Service Overviews Background: Humanized single domain antibody can greatly reduce the immunogenicity of alpaca-derived antibodies and accelerate the launch of antibody-based drugs. Sanyou Bio provides sub-library of super-trillion single domain antibody library screening service with the humanization level of 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 attenable. Methods: Sanyou sub-library of super-trillion single domain antibody library(ST-SDAL) is constructed by using the cutting-edge technology of primer synthesis for human framework with excellent druggability. With a library capacity of up to 2.50E+11 cfu, ST-SDAL has independent intellectual property rights and is screened with comprehensive physiochemical and biochemical analysis. Advantages: It takes only 5-6 weeks to obtain humanized monoclonal antibodies with the humanization level of up to 98%, which have good specificity, high affinity, and clear sequence and have been verified through eukaryotic expression system. Cases: ST-SDAL has completed the verification of 12 targets of different difficulties since its release; 227 lead antibody clones were obtained on average with unique sequence, good specificity and verified affinity. Fig. 1 Statistics of the number of lead molecules for targets of different difficulties Service Contents Service Service Details Client Provides Deliverables and Standards Time Sub-library of super-trillion single domain antibody library screening service 1. Antigen identification 2. Antigen biotin labeling 3. Sub-trillion Humanized Single Domain Antibody Library Screening Service 4. Antibody full-length construction, expression and purification 5. Affinity assay and preliminary druggability analysis Target antigens (Over- expression cell lines will be provided as required) Deliverables: 1. 100 sequence-specific lead antibodies 2. 30 selected full-length antibodies Delivery standards: 1. Antibody affinity ranking 2. Molecules through affinity kinetics or FACS verification 6-8 weeks MIT alpaca recombinant monoclonal antibody customization services 1. Alpaca immune 2. Library construction 3. High-throughput screening 4. Eukaryotic expression verification Target name, target antigen, or cell line Deliverables: 1. Dozens of lead antibody molecules 2. Sample antibodies as needed Delivery standards: 1. Antibody affinity ranking 2. Molecules through affinity kinetics or FACS verification Approximately 4 months Service Highlights 1. High Level of Antibody Humanization Based on the accumulated experience in more than 130 molecules of humanization projects, the human framework with good druggability is used to build the library, and the humanization degree of single domain antibodies obtained is up to 98%. 2. A Large Number of Lead Antibodies After the verification of 12 projects, each project yields an average of 200-300 lead antibody molecules with antibody affinity reaching the level of pM. 3. Suitable for a Wide Array of Difficult Targets A large number of antibody molecules are obtained for a wide array of difficult targets, such as single-pass membrane, multi-pass membrane and cytokine targets, providing perfect solutions to antibody drug screening confronted with highly challenging. 4. Diversified and High-throughput Screening For different antigens, a variety of screening strategies, such as Fc/His label crossover, solid-liquid crossover, cell-protein crossover, competition and blocking for screening, may be developed, and lead antibody molecules can be rapidly obtained through high throughput solid-liquid phase. 5. Eukaryotic Expression System and Cellular Level Activity Verified The full-length lead antibodies are constructed, and then expressed through CHO/293 eukaryotic expression system, followed by physiochemical analysis as well as functional verification of cellular level in order to obtain more authentic and meaningful druggability data. Service Features 1. High Level of Antibody Humanization The ST-SDAL established by Sanyou 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, every target can obtain single domain antibodies with a humanization level of up to 98%, thus improving the druggability of antibodies. 2. A Large Number of Lead Antibodies A large number of nano-antibody lead molecules were obtained through the screening of the ST-SDAL. The ST-SDAL was validated by screening 12 different targets (as shown in Fig. 3), a total of 2734 humanized antibody clones with unique sequence were obtained, and the median number of clones was 235. Fig. 3 Antibody number per project 3. High Affinity of Lead Antibodies Most of the nano-antibody candidate molecules obtained through the screening of the ST-SDAL have excellent affinity activity. The affinity of the antibodies could often reach pM. According to Fig. 4, the molecule affinity analysis after full-length construction shows that most of the antibody clones had good affinity. Fig. 4 Kinetics determination by ForteBio 4. Overall druggability analysis After the construction of full-length sequence of the molecules obtained through the ST-hSDAL screening service, the expression level and physiochemical characteristics of the antibody were comprehensively analyzed. As shown in Table 1, the analysis covere multiple aspects such as purity and concentration determination, primary structure analysis, affinity, and affinity kinetics. Table 1 Druggability of Antibodies from ST-hSDAL Category Test Test method Purity and concentration detection Purity identification SDS-PAGE/SEC/CE-SDS Purity and concentration detection Concentration identification Protein A-HPLC/UV280 Primary structure analysis Molecular weight analysis LC/MS Primary structure analysis Isoelectric point iCIEF Primary structure analysis Hydrophobicity identification HIC-HPLC Primary structure analysis Charge heterogeneity determination CEX Primary structure analysis Peptide mapping analysis LC-UV-MS/MS Primary structure analysis N-glycan mapping analysis LC/MS Affinity and affinity kinetics Affinity ELISA Affinity and affinity kinetics Affinity kinetics BLI/SPR Affinity and affinity kinetics Cellular binding assay (demand-based) FACS Case Stastics 1. R&D of Anti-CLDN18.2 Nanobodies 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%. Clinical data show that 70% of gastric cancer patients have highly expressed CLDN18.2 with poor prognosisr; CLDN18.2 is specifically expressed in gastric epithelial cells, but not in other healthy tissues. Therefore, the development of antibody drugs that specifically recognize CLDN18.2 is expected to provide precise and safe treatment for gastric cancer patients. 1.1. Clinical competition pattern 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. 1.2. Monoclonal antibody MOA 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. 2. Critical results of anti-CLDN18.2 antibody 2.1. Analysis of binding activity at the cellular level Most of the nano-antibody candidates obtained through the screening of the Sanyou Sub-trillion Library have excellent cellular-level binding activity. Fig. 6 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 control antibody. Fig. 5 Binding affinity determination by FACS 2.2. In vitro validation of drug efficacy 2.2.1. ADCC & CDC assays The candidate antibodies contain an IgG1 Fc fragment, which can kill the target cells through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). As shown in Fig. 7, the ADCC effect of candidate antibody C2 on Human CLDN18.2-HEK293T cells was equivalent to that of the control antibody; as shown in Fig. 8, the CDC effect of candidate antibody C2 on Human CLDN18.2- HEK293T cells was equivalent to that of the control antibody. Fig. 6 ADCC assay on Human CLDN18.2-HEK293T cell Fig. 7 CDC assay on Human CLDN18.2-HEK293T cell 2.2. Endocytosis assays Endocytosis is the process by which a membrane surface antigen binds to an antibody and then endocytosis the antibody into the cell. Fig. 9 showed the toxicity analysis of anti-CLDN18.2 antibody-mediated endocytosis after toxin delivery into cells. 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. The endocytosis results indicated that candidate antibody C2 may be used in the development of ADC antibody drugs. Fig. 8 Internalization assay on Human CLDN18.2-HEK293T cell 2.3. In vivo validation of drug efficacy The nano-antibody candidate molecules obtained through screening of the ST-SDAL have excellent in vivo efficacy, even better than that of the control antibodies. As shown in Fig. 10, there is a case of the in vivo 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. 9 Tumor growth inhibition