka (1/Ms) kd (1/s) R2 Rmax (nm) Ratio (WT/Ab) Parental Ab 6.53E-08 3.94E+04 2.57E-03 0.99 1.28 1 Engineered Ab 2.37E-10 3.70E+04 8.77E-06 0.99 1.22 276 2.2. Biacore-based assay Background: While benchmark-comparable antibody had been obtained, a 10-fold increase in KD was desired.. Methods: 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. 4, while the activity of the Parental Ab was comparable to that of the Benchmark antibody, the KD value of Engineered Ab increased from 1.98 x 10-9 M to 1.01 x 10-10 M (20-fold increase). Fig. 4A Parental Ab Fig. 4B Engineered Ab Fig. 4C Benchmark Samples ID KD (M) ka (1/Ms) kd (1/s) Rmax (RU) Chi2 (RU2) Ratio (WT/Ab) Parental Ab 1.98E-09 5.31E+05 1.05E-03 68.97 1.49 1 Engineered Ab 1.01E-10 7.57E+05 7.62E-05 92.83 0.07 20 Benchmark 1.56E-09 2.85E+05 4.46E-04 237.70 2.04 1

Service Contents

Service

Service Details

Client Provides

Deliverables and Standards

Time

Affinity improvement

1. Quality control for antigen

2. Quality control for parental antibody

3. Library construction based on multiple mutagenesis strategies

4. Customized panning and screening plans

5. Eukaryotic expression of antibodies

6. Quality control of engineered antibody

7. Project-specified assays

1. Antibody sequence

2. Benchmark

3. Antigen

2-5antibodies 

(≥5-fold higher affinity than the Parental Ab )

6 weeks

Developability improvement

1. Antibody sequence

2. Benchmark

3. Antigen

2-5 optimized antibodies

5-6 weeks

Block activity improvement

1. Antibody sequence

2. Benchmark

3. Antigen

4.Acceptor/ligand

2-5 antibodies (≥5-fold greater blocking activity than the Parental Ab )

6 weeks

Species cross reactivity improvement

1. Antibody sequence

2. Benchmark

3. Human antigen

4. Rat/monkey antigen

2-5 antibodies ( ≥5-fold enhanced species cross reactivity compared to the Parental Ab)

6 weeks



Service Highlights
  • 1. Extreme enhancement of affinity activity
    1. By applying advanced structure simulation and multiple mutagenesis strategies, the affinity of antibodies can be enhanced, and the average enhancement ranges from 5 to100 folds.
  • 2. Comprehensive verification of eukaryotic expression
    1. With mammalian cell expression system (CHO/HEK293), our platform can provide natural conformation-like and highly bioactive recombinant proteins. Moreover, comprehensive QC will be conducted to ensure the quality of the produced protein.
  • 3. Unlimited competencies for various target types
    1. 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.
  • 4. Extensive experience project development
    1. Our SY-FLAM platform has performed more than 180 affinity maturation projects. Our core R&D experts have more than 10 years of experience, and will offers the best service based on the requirements of the project.
  • 5. Rapid and qualified delivery in 6 weeks
    1. Our SY-FLAM platform can achieve a single round affinity maturation within 6 weeks. In coordination with comprehensive QC system, we are able to accomplish delivery with high efficiency and quality.

Case Studies
1. Validation of full-length antibody based on ELISA

1.1. 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.

Methods: 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. 1, the Engineered Ab showed comparable binding and blocking activity to the benchmark, indicating a significant improvement on the Parental Ab.


Fig. 1A Affinity


Fig. 1B Blocking


1.2. 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.

Methods: 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. 2, 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 695.900 nM to 5.810 nM (120-fold increase).


Fig. 2A Human antigen binding activity


Fig. 2B Mouse antigen binding activity

2. Full-length antibody kinetics assay

2.1. Fortebio-based assay

Background: The fast dissociation rate (KD) of the Parental Ab results in unsatisfying in vivo pharmacokinetics.

Methods: 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. 3, the KD value of Engineered Ab increased from 6.53 x 10-8 M to 2.37 x 10-10 M (276 fold increase).


Fig. 3A Parental Ab


Fig. 3B Engineered Ab


Samples ID

KD (M)

ka (1/Ms)

kd (1/s)

R2

Rmax (nm)

Ratio (WT/Ab)

Parental Ab

6.53E-08

3.94E+04

2.57E-03

0.99

1.28

1

Engineered Ab

2.37E-10

3.70E+04

8.77E-06

0.99

1.22

276


2.2. Biacore-based assay

Background: While benchmark-comparable antibody had been obtained, a 10-fold increase in KD was desired. Methods: 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. 4, while the activity of the Parental Ab was comparable to that of the Benchmark antibody, the KD value of Engineered Ab increased from 1.98 x 10-9 M to 1.01 x 10-10 M (20-fold increase).


Fig. 4A Parental Ab


Fig. 4B Engineered Ab


Fig. 4C Benchmark


Samples ID

KD (M)

ka (1/Ms)

kd (1/s)

Rmax (RU)

Chi2 (RU2)

Ratio (WT/Ab)

Parental Ab

1.98E-09

5.31E+05

1.05E-03

68.97

1.49

1

Engineered Ab

1.01E-10

7.57E+05

7.62E-05

92.83

0.07

20

Benchmark

1.56E-09

2.85E+05

4.46E-04

237.70

2.04

1