Service Overview
The target to PCC integrated R&D service of Sanyou Bio's innovative antibody drugs covers various stages of antibody drug development such as collaborative investigation on project approval, overall solution of raw materials, candidate molecule production based on super-trillion phage display antibody library, one-stop in vitro efficacy evaluation, structure simulation + antibody engineering, and animal model-based efficacy evaluation. Sanyou Bio's one-stop R&D service of innovative antibody drugs allows rapid discovery of lead molecules as well as in vitro and in vivo efficacy validation to help customers take the lead in drug R&D.
As of September 2022, Sanyou Bio has accumulated experience in more than 100 Pre-PCC, PCC and preclinical development projects, and has rich experience in development of antibody drugs across different disease fields, different molecular types, and different mechanism targets. The company's professional team has grown to a scale of nearly 300 employees with over 70% staff possessing Ph.D. and master's degree in the R&D team. The team has comprehensive experience in innovative drug development and industrialization, supporting customers' projects to move forward.
Service Advantages
Advantage 1: Integrated innovative antibody drug discovery platforms
  • 10 functional modules and 50+ teams covering the whole process of drug discovery.
  • Complementary project management for administrative affairs and seamless connection of all platforms, to enable completion of PCC within 12 months.
Advantage 2: Distinctive technologies for various antibody generation
  • Seven trillion antibody libraries; thousands of lead antibodies can be obtained for conventional targets.
  • Various sources of antibodies: naive B cell antibody, B cell recombinant antibody, synthetic and semi-synthetic antibody, mouse immunization library, and alpaca immunization library.
  • Full coverage of drug modalities: fully human antibody, single-domain antibody, bispecific antibody, mouse monoclonal antibody and rat monoclonal antibody.
Advantage 3: Rich R&D experience through IND applications
  • 5+ projects have entered into IND application.
  • Successful R&D experience in 30+ PCC.
  • 100+ Pre-PCC R&D experience of various targets.
  • Successful cooperation with more than 300 pharmaceutical companies worldwide.
Advantage 4: Successful cooperation with more than 300 pharmaceutical companies worldwide
  • Carry out flexible and diverse business cooperation models based on the concept of customer first and oriented to the success of projects.
Service Content
Service Process
Obtain perfect PCC molecule with only seven steps!
STEP.1 Sanyou Bio's collaborative investigation on project approval
——– Professional scientists team providing differentiated projects matching customer needs with pipeline layout
  • 80+ target recommendation successful cases
  • 500+ target research and development experience
  • 1400+ target information summary database
  • Target libraries cover autoimmune diseases, tumors, chronic diseases
Based on the professional scientist team and rich development experience, Sanyou Bio explores targets information based on unmet clinical needs and establish an "ad libitum target library" for tumors, autoimmune diseases, cardiovascular diseases, ophthalmic and other diseases. Until now, Sanyou Bio's scientist team has investigated more than 500 targets cumulatively, successfully recommended and developed more than 80 targets covering various disease types and molecular forms. Targets can also be explored based on customer needs to find out the optimal targets for certain disease and the optimal indication layout based on target mechanism.
Sanyou Bio has also collated a complete project approval report for each member of the "target library", and conducted comprehensive research and analysis on target characteristics in terms of target structure, homology, specificity, marketing, clinical progress, patent analysis, etc., and formulated a targeted project development plan, to avoid the whirlpool of "involution" in advance through professional and in-depth target research.
STEP.2 Sanyou Bio's integrated raw material solution
—— Integrated solution for overexpressing cell line, secretory cell line, functional cell line, antigen, antibody and membrane protein
  • 14-day rapid protein preparation
  • 8-week monoclonal cell line construction
  • 800+ target overexpression cell bank
  • 2500+ antigen and control antibody libraries
Sanyou Bio has various eukaryotic expression systems such as Expi-CHO transient expression, Expi-293F transient expression and 293F transient expression, which can match with the expression requirements of most proteins. In addition, quality control can be conducted from multiple dimensions such as concentration, purity, molecular weight, stability, safety, charge heterogeneity, sequence variants and structure analysis, binding or blocking biological activity to ensure the quality of antigen antibody proteins.
