Service Overview
Background:Humanized single domain antibody can greatly reduce the immunogenicity of alpaca-derived antibodies and accelerate the launch of antibody drugs. Sanyou Bio provides screening service via the sub-library of sub-trillion single domain Ab Library with a humanization level up to 98% to address the problems faced by conventional single domain antibody preparation technologies, such as long preparation time, heavy screening workload, and a small number of candidate antibodies obtained.
Service Highlights
Super large library capacity enables hundreds of lead antibodies
With the trillion-level library capacity, a large amount of single domain antibodies with up to pM level affinity can be obtained for targets of different characters, including single transmembrane, multiple transmembrane proteins, cytokine, etc.
The superior humanized framework ensures drug developability
Based on the accumulated experience of over130 humanization projects of single domain antibodies and thorough investigation of the template humanized framework, single domain antibodies with up to 98% humanization degree and improved drug developability can be obtained.
Automation supports diversified cross screenings
A variety of screening strategies can be customized with different antigens, such as crossing screening with FC and his tagged antigens, with solid and liquid panning phases, with cell-expressing and protein antigens, and competition and blocking screenings. The lead antibodies can be rapidly obtained by high-throughput screenings with solid and liquid phases, and the whole process takes only several weeks.
Comprehensive validation of pharmaceutical properties with the mammalian cell expressed antibodiesThe full-length lead
The full-length lead antibodies are expressed by CHO / 293 eukaryotic expression system and purified. After systematic and comprehensive physicochemical and biochemical tests and multi-dimensional affinity kinetic tests, authentic and effective proprietary drug data can be obtained.
Service Content
Service Features
1. High level of antibody humanization
The ST-SDAL established by Sanyou Bio has a high level of antibody humanization. Through the accumulated experience in more than 130 humanization projects of single domain antibody molecules, by humanizing the framework region ( FR ) of single domain antibodies, we can obtain single domain antibodies with a humanization level up to 99%, improving the druggability of antibodies.
2. A large number of lead antibodies
A large number of lead molecules were obtained through the screening with the sub-library of ST-SDAL After validated by screening 14 different targets, as shown in Fig. 1, a total of 9423 humanized antibody clones with unique sequence were obtained, and the median number of clones was 606.
Fig. 1 Antibody number per project
3. High affinity of lead antibodies
Most of the nanobody candidate molecules obtained through the screening of the ST-SDAL have optimal affinity activity. The affinity of the antibodies could reach pM. As shown in Fig. 2, the molecule affinity analysis after full-length construction showed that most antibody clones had superior affinity.
Fig. 2 Kinetics determination by ForteBio
4. Comprehensive druggability analysis
After full-length construction of the molecules obtained through ST-SDAL screening, the expression level and physiochemical characteristics of the antibody were comprehensively analyzed. As shown in Table 1, the analysis covers multiple aspects such as purity and concentration determination, primary structure analysis, affinity, and affinity kinetics.
Table 1 Druggability of Antibodies from ST-SDAL
Service Process
Case Study
Case 2: Development of Nanobody Against SARS-CoV-2 Spike
1. Background
The SARS-CoV-2 Spike protein is composed of three identical monomers which include the S1 subunit and the S2 subunit. The S1 subunit domain includes NTD and RBD, and the S2 subunit domain includes FP, HR1, HR2, TM, and CP. The SARS-CoV-2 binds to angiotensin-converting enzyme II (ACE2) on the host cell, through the spike protein (S protein), to mediate virus entering the host cell. The mainstream variants of SARS-CoV-2 include Alpha, Beta, Delta, and Omicron (widespread currently). Most neutralizing antibodies that have been approved for emergency use are currently ineffective. Therefore, it is important to develop antibodies that bind epitopes with relatively conserved amino acid sequences and posses good neutralizing effects.
1.1 Clinical Competition Landscape
A total of 17 SARS-CoV-2-associated therapeutic antibodies are launched or in the late clinical phase worldwide. The US FDA has granted AstraZeneca‘s long-acting SARS-CoV-2 neutralizing antibody Evusheld (AZD7442) with emergency use authorization (EUA). Evusheld is a SARS-CoV-2 pneumonia antibody combining two long-acting monoclonal antibodies (150 mg tixagevimab and 150 mg cilgavimab). The first SARS-CoV-2 neutralizing antibody combination therapeutic agent with independent intellectual property rights in China is developed by TSB Therapeutics (Beijing) Co., Ltd., which is composed of amubarvimab injection (BRII-196) and romlusevimab injection (BRII-198).
