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Service Overviews

Background: Bispecific antibodies with common light chain can reduce the mispair of light and heavy chains, and are highly consistent with natural IgG structure. Compared with various bispecific antibody formats that do not exist in human body, bispecific antibodies with common light chain have better druggability and higher safety. In order to obtain common light chain antibodies with good druggability, high affinity and high diversity, Sanyou Bio has launched a sub-library of super-trillion common light chain antibody library screening service.


Methods: Common light chain Fab-phage display libraries were constructed through the combination of fully human light chain sequences with good druggability and fully human heavy chain sequences with high diversity. The common light chain antibodies can be generated rapidly by high-throughput panning, screening, eukaryotic expression and identification.


Advantages: Common light chain antibodies targeting different epitopes of the same target or different targets can be generated in one step, and the obtained fully human common light chain antibodies can be directly used for bispecific antibody construction.


Cases: Sanyou Bio has completed the verification of dozens of projects through the human recombinant common light chain library, On Average, hundreds of leads with unique sequence and high affinity can be obtained for each target.


Service Overviews Background: Bispecific antibodies with common light chain can reduce the mispair of light and heavy chains, and are highly consistent with natural IgG structure. Compared with various bispecific antibody formats that do not exist in human body, bispecific antibodies with common light chain have better druggability and higher safety. In order to obtain common light chain antibodies with good druggability, high affinity and high diversity, Sanyou Bio has launched a sub-library of super-trillion common light chain antibody library screening service. Methods: Common light chain Fab-phage display libraries were constructed through the combination of fully human light chain sequences with good druggability and fully human heavy chain sequences with high diversity. The common light chain antibodies can be generated rapidly by high-throughput panning, screening, eukaryotic expression and identification. Advantages: Common light chain antibodies targeting different epitopes of the same target or different targets can be generated in one step, and the obtained fully human common light chain antibodies can be directly used for bispecific antibody construction. Cases: Sanyou Bio has completed the verification of dozens of projects through the human recombinant common light chain library, On Average, hundreds of leads with unique sequence and high affinity can be obtained for each target. Service Contents Services Service Details Client Provides Deliverables and Standards Time Sub-library of super-trillion common light chain antibody library screening services 1.Antigen identification 2.Antigen biotin labeling 3. screening of sub-trillion antibody library 4.Full-length construction, expression and purification of candidate antibody 5. Affinity determination and preliminary analysis of druggability Target name, or target antigen, or cell line Deliverables: 1. Dozens of antibody leads 2. Antibody samples as required Standards: 1. Affinity ranking of the antibodies 2. Affinity kinetics and FACS detection of preferred antibodies. 6-10 weeks Service Highlights 1. Preferable light chain, better druggability The light chain with better druggability was selected from thousands of human light chain germline sequences. 2. Fully human antibody, high safety Fully human antibody can be obtainedby phage display technology directly and with high safety. 3. Diverse heavy chains, more candidates The well-chosen light chain germline was combined with ten billions humanized heavy chain sequences, which has been validated with dozens of targets, hundreds of leads can be obtained for each target averagely. 4. Multiple panning strategies, high quality candidates Multiple panning strategies including cross-panning with Fc/His-tag antigen, cross-panning with solid-phase and liquid-phase antigens, cross-panning with over-expressed cell line and recombinant protein antigens, and competition or blocking panning were adopted to obtain preferable candidates efficiently. Service Features 1. Combination with Preferable light chain and heavy chain As shown in Fig. 1, the light chains from IgVK3-11, IGVK3-20 and IGVK1- 39 families have the highest proportion among the light-chain germline of natural human antibodies. Moreover, the heavy chains from IGHV3, IGHV4 and IGHV1 families are the most abundant in natural human heavy-chain germline in Fig. 2. SanyouBio constructed the sub-trillion human recombinant common light chain library by combining the heavy and light chain germline with high natural frequency. Fig. 1 frequency distribution of VL germline Fig. 2 frequency distribution of VH germline 2. High diversity of antibodies The length distribution of CDR3 is an important indicator for antibody diversity. Hundreds of clones were selected for sequencing and analysis, the CDR-H3 lengths range from 6 to 27 amino acids with extremely high diversity and shows a normal distribution, which was consistent with data from the database. Fig. 3 Comparison of CDR-H3 length distribution frequency 3. The prokaryotic expression rate was high Thirty-two clones were randomly selected for prokaryotic expression to detect the expression of antibody sequences from the sub-library of super-trillion common light chain antibody library constructed by Sanyou Bio. The results showed that more than 85% clones expressed no less than 1 μg/mL, and more than 50% clones expressed no less than 5 μg/mL, indicating that antibodies from the human recombinant sub-trillion library showed a good prokaryotic expression profile. Fig. 4 CLC clone expression in Escherichia coli 4. High expression in eukaryotic expression A total of 68 clones were randomly selected for eukaryotic expression to detect the expression ofthe antibody sequences from Sanyou Bio sub-library of super-trillion common light chain antibody library. The results showed the full-length antibody expression in CHO cell of almost all clones was above 10 μg/mL and showed a normal distribution, with a median peak value ranging from 30 to 40 μg/mL. These data indicates that the antibodies from SanyouBio sub-trillion human recombinant common light chain library exhibit high expression levels in eukaryotic system. Fig. 5 CLC antibody expression in CHO cell 5. Comprehensive evaluation system for medicinal property In the panning service of Sanyou Bio sub-trillion common light chain library, the expression level and the biochemical and physicochemical properties of candidates will be comprehensively analyzed after full-length antibody construction. As shown in Table 1, it includes the purityand concentration determination, primary structure analysis, affinity and affinity kinetics, etc. Table 1 Drug developability of Antibody from ST-CLCL category Test content Detection method Determination of purity and concentration Purity identification SDS-PAGE/SEC/CE-SDS Determination of purity and concentration Concentration determination Protein A HPLC/UV280 Primary structure analysis Molecular weight analysis LC/MS Primary structure analysis Isoelectric point iCIEF Primary structure analysis Hydrophobic identification HIC-HPLC Primary structure analysis Charge heterogeneity determination CEX Primary structure analysis Peptide mapping analysis LC-UV-MS/MS Primary structure analysis N glycogram analysis LC/MS Affinity and affinity dynamics Affinity detection ELISA Affinity and affinity dynamics Affinity kinetics detection BLI/SPR Affinity and affinity dynamics Cell binding test(if any) FACS 6. High diversity of lead antibodies Diverse leads from the SanyouBio common light chain antibody library. As shown in Fig. 6, evolutionary tree analysis of heavy chain sequences of a representative projectthe results showed that the sequences of heavy chain from each leads were significantly different, which indicatedthat the heavy chain sequences of the Sanyou Bio These data indicates that the antibodies from Sanyou Bio sub-trillion human recombinant common light chain library exhibit high expression levels in eukaryotic system. exhibit extremely high diversity. Fig. 6 Statistics of Binder diversity Case Stastics 1. Efficacy evaluation of bispecifc antibody The anti-tumor efficacy of common light chain bispecific antibody was validated in a nude mouse CDX model. As shown in Fig.7. The experimental groups were divided into PBS, monoclonal antibody control (Ab1), monoclonal antibody combination control (Ab1+Ab2), bispecific antibody control (BsAb BM) and treatment (CLC BsAb), and the above groups were injected with antibodies twice weekly, 8 times in total. The bispecific antibody (CLC BsAb) showed a better anti-tumor efficacy than the control bispecific antibody (BsAb BM) at the dose of 20 MPK, and similar withthe combination of monoclonal antibodies. Fig. 7 Tumor growth curve