Through 7-year development, Sanyou Bio has successfully completed the preparation of thousands of proteins, covering various molecular types such as nano-antibodies, human-derived or humanized IgG-type antibodies, cytokines and other types of recombinant proteins, and has accumulated rich experience in protein preparation.
Overexpression cell lines for research are important materials for antibody drug development. Overexpression cell lines are constructed with CHO, HEK293, and various tumor cells by electroporation, chemical transformation and lentiviral infection, and are screened by puromycin, hygromycin, G418, and GS high expression screening systems to obtain stable high-expression cell lines. As of Apr. 2022, hundreds of projects had been completed by Sanyou's cell line construction platform for research, covering the cell lines overexpressing secreted antigens, antibodies, membrane proteins, and transmembrane proteins, and more than 1000 overexpression cell lines had been successfully constructed with CHO, HEK293, Jurkat, and other cells, 90% of which can be stably passaged for 20 PDLs.
STEP.3 Sanyou Bio's super-trillion phage display antibody library
—— Core keystone for Sanyou Bio molecule production, trillion-level capacity, guarantee for preclinical research and development
  • Lead molecules obtained in 8 weeks
  • Ten trillion level large-capacity antibody libraries
  • One-stop panning and precise screening combination
  • Proven in depth through nearly 100 projects
Sanyou Bio's molecular discovery integrates animal immunization, fully human or immune-derived B-cell recombinant antibodies, synthetic and semisynthetic antibody libraries construction, phage display antibody libraries, recombinant antibodies mammalian cell expression, automation and high-throughput screening. It covers immune, natural, recombinant, synthetic, and semisynthetic antibody types, suitable for all kinds of antigen cross-screening and difficult targets with high challenge.
Sanyou Bio's "magnetic array high-throughput antibody screening" and rapid protein expression technology covering four key nodes: high-throughput panning, high-throughput screening, high-throughput construction, and high-throughput expression. The seamless connection between "phage display antibody library and mammalian cell protein expression" can help realize automatic, high-throughput, and precise screening. Compared with traditional methods, this platform can accelerate the R&D process by substantially improving the speed of screening and construction. It allows lead molecule discovery within 7 weeks, and allow the analysis of lead molecule early activity and preliminary druggability within 10 weeks, which greatly shorten the time cost of early research and development, and can quickly obtain lead molecules for validation. One-stop panning combined with precise screening has been verified in-depth through nearly 100 projects.
Based on Sanyou Bio's trillion-level fully human/semi-synthetic library, the screening of 16 targets produces the lead molecule at a median of 345 and an average of 317; Using Sanyou trillion humanized single domain antibody library, the screening of 14 targets produces the lead molecule at a median of 606 and an average of 673. It is also possible to obtain a considerable number of lead antibodies for high difficulty target screening and difficult multiple transmembrane protein screening, therefore ensuring sufficient molecules for efficacy screening, improving success rate.
STEP.4 Sanyou Bio's one-stop in vitro efficacy evaluation
——40+ SOPs, covering upstream and downstream signaling and functional validation of various types of targets
  • Primary cells and tumor cells
  • Signal mechanism and cytokine secretion
  • 96+ mature cell-based pharmacodynamic models
  • 200+ in vitro efficacy evaluation experience
In recent years, antibody drugs represented by immune checkpoint inhibitors have become a hot spot in the development of innovative drug. In this process, the in vitro efficacy evaluation model plays an irreplaceable role, which can greatly accelerate the process of antibody drugs discovery, screening and optimization. Sanyou Bio focused on the research and development of innovative antibody drugs, and it took 7 years to establish a stable, efficient and reliable in vitro efficacy evaluation team. Sanyou Bio systematically developed stable cell efficacy models of tumor surface antigens, suppressive immune checkpoints, co-stimulated immune checkpoints, and other immunomodulators to ensure the high-quality and efficient promotion of candidate antibody cell efficacy screening.
Sanyou Bio in vitro efficacy evaluation covers primary cell isolation, primary cell induction, flow cytometry screening, luciferase reporting system, cell killing, antibody endocytosis detection, phosphorylation detection, cytokine detection, enzyme bioactivity detection, neutrophil chemotaxis, etc. The platform has experience in in vitro efficacy evaluation of 200+ projects, and has established mature cell pharmacodynamic models of 60+ targets, tumor cell lines of 100+ targets, 10+ human primary cells, 40+ SOP guidance. It only takes 6 days to complete all in vitro efficacy evaluations of candidate molecules. Sanyou Bio's one-stop in vitro efficacy evaluation provides scientific, complete and high-quality services for antibody molecular efficacy evaluation.
