Background

Properties

Key points

Technical background

The antibody library is started from a directional design based on the data analysis of more than 20,000 human naïve antibody genes and more than 150 antibody sequences from approved antibodies or antibodies at clinical phase III. The antibody genes are derived from tens of billions of healthy PBMC cells, and they are eventually constructed into self-developed display vector system.

At first, sanyou established E+10 size of fully human heavy chain antibody sub-library and E+10 size of fully human light chain antibody sub-library. Heavy chain antibodies and light chain antibodies were randomly combined and eventually complete the construction of the 2E+11 size of fully human naïve antibody libraries. Through increasing the proportion of germline genes with high druggability and good expression level, and combined with technological innovation and comprehensive quality control, the constructed antibody library ST-HuNAL can provide excellent performance of high quality and capacity. 

Through the validation by screening against dozens of targets, it shows that hundred or even hundreds of lead antibody molecules could be acquired through the library. The affinity, specificity and diversity of the lead antibodies all meat the international leading levels.

This antibody library ST-HuNAL will bring breakthrough to the field of antibody discovery and it could be comprehensively applied into the fields of therapeutic antibody discovery, diagnostic antibody discovery, antibody engineering and related fields. It will greatly contribute to the research and development of new drugs, disease diagnosis and scientific research, it will provide insights to resolve the current dilemma of “ difficult to develop high-quality innovative antibody drug”, and it will help to improve the accurate diagnosis, treatment of serious diseases, therefore benefit the patients.

Properties

1. Human antibodies are the absolute mainstream of antibody drug development.

Since the approval of the first antibody drug in 1985, 26 of the 66 antibody drugs that were approved for marketing are human antibodies, accounting for 39% (statistics counts to the point that the design of this antibody was completed).

Since 2015, 12 of the 22 monoclonal antibodies approved for marketing are human antibodies, accounting for about 54%. In 2017, 5 of the 6 approved antibodies are human antibodies, accounting for approximately 83%. Thus, humanized antibodies and human antibodies have become the mainstream of antibody drugs.

2. Phage display technology has become an vital way to develop human antibodies.

Among the 25 approved human antibodies, there are 8 antibodies obtained through phage display antibody library technology: Adalimumab, Belimumab, Raxibacumab, Ramucirumab, Necitumumab, Avelumab, Ranibizumab and Durvalumab, which represent 32% of the total. Evidently phage display technology has become an critical way to develop human antibody drugs.

3. The phage display human antibody library has been extensively validated.

Eight approved antibody drugs were obtained by phage display antibody library technology. Six out of these eight antibodies were obtained by phage display human antibody library. In addition to the eight marketed antibody drugs, at least 50 antibody drugs obtained by the phage display human antibody library are at different clinical research stages.

Thus, the phage display human library is the mainstream technology for phage display antibody drug production.

4. Comparative advantage of phage display human antibody library technology over hybridoma monoclonal antibody technology.

Hybridoma mAb technology was used to be the most important technology for antibody generation before the application of phage display technology. Phage display technology has gradually evolved into a mainstream technology for antibody discovery due to its overwhelming advantages.

Hybridoma monoclonal antibody technology is the fusion of myeloma cells and immune lymphocytes (immunized B lymphocytes) aiming for a highly pure monoclonal antibody production.

Phage display technology was first reported in 1990 for the construction and screening of antibody library.

As shown in the table, phage display antibody library technology has shown many advantages that hybridoma antibody technology lacks in many respects.

Number Parameter Hybridoma Phage display antibody library
1 Antibody source Mice or human Fully human
2 Library capacity 103 -104 >1010
3 Genetic diversity Poor Extremely high
4 Antibody screening time investment 3-4 months 3-4 weeks
5 Screening process Complex Simple
6 Antibody affinity Low-high High
7 Animal immunization Need No need
8 Cost High Low
9 Antibody gene cloning Time-consuming Direct access to antibody sequences
10 Subsequent transformation Time-consuming Simple and efficient
11 Light and heavy chain reorganization No Yes

5. Production process of Sanyou one hundred billion human antibody library.

6. Germline distribution comparison between the 2E+11 size of human antibody library ST-HuNAL from Sanyou and marketed antibody drugs.

6.1 Comparison of the heavy chain germline:

The distribution characteristics of of the heavy chain germlines of Sanyou ST-HuNAL library are shown in the left graph, wherein the ratios of IGHV1 and IGHV3 are 25.4% and 61.5% respectively and together sum up to 86.9% of the whole library.

