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Service Overviews

Background: Compared with traditional antibodies, camel derived antibodies with higher affinity, are, easy to be recombined, expressed, humanized and combined with other target antibodies.。 They have great application value and development prospects in disease diagnosis and treatment. Sanyou proudly launched MIT Alpaca monoclonal antibody customized service with all above functions.


Methods: The preparation technology of MIT Alpaca monoclonal antibody integrated four technologies: Alpaca immunity, construction of array phage antibody library, high-throughput screening of magnetic beads and recombinant expression of CHO eukaryotic cells, and matched with comprehensive physical, chemical and biochemical analysis for screening.


Advantages: Alpaca monoclonal antibodies with good specificity, high affinity, clear sequence and verified by eukaryotic expression can be obtained in only 3-6 weeks (from the end of Alpaca immunization).


Cases: Since the launch of the service in 2016, by December 2021, Sanyou biology has completed hundreds of projects through this platform; For each target, 40-60 lead antibody clones with unique sequence, good specificity and verified affinity can be obtained.

Service Overviews Background: Compared with traditional antibodies, camel derived antibodies with higher affinity, are,easy to be recombined, expressed, humanized and combined with other target antibodies.。 They have great application value and development prospects in disease diagnosis and treatment. Sanyou proudly launched MIT Alpaca monoclonal antibody customized service with all above functions. Methods: The preparation technology of MIT Alpaca monoclonal antibody integrated four technologies: Alpaca immunity, construction of array phage antibody library, high-throughput screening of magnetic beads and recombinant expression of CHO eukaryotic cells, and matched with comprehensive physical, chemical and biochemical analysis for screening. Advantages: Alpaca monoclonal antibodies with good specificity, high affinity, clear sequence and verified by eukaryotic expression can be obtained in only 3-6 weeks (from the end of Alpaca immunization). Cases: Since the launch of the service in 2016, by December 2021, Sanyou biology has completed hundreds of projects through this platform; For each target, 40-60 lead antibody clones with unique sequence, good specificity and verified affinity can be obtained. Service Contents Service name Service Details Client Provides Deliverables and Standards Time Customized service of MIT Alpaca monoclonal antibody Alpaca immunity Construction of immune Library High throughput screening Eukaryotic expression verification Target name, or target antigen, or cell line Deliverables: 1. Dozens of lead antibody molecules 2. Provide antibody samples as required Standards: 1. Antibody affinity ranking 2. The preferred molecule is verified by affinity kinetics or FACS About 4 months Humanization of Camel derived antibody Humanized design Eukaryotic expression verification Camel derived antibody sequence, target protein and control antibody, identification cell line, etc Humanized sequence, protein, plasmid and test report 4-6 weeks Service Highlights 1. Clear Antibody Sequence and Reduce Redundant Screening Work This service utilizes genetic engineering and phage display technology to obtain thegene sequence of antibody directly, so as to reduce unnecessary screening work. 2. Deliver Tens to Hundreds of Lead Antibody Molecules This service is adopting the multi group primer array library construction method and multi type screening library strategy, and dozens to hundreds of candidate molecules can be obtained from one target. 3. Full Coverage of Difficult Targets and Hidden Epitopes A large number of antibody molecules can be obtained for difficult targets such as G protein coupled receptors, ion channels and multiple transmembrane proteins, covering most of the protein epitopes. 4. Eukaryotic Expression, Cell Level Verification, Ensuring the Druggability of Delivered Molecules The molecules obtained from this service will be constructed and expressred at the eukaryotic level, and the physical and chemical properties and cell functions of the antibody will be comprehensively tested to ensure that the delivered lead antibody has good druggability. Service Features 1. Dozens of Lead Antibodies A large number of nano antibody lead molecules can be obtained through the customized service of Sanyou MIT Alpaca monoclonal antibody. Based on the verification of MIT Alpaca library with 10 targets, 475 antibody clones with unique sequences were obtained, and the median number of clones was 38. Fig. 1 Statistics of Binder amount/project 2. High