Antibody Therapeutics最新文献

{"title":"REVOLUTIONIZING ANTIBODY DISCOVERY INDUSTRY WITH HIGHLY EFFICIENT AND ACCURATE AI-BASED EPITOPE-SPECIFIC ANTIBODY DE NOVO DESIGN WORKFLOW","authors":"Tianyuan Wang, Xiangrui Gao, Zhe Huai, Zhaohui Gong, Ting Mao, Xuezhe Fan, Xingxing Wu, Zhiyuan Duan, Xiaodong Wang, Jiewen Du, Mengcheng Yao, Xin Li, Min Wu, Zonghu Wang, Lin Zhang, Junjie Zhang, Wenbo Cao, Kai Yan, Yujie Fang, Shixiang Ma, Kun Yang, Lili Wu, F. An, Yezhou Yang, L. Lai, Xiaolu Huang","doi":"10.1093/abt/tbad014.024","DOIUrl":"https://doi.org/10.1093/abt/tbad014.024","url":null,"abstract":"Abstract Background and significance The global antibody drug market is worth over $200 billion in 2021 and is expected to reach $380 billion by 2030. Antibody discovery is one of the most critical steps that determine the crucial properties of antibody drugs, such as efficacy, safety, and developability. Traditional methods based on mouse immunization have many drawbacks limiting drug discovery, which include long time periods, high costs, inability to target function-specific epitopes, unsuitable for low immunogenic and difficult-to-prepare antigens, the need to sacrifice mice, the need for further humanization to reduce immunogenicity, and so on. Here we report an antibody de novo design computational workflow that utilizes high-quality internally produced antibody data and advanced AI models. Using this workflow, we can de novo design antibodies that bind to user-specified functional epitopes with high affinity and specificity. Compared with classical wet-lab methods, the entire process is shortened from several months to several days and suitable for low immunogenicity and difficult-to-prepare antigens. It is particularly noteworthy that due to the use of humanized mouse-generated antibodies (Renlite bearing common light chain from Biocytogen) as training data for AI models, the designed antibodies have a high degree of humanization and good developability, effectively avoiding issues such as ADA and aggregation in subsequent processes. Methods First, with the help of Renlite, we comprehensively combined mouse immunization, B cell sorting with FACS, NGS single-cell sequencing, and bioinformatics analysis to internally generate a large amount of high-quality antibody sequence data. Second, we developed AI models for antigen-specific antibody selection and epitope prediction (bioRxiv, 2022: 2022.12. 22.521634.) to mine antigen-specific antibodies and corresponding antigen epitopes in the data. Based on the processed high-quality data, we trained an affinity prediction model that can accurately predict whether an antigen epitope and antibody sequence pair can bind to each other. Besides, using the sequence data, we trained an antibody sequence pre-training language model (bioRxiv, 2023: 2023.01. 19.524683.), which can generate high-quality antibody sequences to simulate the antibodies produced by mouse immunization. Finally, integrating the above AI models, we established an antibody de novo design computational workflow to simulate the biological process of antibody generation and affinity maturation in the mouse immune system, which can be seen as a “DigitalMouse”. Results In a test case, 1 million antibodies were designed aiming at binding to specific epitope of an antigen. 10 antibodies were selected and expressed. Binding affinity was determined using BLI. Two antibodies out of 10 had KD of 194 nM and 336 nM, respectively, with a concentration dependent signal increase on BLI. These antibodies have great potential as the starting point of ","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43115944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CAMEL NANOBODIES NEUTRALIZE SARS-COV-2 VARIANTS","authors":"Jessica Hong, H. Kwon, R. Cachau, K. J. Butay, Zhijian Duan, Dan Li, Hua Ren, C. Hsieh, V. Dandey, M. Borgnia, Hang Xie, Mitchell Ho","doi":"10.1093/abt/tbad014.001","DOIUrl":"https://doi.org/10.1093/abt/tbad014.001","url":null,"abstract":"Abstract Background and Significance With the emergence of SARS-CoV-2 variants during the global pandemic from 2020 to 2023, there is need for broadly neutralizing antibodies. Due to their small size and unique conformations, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. Methods and Results Here, we used phage display libraries built from dromedary camels to isolate two VHH nanobodies (7A3 and 8A2), which have high affinity for the receptor-binding domain (RBD) of the SARS-CoV-2 spike. Cryo-EM complex structures revealed that 8A2 binds the RBD in its up mode and 7A3 targets a conserved and deeply buried site in the spike regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, nanobody 