1.2 Monoclonal Antibody MOA
The RBD-specific monoclonal antibody binds to the receptor-binding motif in the RBD domain on Spike protein. RBM is responsible for the initial binding of the virus to host cell ACE2, which initiates the virus entring the cell. The RBD-specific monoclonal antibody can block the interaction of RBM and ACE2 and works as a blocking agent of ACE2.
2. Key Results of Anti-Spike Antibody
2.1 Binding Affinity Testing
Candidate antibodies were tested for binding activity to the S protein of SARS-CoV-2 Omicorn variant by ELISA. The results of affinity activity assay are shown in Fig. 1. The binding activity of candidate antibodies S1D-Ab-092 and SID-Ab-102 to the S protein of SARS-CoV-2 Omicorn variant were comparable to that of the control antibody.
Fig. 1 Affinity testing of antibodies
2.2 Blocking Function Assay
Candidate antibodies were tested for the blocking function on the binding of SARS-CoV-2 S protein to receptor ACE2 by ELISA. The results are shown in Fig. 2. Candidate antibodies S1D-Ab-092 and S1D-Ab-102 both efficiently blocked the binding of the SARS-CoV-2 S protein to the receptor ACE2.
Fig. 2 Blocking testing of antibodies
2.3 Pseudovirus Neutralization
The neutralization activity of S1D-Ab-102 and the control antibody Sotrovimab on Omicron pseudovirus were detected by Luciferase Reporter System. As shown in Fig. 3, the results showed that the IC50 of S1D-Ab-102 was similar to that of the positive control, and the complete inhibition rate was superior to that of the positive control.
Fig. 3 Pseudovirus neutralization
Case 2: Development of Nanobody Against Nanobody-A
1. Background
Nanobody-A is a lymphocyte activation gene, a member of the immunoglobulin superfamily. The expression of Nanobody-A is negatively correlated with the immune-modulating function of specific T cells. Inhibiting Nanobody-A function can enhance the anti-tumor effect of specific CD8+ T cells. Nanobody-A is highly expressed in tumor-infiltrating lymphocytes (TIL) in various solid tumors. Nanobody-A is an emerging potential target for cancer immunotherapy.
1.1 Clinical Competition Landscape
Currently, there are no drugs launched worldwide targeting Nanobody-A. More than 30 Nanobody-A antibody drugs are under development worldwide. The fastest progress among those is the benchmark developed by BMS and ONO, which is in clinical II/III phase. The main therapeutic areas of drugs targeting Nanobody-A include cancer and autoimmune diseases. Nanobody-A is currently one of the second-generation targets of immune checkpoints with many clinical data and relatively certain druggability. Antibiotic drugs targeting this target may become important anti-tumor drugs in the future.
1.2 Monoclonal Antibody MOA
Monoclonal antibodies targeting Nanobody-A can promote the proliferation of T cells, as well as increase the levels of IL-2, IL-4, IFN-γ, and TNFα. In the absence of Nanobody-A, Lck binds to CD4 or CD8, eliciting phosphorylation of ITAM in the CD3ζ complex and subsequently recruiting the tyrosine kinase ZAP-70, thereby initiating the TCR signaling cascade and leading to T cell activation.
2. Key Results of Anti-Nanobody-A antibody
2.1 Affinity Activity Assay
The affinity activity of the candidate antibodies on huNanobody-A-CHO cells were tested by FACS and the results are shown in Fig. 1. Most candidate antibodies showed comparable affinity activity to that of the control antibody, with candidate A having an EC50 of 0.035 and affinity activity approximately 10-fold higher than the control antibody (EC50 = 0.342).
Fig. 1 Binding affinity determination by FACS
2.2 Blocking Function Assay
Candidate antibodies were tested for blocking function at Raji cellular level by FACS and luciferase reporter gene systems. As shown in Fig. 2 and Fig. 3, candidate antibody Nanobody-A showed comparable or superior blocking function to that of the control antibody.
Fig. 2 Blocking assay by FACS
Fig. 3 Blocking assay by luciferase reporter assay
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