Affinity of Lead Antibody The nanoantibody candidate molecules screened by this service have high affinity and can reach the pM level. The affinity testing of the full-length IgG constructed from the single domain antibody clone is as follows. The antibodies in Fig. 2 and Fig. 3 come from two different projects. The figures show that multiple molecules obtained from these two projects have significantly better or equivalent affinity than the control. Fig. 2 Affinity ranking assay of SD10 Fig. 3 Affinity ranking assay of SD41 3. Parameters Related to Drug Developability Assessment After the full-length molecular construction obtained from this service, theexpression amount of the antibody and the physical/chemical properties of the antibody were analyzed. The following Table shows a comprehensive evaluation system of drug developability assessment, including purity and concentration determination, primary structure analysis, affinity, and affinity kinetics. Table 1 Drugdevelopability of Antibody from MITA Category Test content Test method Purity and concentration Testing Purity identification SDS-PAGE/SEC/CE-SDS Purity and concentration Testing Concentration identification Protein A-HPLC/UV280 Primary structure analysis Molecular weight analysis LC/MS Primary structure analysis Electric point iCIEF Primary structure analysis Hydrophobicity identification HIC-HPLC Primary structure analysis Charge heterogeneity measurement CEX Primary structure analysis Peptide map analysis LC-UV-MS/MS Primary structure analysis N-sugar spectrum analysis LC/MS Affinity and Affinity Kinetics Affinity detection ELISA Affinity and Affinity Kinetics Affinity kinetic detection BLI/SPR Affinity and Affinity Kinetics Cell binding test (if any) FACS Case Stastics 1. Cell Level Binding Analysis Most of the nano antibody candidates screened throuhg this platform have cell level activity and blocking activity. Fig. 4 shows a case project using FACS to analyze the binding activity of antibodies to cell surface proteins. It can be seen that all lead antibody molecules show good cell level binding activity. Fig. 4 Binding affinity determination by FACS 2. Validation of Efficacy in Vitro The nanoantibody candidate molecules obtained through this service platform exhibits excellent efficacy in vitro, even better than the control antibody. In MLR experiment, CD4+T cells and DC cells with different donors were co-cultured. Anti-PD-L1 antibody can effectively block PD1/PD-L1 inhibition signal, activate T cells and stimulate the secretion of cytokines. As shown in Fig. 5, after treatment with PD-L1 candidate antibodies (A1, A2), the secretion of IL-2 increased significantly, indicating. ’ Fig. 5 In vitro evaluation of SD10 sdAb candidates by MLR 3. Validation of Efficacy in Vivo The nano antibody candidate molecules screened through this service platform reflect excellent efficacy in vivo. The in vivo antitumor efficacy verification cases of target candidate molecules in different mouse CDX models are shown in Fig. 6, Fig. 7 and Fig. 8. The treatment group and control group were administered twice a week for three consecutive weeks. The results showed that each target candidate antibody showed the same antitumor effect as the control antibody at each dose, even better than the positive control. Fig. 6 Tumor growth inhibition Fig. 7 Tumor growth inhibition Fig. 8 Tumor growth inhibition 4. The Progress of Representative Projects Table 2 The Progress of Representative Item No Target Indication Project Difficulties Molecular Form Library Source Progress SYZJ002 NCOV Novel coronavirus Broad spectrum neutralization activity Double antibody Alpaca immune bank In IND SY11-sAb CL18 Digestive tract tumor Multiple cross touch Nano antibody Alpaca immune bank In IND SYDX P003 PL1/H2/VF Digestive tract tumor ADCC Tertiary antibody Alpaca immune bank +Alpaca immune bank +Biosimilars Production cell line SYDX-B019 TR2/C4 Breast cancer, lung cancer Liver toxicity TNFR2 Double antibody Alpaca immune bank + Alpaca immune bank In vitro efficacy SYDX B020 TR2/C18.2 Solid tumor Liver toxicity TNFR2 Double antibody Alpaca immune bank +Alpaca immune bank In vitro efficacy SYDX-B022 TR2/PL1 Solid tumor Dual immunosuppression relief Double antibody Alpaca immune bank +Alpaca immune bank In vitro efficacy LK003 undisclosed Solid tumor Immune regulation related targets Full length antibody Alpaca immune bank In vitro efficacy SYDX B014 I17A/F Rheumatoid arthritis Neutralizing bispecific antibody Double antibody Alpaca immune bank +Alpaca immune bank In vitro efficacy DJ001 undisclosed AMD,DME Blocking antibody Full length antibody Alpaca immune bank In vitro efficacy DJ002 undisclosed AMD,DME Blocking antibody Full length antibody Alpaca immune bank In vitro efficacy