7A3 efficiently protected K18-hACE2 transgenic mice from the lethal challenge of SARS-CoV-2 variants B.1.351 or B.1.617. With the addition of omicron variant, a new VHH nanobody (J1B4) was isolated to target the S2 subunit of the SARS-CoV-2 spike that can bind across many variants including omicron. Trispecific nanobodies were made using 7A3, 8A2, and J1B4 which had increased binding signals compared to the nanobodies alone. Using this method, we hope to create a therapeutic that is able to broadly neutralize not only all pre-existing variants of SARS-CoV-2, but also be effective towards future SARS-CoV related variants. Conclusions and Future Directions By combining nanobodies targeting the RBD of the S1 subunit (7A3+8A2) with a nanobody targeting the S2 subunit (J1B4), we can increase the chance of protection against all SARS-CoV-2 infections. Due to the increased protein binding of the trispecific compared to individual nanobodies alone, it shows great promise that the trispecific may be able to enhance its activity across all variants. Nanobody-based therapeutics may be developed as a nasal spray which can be self-administered and inhaled directly to the lungs to treat the infection at its source.","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46199194","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AB-Amy: machine learning aided amyloidogenic risk prediction of therapeutic antibody light chains.","authors":"Yuwei Zhou,&nbsp;Ziru Huang,&nbsp;Yushu Gou,&nbsp;Siqi Liu,&nbsp;Wei Yang,&nbsp;Hongyu Zhang,&nbsp;Anthony Mackitz Dzisoo,&nbsp;Jian Huang","doi":"10.1093/abt/tbad007","DOIUrl":"https://doi.org/10.1093/abt/tbad007","url":null,"abstract":"<p><p>Over 120 FDA-approved antibody-based therapeutics are used to treat a variety of diseases.However, many candidates could fail because of unfavorable physicochemical properties. Light-chain amyloidosis is one form of aggregation that can lead to severe safety risks in clinical development. Therefore, screening candidates with a less amyloidosis risk at the early stage can not only save the time and cost of antibody development but also improve the safety of antibody drugs. In this study, based on the dipeptide composition of 742 amyloidogenic and 712 non-amyloidogenic antibody light chains, a support vector machine-based model, AB-Amy, was trained to predict the light-chain amyloidogenic risk. The AUC of AB-Amy reaches 0.9651. The excellent performance of AB-Amy indicates that it can be a useful tool for the <i>in silico</i> evaluation of the light-chain amyloidogenic risk to ensure the safety of antibody therapeutics under clinical development. A web server is freely available at http://i.uestc.edu.cn/AB-Amy/.</p>","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":"6 3","pages":"147-156"},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/07/8d/tbad007.PMC10365155.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9875993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ENGINEERING T CELLS TARGETING GPC2 FOR TREATING NEUROBLASTOMA","authors":"Alex Quan, Nan Li, Dan Li, Madeline R. Spetz, Hongbing Zhang, Cheng Liu, Mitchell Ho","doi":"10.1093/abt/tbad014.023","DOIUrl":"https://doi.org/10.1093/abt/tbad014.023","url":null,"abstract":"Abstract Background and Significance Neuroblastoma is a rare pediatric cancer that forms in immature nerve tissue of infants and accounts for 10 to 15 percent of cancer-related deaths in children. The five-year survival for high-risk neuroblastoma is 50% with current treatment practices being a combination of surgery, chemotherapy, and radiation. A more effective therapy is therefore needed to improve overall patient outcomes. Methods The CT3 mouse antibody that targets GPC2 was previously identified in the lab and has shown activity in the chimeric antigen receptor (CAR) T cell format against neuroblastoma. Humanization of the CT3 antibody was also done through CDR grafting in human germline sequences to prevent potential adverse immunogenic effects when treating patients. In the present study, the CT3 antibody and humanized CT3 (hCT3) antibody were engineered into T cells based on the engineered gamma/delta TCR scaffold (called AbTCR). The activities of the CT3 and hCT3 AbTCRs were tested in luciferase-based cell killing assays and xenograft mouse models. Results Humanized CT3 retains a comparable binding affinity for GPC2. The hCT3 CAR T cell showed its ability to regress tumor expression in mice. Furthermore, the mice treated with the CT3 AbTCR showed tumor regression while the mice treated with the hCT3 AbTCR became tumor free three weeks after treatment. Conclusions Overall, the hCT3 AbTCR