Background

Introduction

Case

Work flow

Background

Monoclonal antibodies(mAbs) are one of the fastest growing pharmaceutical products in the near future. The world's first murine antibody named Ortholone OTK3 for the treatment of renal transplant rejection was approved in 1986. Since 2017, a total of 79 therapeutic mAbs have been approved in the European and U.S. market, which was valued at more than hundred billion dollars.Currently, there are more than 200 mAbs are in clinical study and more than 600 in preclinical development. They play a major role in treatment of a wide variety of diseases, including cancer, infectious disease, allergy, autoimmune disease and inflammation.

Monoclonal antibodies are produced mainly in the following forms:

1. Murine–100% mouse origin

2. Chimeric–approximately 65% human and 35% mouse origin

3. Humanized–95% human and 5% mouse origin

4. Fully human–100% human origin

Human-derived antibodies and fully humanized antibodies are the trend of mAbs in the future. However, there are many difficulties during the screening using human naïve library, such as the low affinity, or lack of antibodies with species cross-activity, and so on. Therefore, mouse immunization is also a preferred alternative to obtaining antibodies. In order to obtain numerous high affinity antibodies, sanyou has launched a Magnetic & Immunization & Tandem (MIT) Antibody Library service.


Introduction

The MIT antibody library is an antibody generation method combining mouse immunization, tandem construction of antibody library and magnetic bead based high-throughput screening technology. The process of this method is as follows:


01
Selection and Validation of antigen                                    

Multiple types of antigens could be selected as immunogen such as proteins, peptides, cells and DNA, according to specific project requirements .


02

Mouse immunization                                                

Usually, 3-15 mice are selected for multi-point, multi-pathway or multiple immunogen cross-immunization to obtain high titer.


03

Tandem construction of phage display libraries                              

Multiple mouse spleens are taken, and then RNA isolation and cDNA synthesis by reverse transcription using random primers. After that, dozens of primers are used to amplify all the light and heavy chain antibody genes and inserted into the phage display vector, finally, the arrayed antibody libraries are completely constructed.



04
Magnetic beads based high-throughput screening                                         

First, the target antigen is initially biotinylated and coupled to magnetic beads. Then, 3-4 rounds of panning will be applied through magnetic beads based high-throughput screening instrument. After than, numerous clones are picked for preliminary ELISA screening. Finally, positive antibody clones are acquired and sent for sequencing to obtain high-affinity lead antibodies with clear sequences.

Highlights

Save time compared with hybridoma technology

This service only takes about two months throughout the construction and screening process. Compared with the screening of hybridoma technology, it saves 1-2 months!


Antibody sequence clear

Genetic engineering and phage display technology are applied in this service, so the antibody sequences are clear.


Numerous antibodies

Multiple immunization methods, abundant immunogens and cross immunization by different species of antigen make the generated antibodies excellent. Coupled with a variety of screening strategies, dozens to hundreds of candidate molecules could be acquired for any target.


High affinity,better for screening

The candidate molecules obtained from MIT library have higher affinity than that from other sources like human naïve library, thus it is suitable for post functional screening.

Case

The number of antibodies obtained is extremely large

Method: Through antibody panning, preliminary screening, sequencing analysis through MIT library, more than 60 lead antibodies with unique sequences were obtained.

Result: As shown in Figure 1, the EC50 values of the lead antibodies are mostly at the sub-nM level.

Figure 1. Affinity ranking of the candidates from the MIT library

The antibodies obtained from MIT library show higher affinity

Method: Candidate antibodies against the same antigen were generated by MIT library and hybridoma technology, respectively. Affinity of the antibodies was tested by ELISA.

Figure 2. Affinity comparison of candidates from either MIT library or hybridoma technology

Result: As shown in Figure 2, the preferred antibody clones B1, B3 and C4 derived from the MIT library have significantly higher affinity than antibodies ( Project No.JK02) derived from hybridoma technology.

Antibodies from MIT library have species cross-activity

Method: Candidate antibody (Sample A) obtained from the MIT library was tested for the species cross activity by ELISA.

Result: As shown in figure 3, this candidate antibody has significant human, monkey cross-activity and low murine cross-activity.

Figure 3. Species cross-activity assay of sample A from MIT library

Antibodies from MIT library are more suitable for FACS detection

Method: The antigen overexpressed cells are sequentially incubated with different concentrations of the candidate antibodies and then the fluorescent secondary antibody.

Result: The binding activity of the candidate antibodies are shown in Figure 4, Figure 5 and table1. The result showed that the affinity of preferred antibodies (P4-C and P4-D) from MIT library is significantly higher than preferred antibodies (P4-F and P4-G) from hybridoma method. The details can be seen in Table 1.

Figure 4. Affinity Determination of antibodies from MIT library by FACS

Figure 5. Affinity Determination of antibodies from hybridoma by FACS

Table 1. EC50 summary of the antibodies from two different source

MIT library feature

Multiple immunization methods and abundant kinds of immunogens

Combination of phage display and mouse immunization

High-throughput screening

Sanyou has numerous successful cases through the MIT library. Our immune MIT library has the advantages of mature technology, experienced platform staff and a extreme optimization system. By March 2019, a total number of 352 immunization libraries had been successfully constructed by sanyou, and the cumulative library capacity exceeds 4.0×1010. Up to now, through MIT Library, sanyou has completed more than 30 projects, and totally obtained 770 high-quality candidate antibodies.

Work flow

Benefit

You can provide any type of antigen, such as protein, peptide, cell or DNA; or you can provide the protein sequence of antigen only; or you can provide the antigen sample only without telling the target name.


You will obtain dozens of candidate antibodies!


You will obtain antibodies with high affinity at sub-nM level!

 

You will receive the exact sequences of the antibodies!


Copyright © 2015 All rights reserved Sanyou Biopharmaceuticals Co., Ltd. Inc Shanghai ICP for:no.15044189-1