Service Contents

Service name

Service Details

Client Provides

Deliverables and Standards

Time

Customized service of MIT Alpaca monoclonal antibody

Alpaca immunity

1. Construction of immune Library

2. High throughput screening Eukaryotic expression verification

Target name, or target antigen, or cell line

Deliverables:

1. Dozens of lead antibody molecules

2. Provide antibody samples as required

Standards:

1. Antibody affinity ranking

2. The preferred molecule is verified by affinity kinetics or FACS

About 4 months

Humanization of camel derived antibody

Humanized design Eukaryotic expression verification

Camel derived antibody sequence, target protein and control antibody, identification cell line, etc.

Humanized sequence, protein, plasmid and test report

4-6 weeks


Service Highlights
  • 1. Clear Antibody Sequence and Reduce Redundant Screening Work
    1. This service utilizes genetic engineering and phage display technology to obtain thegene sequence of antibody directly, so as to reduce unnecessary screening work.
  • 2. Deliver Tens to Hundreds of Lead Antibody Molecules
    1. This service is adopting the multi group primer array library construction method and multi type screening library strategy, and dozens to hundreds of candidate molecules can be obtained from one target.
  • 3. Full Coverage of Difficult Targets and Hidden Epitopes
    1. A large number of antibody molecules can be obtained for difficult targets such as G protein coupled receptors, ion channels and multiple transmembrane proteins, covering most of the protein epitopes.
  • 4. Eukaryotic Expression, Cell Level Verification, Ensuring the Druggability of Delivered Molecules
    1. The molecules obtained from this service will be constructed and expressred at the eukaryotic level, and the physical and chemical properties and cell functions of the antibody will be comprehensively tested to ensure that the delivered lead antibody has good druggability.

Service Features
1. Dozens of lead antibodies

A large number of nano antibody lead molecules can be obtained through the customized service of Sanyou MIT Alpaca monoclonal antibody. Based on the verification of MIT Alpaca library with 10 targets, 475 antibody clones with unique sequences were obtained, and the median number of clones was 38.


Fig. 1 Statistics of Binder amount/project

2. High affinity of lead antibody

The nanoantibody candidate molecules screened by this service have high affinity and can reach the pM level. The affinity testing of the full-length IgG constructed from the single domain antibody clone is as follows. The antibodies in Fig. 2 and Fig. 3 come from two different projects. The figures show that multiple molecules obtained from these two projects have significantly better or equivalent affinity than the control.

Fig. 2 Affinity ranking assay of SD10


Fig. 3 Affinity ranking assay of SD41

3. Parameters related to drug developability assessment

After the full-length molecular construction obtained from this service, theexpression amount of the antibody and the physical/chemical properties of the antibody were analyzed. The following Table shows a comprehensive evaluation system of drug developability assessment, including purity and concentration determination, primary structure analysis, affinity, and affinity kinetics.