T cells are very active when combating neuroblastoma tumors in mice. The efficacy at a low treatment dosage indicates that the GPC2 targeted hCT3 AbTCRs are a promising therapeutic for the treatment of neuroblastoma and other GPC2 positive cancers in patients.","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47789345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DISCOVERY OF A CYNOMOLGUS MONKEY-CROSS-REACTIVE ANTI-HUMAN CD3 MAB FOR T CELL ENGAGERS","authors":"Qin Mei, George Wang, JieYing Liu, Yunying Chen, J. Gu, Siwei Nie","doi":"10.1093/abt/tbad014.004","DOIUrl":"https://doi.org/10.1093/abt/tbad014.004","url":null,"abstract":"Abstract Background Anti-CD3 based T cell engager antibodies can redirect cytotoxic activity of T cells in a non-MHC restricted fashion to kill tumor cells effectively. Therefore, the discovery of an anti-CD3 antibody capable of activating T cells in the presence of tumor cells is highly desirable. Recently, many anti-CD3 bispecific antibodies (bsAbs) entered clinical trials. Despite the promising efficacy of anti-CD3 bsAbs, safety issues arose and establishing a proper therapeutic window between efficacy and safety became a challenge. One of the safety concerns for anti-CD3 bsAbs is the cytokine release syndrome due to T-cell activation. Recent studies have shown that this safety challenge can be mitigated by selecting an anti-CD3 antibody with the appropriate binding epitope, CD3 affinity and binding kinetics (on and off rate). Methods by using WuXi Biologics’ state-of-the-art hybridoma platform, an anti-CD3 Ab was discovered through a combination of immunization and screening strategies. Results the selected anti-CD3 Ab demonstrates moderate affinity and fast-on fast-off binding kinetics against both human and cynomolgus CD3 molecules. Once constructed into T cell engagers (TCEs) using this anti-CD3 Ab with TAA binding arms in WuXiBody® format, the obtained TCEs mediated efficient anti-tumor activity, but induced low levels of cytokine production by T cells. Conclusions WuXi Biologics has discovered an anti-CD3 Ab with desired binding properties to human CD3. As shown in two showcases, the TCEs constructed using this anti-CD3 Ab can elicit efficient T cell cytotoxicity against tumor cells but low levels of cytokine release. The cross-reactivity of the anti-CD3 Ab enables preclinical assessments of toxicity in NHP.","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49649809","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient production of bispecific antibodies-optimization of transfection strategy leads to high-level stable cell line generation of a Fabs-in-tandem immunoglobin.","authors":"Shiyong Gong,&nbsp;Chengbin Wu","doi":"10.1093/abt/tbad013","DOIUrl":"https://doi.org/10.1093/abt/tbad013","url":null,"abstract":"<p><p>Bispecific antibodies (bsAbs) are often composed of more than two component chains, such as Fabs-in-tandem immunoglobin (FIT-Ig) comprising three different component chains, which bring challenges for generating a high proportion of the correctly assembled bsAbs in a stable cell line. During the CHO-K1 stable cell line construction of a FIT-Ig, we investigated the FIT-Ig component chain ratio in transfection, where two sets of expression vectors were designed. Both designs utilized two vectors for co-transfection. Multiple transfections with plasmid ratio adjustment were applied, and the resultant minipools were evaluated for expression titer and quality of produced FIT-Ig. The results suggested that abundant outer Fab short chains (twofold chain genes versus other chains) can promote complete FIT-Ig assembly and therefore reduce the fragmental impurities of FIT-Ig. This adjustment of the component chain ratios at the beginning is beneficial to FIT-Ig stable cell line generation and brings favorable clones to process development.</p>","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":"6 3","pages":"170-179"},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/88/39/tbad013.PMC10365153.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9878120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction to: A mammalian cell display platform based on scFab transposition.","authors":"","doi":"10.1093/abt/tbad015","DOIUrl":"https://doi.org/10.1093/abt/tbad015","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1093/abt/tbad009.].</p>","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":"6 3","pages":"181"},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10365151/pdf/tbad015.