Service Contents

Services

Service Details

Client Provides

Deliverables and Standards

Time

Sub-library of super-trillion common light chain antibody library screening services

1.Antigen identification

2.Antigen biotin labeling

3. screening of sub-trillion antibody library

4.Full-length construction, expression and purification of candidate antibody 

5. Affinity determination and preliminary analysis of druggability



Target name, or target antigen, or cell line

Deliverables:

1. Dozens of antibody leads

2. Antibody samples as required

Standards: 

1. Affinity ranking of the antibodies

2. Affinity kinetics and FACS detection of preferred antibodies.

6-10

weeks


Service Highlights
  • 1. Preferable light chain, better druggability
    1. The light chain with better druggability was selected from thousands of human light chain germline sequences.
  • 2. Fully human antibody, high safety
    1. Fully human antibody can be obtainedby phage display technology directly and with high safety.
  • 3. Diverse heavy chains, more candidates
    1. The well-chosen light chain germline was combined with ten billions humanized heavy chain sequences, which has been validated with dozens of targets, hundreds of leads can be obtained for each target averagely.
  • 4. Multiple panning strategies, high quality candidates
    1. Multiple panning strategies including cross-panning with Fc/His-tag antigen, cross-panning with solid-phase and liquid-phase antigens, cross-panning with over-expressed cell line and recombinant protein antigens, and competition or blocking panning were adopted to obtain preferable candidates efficiently.