Table 1 Drugdevelopability of Antibody from MITA

Category

Test Content

Test Method

Purity and concentration Testing

Purity identification

SDS-PAGE / SEC / CE-SDS

Purity and concentration Testing

Concentration identification

Protein A-HPLC / UV280

Primary structure analysis

Molecular weight analysis

LC / MS

Primary structure analysis

Electric point

iCIEF

Primary structure analysis

Hydrophobicity identification

HIC-HPLC

Primary structure analysis

Charge heterogeneity measurement

CEX

Primary structure analysis

Peptide map analysis

LC-UV-MS / MS

Primary structure analysis

N-sugar spectrum analysis

LC / MS

Affinity and Affinity Kinetics

Affinity detection

ELISA

Affinity and Affinity Kinetics

Affinity kinetic detection

BLI / SPR

Affinity and Affinity Kinetics

Cell binding test (if any)

FACS


Case Studies
1. Cell level binding analysis

Most of the nano antibody candidates screened throuhg this platform have cell level activity and blocking activity. Fig. 4 shows a case project using FACS to analyze the binding activity of antibodies to cell surface proteins. It can be seen that all lead antibody molecules show good cell level binding activity.

Fig. 4 Binding affinity determination by FACS
2. Validation of efficacy in vitro

The nanoantibody candidate molecules obtained through this service platform exhibits excellent efficacy in vitro, even better than the control antibody. In MLR experiment, CD4+T cells and DC cells with different donors were co-cultured. Anti-PD-L1 antibody can effectively block PD1/PD-L1 inhibition signal, activate T cells and stimulate the secretion of cytokines. As shown in Fig. 5, after treatment with PD-L1 candidate antibodies (A1, A2), the secretion of IL-2 increased significantly, indicating.


Fig. 5 In vitro evaluation of SD10 sdAb candidates by MLR

3. Validation of efficacy in vivo

The nano antibody candidate molecules screened through this service platform reflect excellent efficacy in vivo. The in vivo antitumor efficacy verification cases of target candidate molecules in different mouse CDX models are shown in Fig. 6, Fig. 7 and Fig. 8. The treatment group and control group were administered twice a week for three consecutive weeks. The results showed that each target candidate antibody showed the same antitumor effect as the control antibody at each dose, even better than the positive control.


Fig. 6 Tumor growth inhibition


Fig. 7 Tumor growth inhibition


Fig. 8 Tumor growth inhibition

4. The progress of representative projects
Table 2 The Progress of Representative

Item No

Target

Indication

Project Difficulties

Molecular Form

Library Source

Progress

SYZJ002

NCOV

Novel coronavirus

Broad spectrum neutralization activity

Double antibody

Alpaca immune bank

In IND

SY11-sAb

CL18

Digestive tract tumor

Multiple cross touch

Nano antibody

Alpaca immune bank

In IND

SYDX P003

PL1 / H2 / VF

Digestive tract tumor

ADCC

Tertiary antibody

Alpaca immune bank + Alpaca immune bank + Biosimilars

Production cell line

SYDX-B019

TR2 / C4

Breast cancer,

 lung cancer

Liver toxicity TNFR2

Double antibody

Alpaca immune bank + Alpaca immune bank

In vitro efficacy

SYDX B020

TR2 / C18.2

Solid tumor

Liver toxicity TNFR2

Double antibody

Alpaca immune bank + Alpaca immune bank

In vitro efficacy

SYDX-B022

TR2 / PL1

Solid tumor

Dual immunosuppression relief

Double antibody

Alpaca immune bank + Alpaca immune bank

In vitro efficacy

LK003

undisclosed

Solid tumor

Immune regulation related targets

Full length antibody

Alpaca immune bank

In vitro efficacy

SYDX-B014

I17A / F

Rheumatoid arthritis

Neutralizing bispecific antibody

Double antibody

Alpaca immune bank + Alpaca immune bank

In vitro efficacy

DJ001

undisclosed

AMD, DME

Blocking antibody

Full length antibody

Alpaca immune bank

In vitro efficacy

DJ002

undisclosed

AMD, DME

Blocking antibody

Full length antibody

Alpaca immune bank

In vitro efficacy