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10229150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"JSKN003, A NOVEL BIPARATOPIC ANTI-HER2 ANTIBODY-DRUG CONJUGATE, EXHIBITS POTENT ANTITUMOR EFFICACY","authors":"Pilin Wang, K. Guo, Jianjian Peng, Jie Sun, Ting Xu","doi":"10.1093/abt/tbad014.009","DOIUrl":"https://doi.org/10.1093/abt/tbad014.009","url":null,"abstract":"Abstract In human advanced solid tumors, expression of HER2 protein has been reported in various tumor tissues and a variety of cultured tumor cell lines including breast cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, and ovarian cancer. Due to the critical roles of HER2 in carcinogenesis, two main targeted therapies have been developed in the past two decades to block the HER2-driven pathways, which include small molecule compounds that inhibit the tyrosine kinase activity of the intracellular domain, and mono-antibodies (mAbs) that target the extracellular domain (ECD) of the receptors. JSKN003 is an antibody-drug conjugate (ADC) comprised of a recombinant, humanized anti-human epidermal growth factor receptor 2 (HER2) bispecific antibody conjugated to a topoisomerase I inhibitor via a dibenzocyclooctyne tetrapeptide linker. The anti-HER2 component, KN026, is a recombinant, humanized bispecific antibody that targets both extra-cellular domains II (pertuzumab binding site) and IV (trastuzumab binding site) of HER2. JSKN003 showed high affinity binding to human HER2 with KDs of 2.209 E-10M, which is comparable to its parental antibody KN026 and bound to NCI-N87 and BxPC-3 cells in a concentration-dependent manner. At the same time JSKN003 showed more extensive and faster internalization than DS8201 on NCI-N87 cells. As expected, JSKN003 showed directly inhibits growth by targeting HER2 positive tumor model (NCI-N87 and BT474 cell models). The single dose and multiple dose pharmacokinetics study in cynomolgus monkey indicated that JSKN003, total antibody and DXd had general linear dynamic characteristics, and pharmacokinetics parameters showed no significant differences between males and females in the range 0.3-30 mg/kg. The HNSTD (highest non-severely toxic dose) of JSKN003 was determined as 30mg/kg in cynomolgus monkeys. These preclinical data suggest that JSKN003 could potentially benefit patients with tumors co-expressing HER2 through improved drug selectivity and efficacy. JSKN003’s safety, tolerability and preliminary anti-tumor activity are currently being evaluated in a first-in-human phase I study in advanced stage solid tumors in Australia (NCT05494918) using a BOIN design. This ADC is targeted on the subjects who has HER2 expression and/or HER2-gene mutation and may address an unmet medical need for these patients.","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45928177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A NOVEL MSLN×4-1BB BISPECIFIC ANTIBODY FOR SOLID TUMOR","authors":"Liansheng Cheng, Dayan Zhang, Wenting Liu, Wei Zhou, Xiaoli Zeng, Qun Zhao, G. Shen","doi":"10.1093/abt/tbad014.003","DOIUrl":"https://doi.org/10.1093/abt/tbad014.003","url":null,"abstract":"Abstract Background Mesothelin (MSLN) is a 70 KD glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in a variety of cancers, including mesothelioma, ovarian cancer, pancreatic cancer and breast cancer et.al. 4-1BB is a member of the tumor necrosis factor receptor superfamily that functions as a co-stimulatory molecule. Agonistic antibodies bind to 4-1BB, triggering a signaling cascade leading to T-cell activation and expansion of cytotoxic CD8+ T lymphocytes. Here, we developed two bispecific antibodies (bsAbs) targeting both MSLN and 4-1BB with an intact Fc fragment from human IgG1 or IgG4, named HK013-G1 and HK013-G4 respectively. We suspected that HK013-G1 can simultaneously exert the cytotoxic effect of CD8+T cells and NK cells on tumor cells expressing MSLN to achieve better antitumor efficacy. Methods Both HK013-G1 and HK013-G4 were constructed by fused a single-chain variable fragment (scFv) targeting hu4-1BB to the C terminus of an anti-MSLA nanobody. And their affinity was optimized to making it highly effective in tumor localization. Next, we tested the killing ability of bsAbs-mediated PBMC or NK92 against tumor cells with different expression levels of MSLN in vitro. And the IFN-γ secretion was detected when CD8+T cells co-cultured with MSLN+ or MSLN- cells in the presence of antibodies. Also, the 4-1BB agonist activity of bsAbs was measured in a luciferase report gene assay. To confirm the safety of HK013-G1, non-specific activation of 4-1BB signal mediated by Fc receptor and CRS was evaluated in vitro. Finally, we compared the antitumor activity of two bispecific antibodies in both MC38/hMSLN and CT26/hMSLN tumor model and hepatotoxicity as well as cardiotoxicity was