Abstract The single-chain variable fragment (scFv) and antigen-binding fragment (Fab) are widely used in yeast surface display for antibody engineering. However, these non-IgG antibody fragments are not always well-expressed or functional. We developed a novel approach to display full-length IgG on yeast surfaces for engineering monoclonal and bispecific antibodies. The yeast surface display of full-length IgG was generated by transforming glycoengineered yeast P. pastoris with plasmids encoding for anchor protein, heavy chains, and light chains. IgG-displaying yeast cells were incubated with the fluorophore antigen and secondary detection antibody for FACS sorting. The displayed antibody with bound antigen and detection antibody was detected by FACS sorting based on both antibody affinity and display level. FACS sorting could eliminate the deviation caused by expression levels. The yeast surface display of IgG-like bispecific antibodies was also constructed, which exhibited specific binding of two distinct fluorophore antigens in flow-cytometry analysis. Thus, our yeast surface display of full-length IgG can be used to screen light chain libraries to isolate common light chains for bispecific antibodies. To assess antigen-binding efficiency between different displaying formats, an antibody was respectively displayed as full-length IgG or Fab on the surface of yeast cells for flow-cytometry analysis. Cells displaying full-length IgG showed a higher level of fluorescence than those displaying Fab fragments, indicating that yeast surface display of full-length IgG is superior for high-throughput screening. To test the specificity of the displayed antibody, we performed a demo experiment in which TNF-binding adalimumab-displaying cells were mixed with trastuzumab-displaying cells at a 1 to 1,000,000 ratio, mimicking the immune library. After three consecutive rounds of sorting, the yeast cells with high fluorescence were plated on a selective medium and the individual antibody clones were sequenced. All ten sequenced clones were confirmed to be adalimumab, demonstrating the maintenance of genotype-phenotype linkage for library screening in yeast surface display of full-length IgG. A mutation library was also generated from the initial hit and displayed at the surface of yeast cells for the screening of higher affinity maturation antibodies by FACS. A pool of high-affinity binders was plated on a selective medium. Individual clones were analyzed by flow cytometry and sequenced to identify unique antibodies with higher affinity. In summary, full-length IgG antibodies can be displayed on the yeast cell surface, mimicking their native forms in molecular structure and biophysical properties. The technique combines the high throughput of yeast display with mammalian-cell quality control. This novel approach can be used to engineer monoclonal and bispecific antibodies for high affinity and improved developability profiles.
{"title":"YEAST SURFACE DISPLAY OF FULL-LENGTH IGG FOR ENGINEERING MONOCLONAL AND BISPECIFIC ANTIBODIES","authors":"Youwei Jiang","doi":"10.1093/abt/tbad014.022","DOIUrl":"https://doi.org/10.1093/abt/tbad014.022","url":null,"abstract":"Abstract The single-chain variable fragment (scFv) and antigen-binding fragment (Fab) are widely used in yeast surface display for antibody engineering. However, these non-IgG antibody fragments are not always well-expressed or functional. We developed a novel approach to display full-length IgG on yeast surfaces for engineering monoclonal and bispecific antibodies. The yeast surface display of full-length IgG was generated by transforming glycoengineered yeast P. pastoris with plasmids encoding for anchor protein, heavy chains, and light chains. IgG-displaying yeast cells were incubated with the fluorophore antigen and secondary detection antibody for FACS sorting. The displayed antibody with bound antigen and detection antibody was detected by FACS sorting based on both antibody affinity and display level. FACS sorting could eliminate the deviation caused by expression levels. The yeast surface display of IgG-like bispecific antibodies was also constructed, which exhibited specific binding of two distinct fluorophore antigens in flow-cytometry analysis. Thus, our yeast surface display of full-length IgG can be used to screen light chain libraries to isolate common light chains for bispecific antibodies. To assess antigen-binding efficiency between different displaying formats, an antibody was respectively displayed as full-length IgG or Fab on the surface of yeast cells for flow-cytometry analysis. Cells displaying full-length IgG showed a higher level of fluorescence than those displaying Fab fragments, indicating that yeast surface display of full-length IgG is superior for high-throughput screening. To test the specificity of the displayed antibody, we performed a demo experiment in which TNF-binding adalimumab-displaying cells were mixed with trastuzumab-displaying cells