STEP.5 Sanyou Bio's structure simulation + antibody engineering
—— Structure simulation combined with antibody engineering for ultmate affinity maturation and humanization
  • 95% humanization level of candidate molecule
  • Several times to nearly a hundred times increase in the affinity
  • AI-enabled structural simulation and antibody design
  • More than 40 designed antibodies are in the IND stage, and 7 are in the clinical trial stage
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, covering single point saturation mutation, multi-point sequential mutations, and fixed-point sequential mutations in each CDR. Our SY-FLAM platform (established in 2017) has completed more than 180 affinity maturation projects, and the antibody affinity can be improved up to 100 times.
Background: The fast dissociation rate (KD) of the Parental Ab results in unsatisfying in vivo pharmacokinetics .
Method: 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. 5, the KD value of Engineered Ab increased from 6.53x10-8 M to 2.37x10-10 M (276 fold increase).
The in-depth antibody humanization service integrates the technologies of non-human sequence 3D structure simulation and analysis, eukaryotic expression and purification identification, affinity and functional assay, etc. While maintaining the affinity and specificity of the antibody, the degree of humanization of the antibodies can reach more than 95%, and back mutations are less than 6. Since the launch of the service in 2016, SanyouBio has completed more than 200 cases of humanized antibody design, of which more than 40 designed antibodies are in the clinical application stage, and 7 are in the clinical trial stage.
STEP.6 Sanyou Bio's animal model-based efficacy evaluation
—— 80+ prefabricated tumor models, multi-pathway efficacy evaluation, comprehensive testing indicators
  • CDX, PDX
  • Monoclonal/bispecific antibodies, ADCs
  • Tumor, autoimmune diseases, cardiovascular diseases
  • 100+ animal pharmacodynamic models experience
动In vivo efficacy evaluation plays a vital role in basic research and therapeutic drug development. Sanyou Bio is committed to provide you with alternative strains for in vivo efficiency evaluation, corresponding to mechanisms of action, molecular types, mouse strains. A variety of pharmacological indicators such as cytokines, cells distribution and in vivo tumor imaging can be detected via immunohistochemistry, multi-color flow cytometry, and in vivo imaging.
By September 2022, we have successfully developed 80+ tumor models, covering major tumors, hematoma, autoimmunity (asthma), cardiovascular disease. And we have successfully completed efficacy evaluation of a series of targets such as TNFR2 and CD39, and accumulated extensive project experience for different molecular types.
STEP.7 Sanyou Bio's PCC delivery
—— Deliver validated in vitro and in vivo efficacy PCC within 12 months
  • 3+ molecules delivery
  • 4+ projects are in the IND application stage
  • 45+ PCCs R&D successful experience
  • 200+ Pre-PCC R&D successful experience
Based on "super-trillion phage display antibody library" and "integrated intelligent R & D platform for preclinical innovative antibody drugs", Sanyou Bio provides a one-stop R & D service from target to PCC and delivers preclinical candidate molecules (PCC) with favorable druggability potential.
As of September 2022, Sanyou Bio has accumulated nearly 100 projects’ experiences in PCC drug development, covering various antibody forms and indications, and are expected to complete 70+ PCC projects in 2022.
PCC molecular forms include monoclonal antibodies, bispecific antibodies, trispecific antibodies, and ADCs. The indications for the targets under development focus on oncology, autoimmune, infection, ophthalmology, and metabolism aspects.
After 7-years continuous innovation, Sanyou Bio's technical teams formed unique technical barriers and core competence through collaborative investigation on project approval, integrated raw material solution, super-trillion phage display antibody library, one-stop in vitro efficacy evaluation, structure simulation + antibody engineering, and animal model-based efficacy, which is a solid support for the integrated intelligent R&D service of innovative drugs.
