Zuoan Yi, C. He, Yueh-Mei Hso, M. Strainic, Yong Yin, W. Zhai
Abstract Background Tumor necrosis factor receptor 2 (TNFR2) is considered an appealing target due to its low-level expression on immune cells, but it could be upregulated on regulatory T cells (Tregs) in the tumor microenvironment which plays key roles in Treg proliferation and function. It has been demonstrated that Tregs with high TNFR2 expression were the most suppressive subsets among all Treg populations in the tumor. While early studies showed that TNFR2 co-stimulates naïve T cells, it has been revealed later that TNFR2 also limits CD8+ T-cell-mediated viral clearance and anti-tumor immunity by inducing rapid contraction of CD8+ T cells. Consistent with these findings, several publications reported that anti-TNFR2 antibody treatment exhibited robust antitumor efficacy. In short, TNFR2 signaling is critical in regulating immune response in different diseases. In the current study, the anti-human TNFR2 antibody SBT-1901 was developed and the anti-tumor activity was evaluated. Materials and methods SBT-1901 was generated through hybridoma and humanization technologies. The binding affinity and specificity were tested by ELISA, FACS, and OCTET. The function of SBT-1901 was tested in TNFR2-Fas overexpressing Ramos cells and in Treg proliferation assay. The in vivo anti-tumor activity and pharmacokinetics of SBT-1901 were evaluated in the human TNFR2 transgenic mouse model (Biocytogen) bearing MC38 tumor. Results SBT-1901 binds to the extracellular domain of human and cynomolgus TNFR2 with the affinity of single digit-nanomolar. It competes with TNFα for binding to TNFR2 receptor and inhibits TNFα-induced TNFR2-Fas overexpressing RAMOS cell death. SBT-1901 also blocks TNFα induced Treg proliferation in human PBMC. In addition, SBT-1901 significantly inhibits MC38 tumor growth in a dose-dependent manner as a monotherapy and enhances anti-tumor efficacy of mPD-1 antibody in a combination study in human TNFR2 transgenic mouse model. SBT-1901 is currently under preclinical development. Conclusions Our studies show that SBT-1901 exhibits a very potent anti-tumor efficacy in vivo as a single agent or the combination. Therefore, it is highly valuable to further develop SBT-1901 for human cancer treatment.
{"title":"TARGETING TREG CELLS BY TNFR2 ANTIBODY INDUCES TUMOR REGRESSION IN VIVO","authors":"Zuoan Yi, C. He, Yueh-Mei Hso, M. Strainic, Yong Yin, W. Zhai","doi":"10.1093/abt/tbad014.015","DOIUrl":"https://doi.org/10.1093/abt/tbad014.015","url":null,"abstract":"Abstract Background Tumor necrosis factor receptor 2 (TNFR2) is considered an appealing target due to its low-level expression on immune cells, but it could be upregulated on regulatory T cells (Tregs) in the tumor microenvironment which plays key roles in Treg proliferation and function. It has been demonstrated that Tregs with high TNFR2 expression were the most suppressive subsets among all Treg populations in the tumor. While early studies showed that TNFR2 co-stimulates naïve T cells, it has been revealed later that TNFR2 also limits CD8+ T-cell-mediated viral clearance and anti-tumor immunity by inducing rapid contraction of CD8+ T cells. Consistent with these findings, several publications reported that anti-TNFR2 antibody treatment exhibited robust antitumor efficacy. In short, TNFR2 signaling is critical in regulating immune response in different diseases. In the current study, the anti-human TNFR2 antibody SBT-1901 was developed and the anti-tumor activity was evaluated. Materials and methods SBT-1901 was generated through hybridoma and humanization technologies. The binding affinity and specificity were tested by ELISA, FACS, and OCTET. The function of SBT-1901 was tested in TNFR2-Fas overexpressing Ramos cells and in Treg proliferation assay. The in vivo anti-tumor activity and pharmacokinetics of SBT-1901 were evaluated in the human TNFR2 transgenic mouse model (Biocytogen) bearing MC38 tumor. Results SBT-1901 binds to the extracellular domain of human and cynomolgus TNFR2 with the affinity of single digit-nanomolar. It competes with TNFα for binding to TNFR2 receptor and inhibits TNFα-induced TNFR2-Fas overexpressing RAMOS cell death. SBT-1901 also blocks TNFα induced Treg proliferation in human PBMC. In addition, SBT-1901 significantly inhibits MC38 tumor growth in a dose-dependent manner as a monotherapy and enhances anti-tumor efficacy of mPD-1 antibody in a combination study in human TNFR2 transgenic mouse model. SBT-1901 is currently under preclinical development. Conclusions Our studies show that SBT-1901 exhibits a very potent anti-tumor efficacy in vivo as a single agent or the combination. Therefore, it is highly valuable to further develop SBT-1901 for human cancer treatment.","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":"43558052","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}