at a 1 to 1,000,000 ratio, mimicking the immune library. After three consecutive rounds of sorting, the yeast cells with high fluorescence were plated on a selective medium and the individual antibody clones were sequenced. All ten sequenced clones were confirmed to be adalimumab, demonstrating the maintenance of genotype-phenotype linkage for library screening in yeast surface display of full-length IgG. A mutation library was also generated from the initial hit and displayed at the surface of yeast cells for the screening of higher affinity maturation antibodies by FACS. A pool of high-affinity binders was plated on a selective medium. Individual clones were analyzed by flow cytometry and sequenced to identify unique antibodies with higher affinity. In summary, full-length IgG antibodies can be displayed on the yeast cell surface, mimicking their native forms in molecular structure and biophysical properties. The technique combines the high throughput of yeast display with mammalian-cell quality control. This novel approach can be used to engineer monoclonal and bispecific antibodies for high affinity and improved developability profiles.","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":"43672685","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}
Xiaoling Jiang, Chongbing Wu, Zi Chen, Liusong Yin
Abstract Background and Significance Biological therapies harnessing the adaptive immune system have achieved a great success, given the clinical efficacy of CAR-T therapies and CD3 based T cell engagers in treating hematologic tumors, and the application of T cell immune checkpoint inhibitors in various indications. However, the challenges still exist due to the limitation of CAR-T therapies and T cell engagers in treating solid tumors. Side effects such as cytokine release storm and neurotoxicity remain a concern in the selection of CAR-T therapies and CD3-based T cell engagers as therapeutics. For PD1/L1-based immunotherapy, the relatively low objective response rate (ORR), short progression-free survival (PFS) of patients, and drug resistance and recurrence especially for solid malignancy, are the major clinical challenges. In contrast, innate effector cells such as NK cells and macrophages are naturally existing in human body as front line to defeat general pathogens and cancers，which will have a better safety profile. Also, NK cell activation is not limited by the antigen presentation of the MHC on the cell surface which makes it has broader anti-tumor effects. However, the innate effector cell based therapy is facing the limitation of low cell number, poor in vitro activation, and short effective duration in vivo. SunHo has generated a B7H3/NKp30 NK cell engager bispecific antibody which can directly activate and enrich NK cells to the TME when used alone or enhance the efficacy when used in combination NK cell therapy. NKp30 is a potent NK cell activation receptor with wide and persistent expression compared with other NK markers. B7H3 as a tumor associate antigen is also widely expressed on many kinds of tumor cells with great potential for multiple indications, especially for solid tumors. Methods Three anti-NKp30 VHHs were identified from Alpaca immunized library. The VHHs were fused to the N-terminus or C-terminus of an anti-B7H3 mAb heavy chain with different IgG isotypes (IgG1 or IgG4). The binding activity to NK cells was evaluated by FACS. IFNγ level was detected in the NK cell activation assay. The NK cell mediated target cell killing was evaluated using either NK92MI-cd16a or Primary NK cells as effector cells. To evaluate the non-specific self-killing of NK cell without target cell, the B7H3/NKp30 candidates were incubated with NK92MI-cd16a and cell lysis percentage were calculated. NSG mice bearing Hs.746T tumors were used to evaluate the in vivo efficacy of B7H3/NKp30 candidates. Results The B7H3/NKp30 candidates showed good binding activity and better activation to NK cells compared with benchmarks. Notably, the B7H3/NKp30 candidate IAN0982-VHH25 with NKp30 fused to the C-terminal with an IgG1 isotype showed lowest risk of non-specific NK cell killing. In the in vivo study, B7H3/NKp30 candidates in combination with 1x106 NK cells showed excellent anti-tumor activity with TGI over 95%. And we didn’t observe any significant ch