evaluated. Results Affinity-optimized HK013-G1 has an order of magnitude greater affinity for MSLN(KD≈10−9M) than 4-1BB (KD≈10−8M). HK013-G1 induced stronger PBMC against tumor cells than MOARb009 while HK013-G4 does not. Also, HK013-G1 could only mediate the killing of NK92 on MSLN-positive tumor cells. In co-cultured assay, HK013-G1 had superior ability to stimulate CD8+T cell secretion of IFN-γ than urelumab in the presence of MSLN. In luciferase reporter assay, the bsAbs-induced 4-1BB activation is dependent on expression level of MSLN. In addition, HK013-G1 was shown no stronger ability to inducing non-specific activation of 4-1BB signal mediated by Fc receptor and CRS in vitro. Compared with HK013-G4, HK013-G1 showed a more significant anti-tumor effect in both MC38/hMSLN and CT26/hMSLN tumor model. And, HK013-G4 showed significant hepatotoxicity in mice while HK013-G1 not. Moreover, HK013-G1 can protect mice against tumor re-challenge. Conclusions HK013-G1, an MSLN×4-1BB bsAb with human IgG1 Fc fragment, prevents tumor development by killing tumor cells directly via effector functions mediated by NK and cytotoxic T cells. More importantly, HK013-G1 showed no stronger toxic side effect","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46993982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ENHANCED KINETICS ANALYSIS OF PROTEINS AND LARGE BIOMOLECULES USING NOVEL HIGH SENSITIVITY PROBE","authors":"John Zhang, Weijing Gu, Hongshan Li, Pu Li","doi":"10.1093/abt/tbad014.026","DOIUrl":"https://doi.org/10.1093/abt/tbad014.026","url":null,"abstract":"Abstract Introduction Bio-layer interferometry (BLI) has gained significant interest as a label-free technique for the detection and kinetic analysis of diverse biomolecules such as antibodies, proteins, and small molecules. The technology relies on the phase shift-wavelength correlation generated between interference patterns at the tip of the biosensor probe where molecules associate and dissociate. However, current biosensors face challenges regarding sensitivity with small molecules/peptides and compatibility with large biomolecules like nanomaterials. Traditional BLI often produces inverted signals when nanomaterials bind which hinders accurate kinetics analysis. Overcoming these limitations is crucial for expanding the range of applications and enhancing the performance of BLI-based detection systems. Significance In this study, we have developed an improved BLI sensor, Gator® SA XT, which features newly designed streptavidin-based surface capable of loading biotinylated ligands as small as 1.5 kDa. Compared to traditional BLI streptavidin probes, the SA XT probes exhibit a 3-5 times higher signal intensity. Moreover, the incorporation of a novel optical coating layer enables the detection of large biomolecules such as lipid nanoparticles without signal inversion. This advancement in biosensor technology facilitates the detection of ligands and their analytes at lower concentrations and expands the range of compatible analytes for BLI-based applications. Methods To enhance the sensitivity of the interference patterns, we utilized a proprietary optical coating layer with a refractive index significantly lower than that of proteins and other biomolecules. We assessed the sensitivity and sensing distance of the optical coating layer using a layer-by-layer model system. Binding cycles of biotinylated protein A and human IgG were repeated until the theoretical biolayer thickness reached approximately 700 nm. Results Comparative analysis of binding signals between the newly designed SA XT probes and traditional SA probes were conducted for various biomolecules. The SA XT probes demonstrated significantly higher binding signals for oligos (2.8-fold), peptides (3.0-fold), Protein A (4.1-fold), PDL1 (4.5-fold), and IgG (4.3-fold). Furthermore, the unique optical properties of the SA XT probes prevented signal inversion enabling the detection of biomolecules as large as 2 MDa. Using a layer-by-layer model system, the SA XT probes successfully detected a biolayer thickness of 700nm without signal inversion. Additionally, we demonstrated the detection of lipid nanoparticles and subsequent biomolecule bindings using the SA XT probes. Conclusions In conclusion, we have designed a novel biosensor for BLI that enables the detection of a wider range of biomolecules with high sensitivity. The SA XT probes, coupled with the proprietary optical coating layer, have overcome the limitations of traditional BLI probes and facilitated the generation of reliable and","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43814439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}