Abstract Objective Single-domain antibodies, such as VHH and nanobody, have shown potential for use in therapy and diagnostics. One application of VHHs is the tethering of two fragments to different epitopes on the same target, which is difficult to achieve with conventional antibodies. Synergistic heterologous VHH dimers have higher affinity, better specificity, and broad applications in developing high-affinity monoclonal antibodies, bispecific antibodies, ADCs, and CAR-Ts. However, finding the best pair of VHHs for these applications requires combinational screening, which is traditionally a time-consuming and costly process. The objective of this study is to develop a technology that can quickly screen and pair two synergistic VHHs without the need to express tandem VHH dimers. Methods The researchers developed proprietary tags and specific dockers that, when stabilized on a solid station, can capture any VHHs that the dockers recognize and pull them together to form a non-covalent dimer. This platform is called ExchaBody technology, and the VHH dimers formed this way are ExchaBodies. The researchers used this technology to conduct a bi-epitope screening campaign, where VHHs were first expressed as monomers with tags and then binned and grouped into different categories. VHHs were then paired with all reasonable combinations using ExchaBody technology, and these ExchaBodies were evaluated for their combined activities. Results ExchaBody technology was able to link any two VHHs together within one hour, and the resulting ExchaBodies had bivalent or bifunctional VHH activities. The bi-epitope VHH screening campaign, which would have taken months to complete using traditional methods, was finished within two weeks using ExchaBody technology, saving time and cost. The researchers were able to construct two lead molecules, a bi-specific VHH-Fc fusion protein and a tri-valent VHH molecule, using ExchaBody technology. These lead molecules were found to be superior to their counterparts on the market based on affinity and functional assays. Conclusion ExchaBody technology is a bispecific VHH screening and pairing platform that can quickly and cost-effectively create non-covalent, bispecific VHHs (ExchaBodies) without the need to express them. ExchaBodies possess the binding and cellular activities of a covalently linked, bispecific, tandem VHH dimer. This technology has broad applications in developing high-affinity monoclonal antibodies, bispecific antibodies, ADCs, and CAR-Ts.
{"title":"APPLICATION OF EXCHABODY TECHNOLOGY FOR PAIRING VHHS TO ACHIEVE SYNERGISTIC EFFECTS","authors":"Yi Luo, Xiaoxiao Zhan, Yilong Shen, Ziyang Sheng, Yin Zhu, Mingyue Huang","doi":"10.1093/abt/tbad014.025","DOIUrl":"https://doi.org/10.1093/abt/tbad014.025","url":null,"abstract":"Abstract Objective Single-domain antibodies, such as VHH and nanobody, have shown potential for use in therapy and diagnostics. One application of VHHs is the tethering of two fragments to different epitopes on the same target, which is difficult to achieve with conventional antibodies. Synergistic heterologous VHH dimers have higher affinity, better specificity, and broad applications in developing high-affinity monoclonal antibodies, bispecific antibodies, ADCs, and CAR-Ts. However, finding the best pair of VHHs for these applications requires combinational screening, which is traditionally a time-consuming and costly process. The objective of this study is to develop a technology that can quickly screen and pair two synergistic VHHs without the need to express tandem VHH dimers. Methods The researchers developed proprietary tags and specific dockers that, when stabilized on a solid station, can capture any VHHs that the dockers recognize and pull them together to form a non-covalent dimer. This platform is called ExchaBody technology, and the VHH dimers formed this way are ExchaBodies. The researchers used this technology to conduct a bi-epitope screening campaign, where VHHs were first expressed as monomers with tags and then binned and grouped into different categories. VHHs were then paired with all reasonable combinations using ExchaBody technology, and these ExchaBodies were evaluated for their combined activities. Results ExchaBody technology was able to link any two VHHs together within one hour, and the resulting ExchaBodies had bivalent or bifunctional VHH activities. The bi-epitope VHH screening campaign, which would have taken months to complete using traditional methods, was finished within two weeks using ExchaBody technology, saving time and cost. The researchers were able to construct two lead molecules, a bi-specific VHH-Fc fusion protein and a tri-valent VHH molecule, using ExchaBody technology. These lead molecules were found to be superior to their counterparts on the market based on affinity and functional assays. Conclusion ExchaBody technology is a bispecific VHH screening and pairing platform that can quickly and cost-effectively create non-covalent, bispecific VHHs (ExchaBodies) without the need to express them. ExchaBodies possess the binding and cellular activities of a covalently linked, bispecific, tandem VHH dimer. This technology has broad applications in developing high-affinity monoclonal antibodies, bispecific antibodies, ADCs, and CAR-Ts.","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":"43436208","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}
Hefeng Zhang, Lingling Zhu, Emily Zou, Yu Liang, Linglong Zou