Service Features
1. Combination with Preferable light chain and heavy chain

As shown in Fig. 1, the light chains from IgVK3-11, IGVK3-20 and IGVK1- 39 families have the highest proportion among the light-chain germline of natural human antibodies. Moreover, the heavy chains from IGHV3, IGHV4 and IGHV1 families are the most abundant in natural human heavy-chain germline in Fig. 2. SanyouBio constructed the sub-trillion human recombinant common light chain library by combining the heavy and light chain germline with high natural frequency.


Fig. 1 frequency distribution of VL germline


Fig. 2 frequency distribution of VH germline

2. High diversity of antibodies

The length distribution of CDR3 is an important indicator for antibody diversity. Hundreds of clones were selected for sequencing and analysis, the CDR-H3 lengths range from 6 to 27 amino acids with extremely high diversity and shows a normal distribution, which was consistent with data from the database.


Fig. 3 Comparison of CDR-H3 length distribution frequency

3. The prokaryotic expression rate was high

Thirty-two clones were randomly selected for prokaryotic expression to detect the expression of antibody sequences from the sub-library of super-trillion common light chain antibody library constructed by Sanyou Bio. The results showed that more than 85% clones expressed no less than 1 μg/mL, and more than 50% clones expressed no less than 5 μg/mL, indicating that antibodies from the human recombinant sub-trillion library showed a good prokaryotic expression profile.


Fig. 4 CLC clone expression in Escherichia coli

4. High expression in eukaryotic expression

A total of 68 clones were randomly selected for eukaryotic expression to detect the expression ofthe antibody sequences from Sanyou Bio sub-library of super-trillion common light chain antibody library. The results showed the full-length antibody expression in CHO cell of almost all clones was above 10 μg/mL and showed a normal distribution, with a median peak value ranging from 30 to 40 μg/mL. These data indicates that the antibodies from SanyouBio sub-trillion human recombinant common light chain library exhibit high expression levels in eukaryotic system.


Fig. 5 CLC antibody expression in CHO cell

5. Comprehensive evaluation system for medicinal property

In the panning service of Sanyou Bio sub-trillion common light chain library, the expression level and the biochemical and physicochemical properties of candidates will be comprehensively analyzed after full-length antibody construction. As shown in Table 1, it includes the purityand concentration determination, primary structure analysis, affinity and affinity kinetics, etc.


Table 1 Drug developability of Antibody from ST-CLCL

category

Test content

Detection method

Determination of purity and concentration

Purity identification

SDS-PAGE / SEC / CE-SDS

Determination of purity and concentration

Concentration determination

Protein A HPLC / UV280

Primary structure analysis

Molecular weight analysis

LC / MS

Primary structure analysis

Isoelectric point

iCIEF

Primary structure analysis

Hydrophobic identification

HIC-HPLC

Primary structure analysis

Charge heterogeneity determination

CEX

Primary structure analysis

Peptide mapping analysis

LC-UV-MS / MS

Primary structure analysis

N glycogram analysis

LC / MS

Affinity and affinity dynamics

Affinity detection

ELISA

Affinity and affinity dynamics

Affinity kinetics detection

BLI / SPR

Affinity and affinity dynamics Cell binding test(if any)

FACS

6. High diversity of lead antibodies

Diverse leads from the SanyouBio common light chain antibody library. As shown in Fig. 6, evolutionary tree analysis of heavy chain sequences of a representative projectthe results showed that the sequences of heavy chain from each leads were significantly different, which indicatedthat the heavy chain sequences of the Sanyou Bio These data indicates that the antibodies from Sanyou Bio sub-trillion human recombinant common light chain library exhibit high expression levels in eukaryotic system. exhibit extremely high diversity.


Fig. 6 Statistics of Binder diversity


Case Studies
1. Efficacy evaluation of bispecifc antibody

The anti-tumor efficacy of common light chain bispecific antibody was validated in a nude mouse CDX model. As shown in Fig.7. The experimental groups were divided into PBS, monoclonal antibody control (Ab1), monoclonal antibody combination control (Ab1+Ab2), bispecific antibody control (BsAb BM) and treatment (CLC BsAb), and the above groups were injected with antibodies twice weekly, 8 times in total. The bispecific antibody (CLC BsAb) showed a better anti-tumor efficacy than the control bispecific antibody (BsAb BM) at the dose of 20 MPK, and similar withthe combination of monoclonal antibodies.


Fig. 7 Tumor growth curve