Service Overviews Background: For monoclonal antibody drugs, the multi-specificity of antibody target also brings great risks to the safety of usage, which is a big challenge for their development. Therefore, SanyouBio launches the service for non-specific binding assessment. Methods: Commonly used methods include recombinant proteins, overexpression cell lines, TCR and computer simulation. Advantages: Sanyou Bio specific detection library has included over 2000 target antigen proteins, over 1600 overexpression cell lines, and over 1000 target reference antibodies, which can be used for the rapid evaluation of binding specificity. In the future, the library will also contain over 4500 membrane protein overexpression cell lines to achieve full-target evaluation and facilitate the selection of optimal antibody molecules. In addition, SanyouBio also provides TCR tests on humans, rhesus monkeys, and rats to assist clients in clinical application. Cases: So far, Sanyou Bio has completed the preliminary non-specific evaluation of pre-PCC molecules at the cellular level for more than 30 projects, and TCR evaluation for several projects. Service Contents Service Service Details Client Provides Deliverables and Standards Time Target recombinant proteins binding tests Binding of antibodies to be tested with over 6500 target recombinant proteins 1. Candidate antibody 2. Positive antibody 3. Target information 1. Free target protein combination 2. Preliminary screening of 1 concentration point 3. Reinspection of 8 concentration points 4. Test report 6–8 weeks* Overexpression cell lines binding tests Binding of antibodies to be tested with over 4500 overexpression cell lines 1. Candidate antibody 2. Positive antibody 3. Target information 1. Free target cell line combination 2. Preliminary screening of 1 concentration point 3. Reinspection of 8 concentration points 4. Test report 6–8 weeks* Tissue cross-reactivity (TCR) Cross combination of antibodies to be tested with various species/ tissues 1. Candidate antibody 2. Positive antibody 3. Target information 1. More than 2 sections of the same species 2. At least 2 concentration levels of antibodies 3. Negative and positive tissues 4. High resolution images and interpretation 5. Test report 6-8 weeks *Protein and cell libraries are under continuous preparation and validation. Stay tuned Service Highlights 1. Over 6500 target recombinant proteins Extensive target protein library and 7-day protein preparation process enable rapid and comprehensive completion of antibody non-specific evaluation. 2. Over 4500 Membrane protein overexpression cell lines Multiple target cell libraries and non-specific antibody evaluation based on flow analysis enables accurate determination of non-specific targets. 3. TCR tests on humans, Rhesus monkeys, and rats 33 human tissues, 30 Rhesus monkey tissues, and 30 rat tissues. 4. Rich project testing experience Accumulated detection experience of dozens of projects, covering proteins, cells, and tissues. Case Stastics 1. Cross binding of antibody to recombinant proteins of the same family The non-specific binding of the candidate antibody of a target to a protein of the same family. Fig. 1 to Fig. 6 show that the candidate antibody has no non-specific binding signal with histones of the same-family target (only part of the data is displayed), which is consistent with the Benchmark, indicating good specificity. Fig. 1 Binding of antibody to target proteins Fig. 2 Binding of antibody to CD27 protein of the same family Fig. 3 Binding of antibody to GITR protein of the same family Fig. 4 Binding of antibody to OX40 protein of the same family Fig. 5 Binding of antibody to 4-1BB protein of the same family Fig. 6 Binding of antibody to BCMA protein of the same family 2. Cross binding of antibodies to membrane proteins on cells The non-specific binding of the candidate antibodies against a specific target on normal human dermal fibroblasts (NHDF) and human umbilical vein endothelial cells (HUVEC) is detected. Fig.7 and Fig.8 show that the candidate antibodies A1, A3, and A6 have no non-specific binding signal with membrane proteins on the surface of NHDF or HUVEC cells, which is consistent with the Benchmark, indicating good specificity. Fig. 7 Binding of antibodies to NHDF Fig. 8 Binding of antibodies to HUVEC Fig. 9 and Fig. 10 show the non-specific binding of the candidate antibodies of a certain target on human embryonic kidney cells (HEK293) and human T lymphocytic leukemia cells (Jurkat). The figures show that the two candidate antibodies have no non-specific binding signal with membrane proteins on the surface of cells, which is consistent with the Benchmark, indicating good specificity. Fig. 9 Binding of antibodies to human embryonic kidney cells Fig. 10 Binding of antibodies to human T lymphocytic leukemia cells 3. Tissue cross-reactivity (TCR) Purposes: To confirm the binding of the tested antibody to the epitope of efficacy target, detect whether the antibody binds to the tissue antigen of the non-target site (such as tissue cross-reaction antigen), identify the animal species related to non-clinical safety tests, and predict the toxic target organs. Results: Fig. 11 and Fig. 12 showed the binding of Claudin18.2 antibody to two gastric cancer tissues, both of which were positive, staining on the cell membrane. The target background information was consistent with the description of Claudin18.2 protein. Fig. 13 and Fig. 14 showed the binding of Claudin18.2 antibody to lung tissue or unrelated solid tumor tissue, without staining. According to the staining intensity and the background staining of interstitial tissue, the optimal working concentration was identified to be 1:4000, and the gastric cancer tissue and the lung tissue were selected as the positive and negative tissues of Claudin18.2 antibody respectively. Fig. 11 Binding of Claudin18.2 antibody to gastric cancer tissue, staining on the cell membrane Fig. 12 Binding of Claudin18.2 antibody to gastric cancer tissue,staining on the cell membrane Fig. 13 Binding of Claudin18.2 antibody to lung tissue,staining negatively Fig. 14 Binding of Claudin18.2 antibody to solid tumor tissue,staining negatively Table 1 TCR species and tissues Species Category of Tissues Human Heart, lung, liver, spleen, kidney, ureter, stomach, hypophysis, adrenal gland, thyroid gland (parathyroid gland), bladder, cerebellum, cerebrum, colon, small intestine, eyeball, lymph gland, mammary gland, pancreas, prostate, skin, muscle, testicle, thymus gland, womb, ovary, oviduct, marrow, blood vessel, hemocyte, spinal cord, placenta, and umbilical cord (umbilical artery and vein endothelium) Rhesus monkey Heart, lung, liver, spleen, kidney, ureter, stomach, hypophysis, adrenal gland, thyroid gland (parathyroid gland), bladder, cerebellum, cerebrum, colon, small intestine, eyeball, lymph gland, mammary gland, pancreas, prostate, skin, muscle, testicle, thymus gland, womb, ovary, oviduct, marrow, blood vessel, hemocyte, and spinal cord Rat Heart, lung, liver, spleen, kidney, ureter, stomach, hypophysis, adrenal gland, thyroid gland (parathyroid gland), bladder, cerebellum, cerebrum, colon, small intestine, eyeball, lymph gland, mammary gland, pancreas, prostate, skin, muscle, testicle, thymus gland, womb, ovary, oviduct, marrow, blood vessel, hemocyte, and spinal cord For more information, please contact Sanyou