{"title":"A NOVEL B7H3/NKP30 BISPECIFIC NK CELL ENGAGER FOR CANCER IMMUNOTHERAPY","authors":"Xiaoling Jiang, Chongbing Wu, Zi Chen, Liusong Yin","doi":"10.1093/abt/tbad014.013","DOIUrl":"https://doi.org/10.1093/abt/tbad014.013","url":null,"abstract":"Abstract Background and Significance Biological therapies harnessing the adaptive immune system have achieved a great success, given the clinical efficacy of CAR-T therapies and CD3 based T cell engagers in treating hematologic tumors, and the application of T cell immune checkpoint inhibitors in various indications. However, the challenges still exist due to the limitation of CAR-T therapies and T cell engagers in treating solid tumors. Side effects such as cytokine release storm and neurotoxicity remain a concern in the selection of CAR-T therapies and CD3-based T cell engagers as therapeutics. For PD1/L1-based immunotherapy, the relatively low objective response rate (ORR), short progression-free survival (PFS) of patients, and drug resistance and recurrence especially for solid malignancy, are the major clinical challenges. In contrast, innate effector cells such as NK cells and macrophages are naturally existing in human body as front line to defeat general pathogens and cancers，which will have a better safety profile. Also, NK cell activation is not limited by the antigen presentation of the MHC on the cell surface which makes it has broader anti-tumor effects. However, the innate effector cell based therapy is facing the limitation of low cell number, poor in vitro activation, and short effective duration in vivo. SunHo has generated a B7H3/NKp30 NK cell engager bispecific antibody which can directly activate and enrich NK cells to the TME when used alone or enhance the efficacy when used in combination NK cell therapy. NKp30 is a potent NK cell activation receptor with wide and persistent expression compared with other NK markers. B7H3 as a tumor associate antigen is also widely expressed on many kinds of tumor cells with great potential for multiple indications, especially for solid tumors. Methods Three anti-NKp30 VHHs were identified from Alpaca immunized library. The VHHs were fused to the N-terminus or C-terminus of an anti-B7H3 mAb heavy chain with different IgG isotypes (IgG1 or IgG4). The binding activity to NK cells was evaluated by FACS. IFNγ level was detected in the NK cell activation assay. The NK cell mediated target cell killing was evaluated using either NK92MI-cd16a or Primary NK cells as effector cells. To evaluate the non-specific self-killing of NK cell without target cell, the B7H3/NKp30 candidates were incubated with NK92MI-cd16a and cell lysis percentage were calculated. NSG mice bearing Hs.746T tumors were used to evaluate the in vivo efficacy of B7H3/NKp30 candidates. Results The B7H3/NKp30 candidates showed good binding activity and better activation to NK cells compared with benchmarks. Notably, the B7H3/NKp30 candidate IAN0982-VHH25 with NKp30 fused to the C-terminal with an IgG1 isotype showed lowest risk of non-specific NK cell killing. In the in vivo study, B7H3/NKp30 candidates in combination with 1x106 NK cells showed excellent anti-tumor activity with TGI over 95%. And we didn’t observe any significant ch","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":"41445974","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}
Wuxiang Liao, Christine Tumanut, Lin Li, A. Corper, D. Challa, Alex Chang, Hydari Begum, Elinaz Farokhi, C. Woods, Xiaomin Fan
Abstract Background and Significance Natural killer (NK) cells play a vital role in the human innate immune system and are being explored as a promising approach for cancer immunotherapy. Of particular interest are NK cell engagers that can target and activate NK cells to attack cancer cells. In this study, we developed novel NK cell engagers by targeting the NK cell activating receptor CD16a using antibodies that selectively distinguish between CD16a on NK cells and CD16b on granulocytes, which are highly homologous to each other. Methods and Results To generate antibodies with high developability, we employed a rational design approach to construct large yeast display libraries of human antibodies. This approach was based on the analysis of a deep sequencing dataset of human antibodies from over 500 individuals, which allowed us to determine the natural amino acid usage patterns of human antibody CDRs and mimic human antibody repertoires. Through screening these libraries, we discovered two classes of antibody clones that selectively recognize CD16a without cross-reactivity to CD16b. Epitope mapping revealed that a single amino acid difference confers over 10,000-fold selectivity for one class of antibody clones, while for the other class a second unique epitope on CD16a was identified. To evaluate the activity of these antibody clones, we produced bispecific antibody clones with one arm targeting CD16a and the other arm targeting a tumor-associated antigen (TAA). Our results demonstrated potent tumor cell-dependent activation of NK cells and effective killing of tumor cells. Several of these antibodies had greatly enhanced resistance to human IgG inhibition in killing target cells. Significantly, our anti-CD16a antibody clones exhibited superior performance compared to leading reference anti-CD16a clones in two distinct NK cell engager formats. This included higher affinity for CD16a, higher thermostability, and more potent killing activity both in the absence and presence of 10 mg/mL human IgGs as competitors. Conclusion Our findings indicate that anti-CD16a antibody-based NK cell engagers have significant potential for cancer immunotherapies.