The distribution characteristics of the heavy chain germline of the antibodies that have been advanced to the clinical phase III or marketed stages are shown in the right figure, wherein the ratios of IGHV1 and IGHV3 are 38.0% and 40.8% respectively and together comprising 78.8%. Concluded from above The distribution characteristics of the heavy chain germlines of Sanyou ST-HuNAL library are consistent with antibody drugs advanced into clinical stage III or marketed stages.

6.2 Comparison of the light chain germline

The distribution characteristics of the light chain germlines of Sanyou ST-HuNAL library comprises 57.3% IGKV1 and 36.4% IGKV3, they together account for 93.7% of the antibody library.

The distribution characteristics of light chain germline from antibody drugs that have been advanced to the clinical phase III or marketed are shown in the right panel, wherein the ratios of IGKV1 and IGKV3 are 45.8.0% and 23.9% respectively, together accounting for 79.7%.

Concluded from above, the distribution characteristics of the light chain germlines from Sanyou ST-HuNAL library are highly similar to the antibody drugs that have advanced into clinical stage III or marketed stages.

7. Comparison of the length of CDR3 between Sanyou ST-HuNAL library and IMGT database

7.1 Comparison of the heavy chain CDR3

The length of the heavy chain CDR3 of Sanyou ST-HuNAL library and the IMGT database are shown in the figure below. The figure indicates that length of the heavy chain CDR3 of the Sanyou ST-HuNAL library and the IGMT database are highly similar.

7.2Comparison of the light chain CDR3

As shown in the distribution frequency graph below, the length of the light chain CDR3 shows similar profile between Sanyou ST-HuNAL library and the IMGT database.

8. Property analysis of antibodies that generated from ST-HuNAL library

8.1 Summary of the lead antibodies

A total of 1711 antibody clones with unique sequences were obtained by screening against 17 targets with ST-HuNAL library. Some of the 17 acquied up to more than 300 antibody clones with unique sequences.

8.2 Affinity analysis of lead antibodies

The affinity of antibodies obtained fromSanyou ST-HuNAL library can often reach pM-Sub-nM. The following table shows the affinity data of several antibodies selected from one project, and they shows the affinity KD are from 1.75nM to 6.07 nM.

8.3 Species Cross activity of lead antibodies against human, murine and monkey proteins

Antibodies screened from ST-HuNAL library shows species cross activity against human, murine and monkey proteins. The following figures shows most of the 18 lead antibodies have good cross-recognizing activity for human, mouse and monkey.

8.4 Analysis of cellular binding, blocking and activation activity of candidate antibodies

The following figures are a case study using FACS to analyze the binding activity of antibodies to cell surface proteins (left), the blocking and activation activities of antibodies to receptor ligand binding(right). As shown in the figure, all of the 16 lead antibody clones exhibited comparable binding activity with reference antibody; 8 of the 14 antibodies exhibited excellent activation activity, and 5 antibodies exhibited good blocking activity. Several of the antibodies exhibit similar blocking and activation activities compared to the control antibodies.

8.5 Other functional and animal model level validation

In vitro functional and animal model level validation for a series of leading antibodies obtained from ST-HuNAL library are currently underway.

Key points for business and screening process

Time cost for screening of ST-HuNAL library: overall 1-2 months.

The business model of ST-HuNAL library : an open platform that can provide personalized services according to customer’s needs.


The flow of the application of ST-HuNAL library:

The procedure flows are antibody library selection, antigen identification, antibody screening (usually 3 rounds), affinity and functional screening

1) Antibody library selection

There are six sub-libraries listed as A, B, C, D, E, and F, and the library size of each sub-library is between 10 billion and 40 billion. Four libraries (A, B, C and D) are launched this time .

2)Antigen identification

Recombinant proteins are often directly used as antigens.

Sanyou applied SDS-PAGE, Elisa or FACS to determine antigen bioactivity in order to ensure the quality of antigen customer provided.

For customers who cannot provide their own recombinant protein, Sanyou Bio has a efficient eukaryotic protein expression platform to help customers prepare the antigen protein.

3)Antibody screening

Sanyou has established a variety of antibody screening methods such as antigen coated immuno-tube, antigen conjugated magnetic beads, and cell-based antibody screening. A suitable antibody screening method can be selected according to the characteristics of the antigen target.

4)Affinity screening and functional screening

Sanyou has established an integrated antibody affinity screening platform. Affinity screening can be performed by applying FACS, Elisa or other methods at the protein or cellular level according to the requirement of the project. After affinity screening, samples are prepared for functional screening based on MOA.


Taking the antigen coated immuno-tube screening method as an example, the process of antibody screening is shown in the figure below:




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