R&D of Anti-CLDN18.2 Nanobodies
1. Background
CLDN18.2 is a membrane protein with a tight junction structure, with a quartic transmembrane structure; its two cleavage variants CLDN18.1 and CLDN18.2 share an amino acid sequence identity up to 91%. CLDN18.2 is exclusively expressed in gastric epithelial cells but not in other healthy tissues. Clinical data show that 70% of gastric cancer patients have highly expressed CLDN18.2 with poor prognosis.
Anti-CLDN18.2 monoclonal antibody stimulates the activation of the cells and soluble immune effectors that exert the effects of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by binding to CLDN18.2 on the surface of neoplasm cells. In addition, the anti-CLDN18.2 monoclonal antibody can also induce cell apoptosis and inhibit cell proliferation.
Currently, there is only one antibody drug targeting CLDN18.2 in Phase III, namely IMAB362; its clinical data in Phase II in combination with paclitaxel showed that the overall survival of patients with gastric and esophageal cancer overexpressed by CLDN18.2 increased from 9 months to 16.7 months.
C2 is a humanized nanobody developed and designed by Sanyou Bio and screened from the trillion-level single-domain antibody library, which shows better ADCC and CDC activity in cellular function experiments and better in vivo efficacy when compared with the control antibody.
Fig.1 Mechanisms of targeting CLDN18.2
2. Technical route for antibody discovery
In order to generate anti-CLDN18.2 antibodies, after 2-3 rounds of panning via trillion-level single-domain antibody library and eukaryotic expression validation, multiple candidate antibodies that specifically recognized CLDN18 with human-mouse cross activity were obtained that specifically recognized CLDN18.2. The detailed screening process is shown in Fig. 2.
Fig. 2 Workflow of anti-CLDN18.2 nanobody discovery
3. Analysis of binding activity at the cellular level
Fig. 3 showed the results of affinity assay of the candidate antibodies to human CLDN18.2-HEK293T cells by FACS analysis, the affinity level of all candidate antibodies was better than that of covers antibody.
Fig. 3 Binding affinity determination by FACS
4. ADCC & CDC assays
The full length candidate antibodies can kill the target cells through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). As shown in Fig. 4-5, the ADCC and CDC effect of candidate antibody (C2) on human CLDN18.2-HEK293T cells was equivalent to that of the reference antibody.
Fig. 4 ADCC assay on Human CLDN18.2-HEK293T cell
Fig. 5 CDC assay on Human CLDN18.2-HEK293T cell
5. Endocytosis assays
Endocytosis is the process by which a membrane surface antigen binds to an antibody and then endocytosis the antibody into the cell. The results showed that candidate antibody (C2) could mediate toxin killing on human CLDN18.2-HEK293T, and the effect is significantly superior to that of the control antibody.
Fig. 6 Cell killing assay on Human CLDN18.2-HEK293T cell
6. In vitro validation of drug efficacy
As shown in Fig. 7, there is a case of the anti-tumor efficacy validation of the candidate molecules targeting inhibitory immune checkpoint A in the MC38 humanized mouse model. MC38 cells were inoculated subcutaneously in humanized mice, the mice were intraperitoneally administered BIW*3 (twice per week for three weeks) after 6 days of tumor bearing. Candidate antibody (C2) dosed at 0.4 MPK showed completely inhibition, which showed excellent anti-tumor effect.
Fig. 7 Tumor growth inhibition
7. Approval for clinical trial with BC008
On April 1, 2022, Dragon Boat Bio announced that its independently-developed Anti-CLDN18.2 Antibody Injection (BC008) obtained implied permission for clinical trials from the National Medical Products Administration for the treatment of CLDN18.2-positive advanced malignant solid tumors.
Preclinical study data of BC008 antibody injection showed that the drug could specifically target and bind to cell surface CLDN18.2 molecules, activating NK cell activity, specifically killing CLDN18.2-positive tumor cells through Fc terminal functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), therefore exerting anti-tumor effects.
In vitro activity studies showed that the affinity of BC008 to CLDN18.2 and ADCC activity were significantly stronger than those of IMAB362, a drug with the same target; in vivo efficacy studies showed that BC008 exhibited dose-dependent anti-tumor effects on CLDN18.2-expressing human gastric cancer NUGC-4, human pancreatic cancer BxPC-3 and HEK293T mouse tumor model.
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