{"title":"DEVELOPMENT OF A SELECTIVE CD16A-BASED NK CELL ENGAGER UTILIZING ANTIBODIES TARGETING A SINGLE AMINO ACID VARIATION","authors":"Wuxiang Liao, Christine Tumanut, Lin Li, A. Corper, D. Challa, Alex Chang, Hydari Begum, Elinaz Farokhi, C. Woods, Xiaomin Fan","doi":"10.1093/abt/tbad014.012","DOIUrl":"https://doi.org/10.1093/abt/tbad014.012","url":null,"abstract":"Abstract Background and Significance Natural killer (NK) cells play a vital role in the human innate immune system and are being explored as a promising approach for cancer immunotherapy. Of particular interest are NK cell engagers that can target and activate NK cells to attack cancer cells. In this study, we developed novel NK cell engagers by targeting the NK cell activating receptor CD16a using antibodies that selectively distinguish between CD16a on NK cells and CD16b on granulocytes, which are highly homologous to each other. Methods and Results To generate antibodies with high developability, we employed a rational design approach to construct large yeast display libraries of human antibodies. This approach was based on the analysis of a deep sequencing dataset of human antibodies from over 500 individuals, which allowed us to determine the natural amino acid usage patterns of human antibody CDRs and mimic human antibody repertoires. Through screening these libraries, we discovered two classes of antibody clones that selectively recognize CD16a without cross-reactivity to CD16b. Epitope mapping revealed that a single amino acid difference confers over 10,000-fold selectivity for one class of antibody clones, while for the other class a second unique epitope on CD16a was identified. To evaluate the activity of these antibody clones, we produced bispecific antibody clones with one arm targeting CD16a and the other arm targeting a tumor-associated antigen (TAA). Our results demonstrated potent tumor cell-dependent activation of NK cells and effective killing of tumor cells. Several of these antibodies had greatly enhanced resistance to human IgG inhibition in killing target cells. Significantly, our anti-CD16a antibody clones exhibited superior performance compared to leading reference anti-CD16a clones in two distinct NK cell engager formats. This included higher affinity for CD16a, higher thermostability, and more potent killing activity both in the absence and presence of 10 mg/mL human IgGs as competitors. Conclusion Our findings indicate that anti-CD16a antibody-based NK cell engagers have significant potential for cancer immunotherapies.","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":"46124421","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}
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
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
{"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}
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
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.
{"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}
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 in silico 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/.
{"title":"AB-Amy: machine learning aided amyloidogenic risk prediction of therapeutic antibody light chains.","authors":"Yuwei Zhou, Ziru Huang, Yushu Gou, Siqi Liu, Wei Yang, Hongyu Zhang, Anthony Mackitz Dzisoo, 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}
Alex Quan, Nan Li, Dan Li, Madeline R. Spetz, Hongbing Zhang, Cheng Liu, Mitchell Ho
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.
摘要背景和意义神经母细胞瘤是一种罕见的儿科癌症，在婴儿未成熟的神经组织中形成，占儿童癌症相关死亡的10-15%。高危神经母细胞瘤的五年生存率为50%，目前的治疗方法是手术、化疗和放疗相结合。因此，需要一种更有效的治疗方法来改善患者的整体预后。方法先前在实验室中鉴定了靶向GPC2的CT3小鼠抗体，该抗体在嵌合抗原受体（CAR）T细胞形式中显示出抗神经母细胞瘤的活性。CT3抗体的人源化也通过在人类种系序列中进行CDR移植来防止治疗患者时潜在的不良免疫原性影响。在本研究中，CT3抗体和人源化CT3（hCT3）抗体基于工程化γ/ΔTCR支架（称为AbTCR）被工程化为T细胞。在基于荧光素酶的细胞杀伤测定和异种移植物小鼠模型中测试CT3和hCT3-AbTCRs的活性。结果人源化CT3对GPC2具有相当的结合亲和力。hCT3 CAR T细胞在小鼠中显示出其抑制肿瘤表达的能力。此外，用CT3-AbTCR处理的小鼠显示出肿瘤消退，而用hCT3-AbTCR处理的鼠在处理后三周变得无肿瘤。结论总的来说，hCT3-AbTCR T细胞在对抗小鼠神经母细胞瘤肿瘤时非常活跃。低治疗剂量下的疗效表明，GPC2靶向的hCT3-AbTCRs是治疗患者神经母细胞瘤和其他GPC2阳性癌症的有前途的治疗方法。
{"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}
Qin Mei, George Wang, JieYing Liu, Yunying Chen, J. Gu, Siwei Nie
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.
{"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}
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.
{"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, 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}
[This corrects the article DOI: 10.1093/abt/tbad009.].
[这更正了文章DOI: 10.1093/abt/tbad009.]。
{"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}
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