Abstract Background Monoclonal antibodies (mAb) comprise of two Fab fragments and one Fc fragment or one F(ab)’2 fragment and one Fc fragment. While a full-length mAb is frequently used as an assay reagent for bioanalysis, mAb fragments are required in certain cases. For example, to build a sandwich assay for detection of anti-drug antibodies (ADA) for therapeutic antibodies, Fab or F(ab)’2 fragment is used instead of a full-length mAb as capture reagent. This is because therapeutic antibodies, either humanized or fully human, are in many ways indistinguishable from the ADA generated in patients, especially in Fc fragment. When ADA detection methods utilizes anti-human Fc antibodies as the detection reagent, the full-length mAb drug will be directly bound by the detection reagent, causing interference. Preparation of a Fab or F(ab)’2 fragment is therefore needed. Methods and results We are developing a method for enzymatic digestion of therapeutic antibodies to generate monovalent Fab or bivalent F(ab’)2 fragments in this study. With such reagents becoming available, a sandwich ADA assay formats can be expanded to allow anti-human Fc antibodies as detection regents. To standardize the method, we explored various enzymatic conditions, including type of enzymes (i.e., pepsin, papain, and IdeS Protease), digestion-time (1, 2, 4, and 6 h), enzyme to antibody ratio (1:10, 1:20, and 1:40 w/w), IgG species and isotypes (human IgG1-κ, IgG1-λ, and IgG4-κ). The enzymatic hydrolysates were quantified by NanoDrop and purified by dialysis (10K MWCO) and Protein A/G/L magnetic bead methods. The effective recovery of truncated antibodies was > 90%, as assessed by reduced/non-reduced SDS-PAGE and ELISA analysis. Digestion of human IgG1 and IgG4 with pepsin resulted into a complete cleavage into F(ab')2 fragments and degradation of Fc fragments. While IdeS Protease produced an equivalent quantity of F(ab’)2 and Fc fragments with a similar efficiency, removal of the intact Fc fragment was required as an additional step. If the Fab fragments were desired, papain could be used with yield being over 90%. We have subsequently utilized either Fab or F(ab’)2 as a capture reagent for ADA detection. Conclusion We have successfully developed the enzymatic digestion method to prepare Fab or F(ab’)2 fragments. The optimized conditions described here are broadly applicable to different IgG isotypes across many therapeutic antibodies.
{"title":"DEVELOPMENT OF A METHOD FOR PRODUCING FAB/F(AB’)2 FRAGMENTS FROM A FULL-LENGTH MONOCLONAL ANTIBODY FOR BIOANALYTICAL ASSAYS","authors":"Hefeng Zhang, Lingling Zhu, Emily Zou, Yu Liang, Linglong Zou","doi":"10.1093/abt/tbad014.019","DOIUrl":"https://doi.org/10.1093/abt/tbad014.019","url":null,"abstract":"Abstract Background Monoclonal antibodies (mAb) comprise of two Fab fragments and one Fc fragment or one F(ab)’2 fragment and one Fc fragment. While a full-length mAb is frequently used as an assay reagent for bioanalysis, mAb fragments are required in certain cases. For example, to build a sandwich assay for detection of anti-drug antibodies (ADA) for therapeutic antibodies, Fab or F(ab)’2 fragment is used instead of a full-length mAb as capture reagent. This is because therapeutic antibodies, either humanized or fully human, are in many ways indistinguishable from the ADA generated in patients, especially in Fc fragment. When ADA detection methods utilizes anti-human Fc antibodies as the detection reagent, the full-length mAb drug will be directly bound by the detection reagent, causing interference. Preparation of a Fab or F(ab)’2 fragment is therefore needed. Methods and results We are developing a method for enzymatic digestion of therapeutic antibodies to generate monovalent Fab or bivalent F(ab’)2 fragments in this study. With such reagents becoming available, a sandwich ADA assay formats can be expanded to allow anti-human Fc antibodies as detection regents. To standardize the method, we explored various enzymatic conditions, including type of enzymes (i.e., pepsin, papain, and IdeS Protease), digestion-time (1, 2, 4, and 6 h), enzyme to antibody ratio (1:10, 1:20, and 1:40 w/w), IgG species and isotypes (human IgG1-κ, IgG1-λ, and IgG4-κ). The enzymatic hydrolysates were quantified by NanoDrop and purified by dialysis (10K MWCO) and Protein A/G/L magnetic bead methods. The effective recovery of truncated antibodies was > 90%, as assessed by reduced/non-reduced SDS-PAGE and ELISA analysis. Digestion of human IgG1 and IgG4 with pepsin resulted into a complete cleavage into F(ab')2 fragments and degradation of Fc fragments. While IdeS Protease produced an equivalent quantity of F(ab’)2 and Fc fragments with a similar efficiency, removal of the intact Fc fragment was required as an additional step. If the Fab fragments were desired, papain could be used with yield being over 90%. We have subsequently utilized either Fab or F(ab’)2 as a capture reagent for ADA detection. Conclusion We have successfully developed the enzymatic digestion method to prepare Fab or F(ab’)2 fragments. The optimized conditions described here are broadly applicable to different IgG isotypes across many therapeutic antibodies.","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":"46882064","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}
Yansong Luan, Hong-Ying Deng, Fengpo Wang, Cuihui Wang, Zhen Zhang, Xun Liu, K. Abuduwaili, Jiajian Liu
Abstract Background Immune checkpoint inhibitors (ICI) PD-1/PD-L1 antibody are key drugs for the treatment of cancer. Bispecific antibody is one of the strategies aimed to meet the clinical needs for cancer patients who are resistant to or refractory from ICI treatment. TIM-3, one of the next generation of ICI targets, co-expressed on exhausted T cells with PD-1. It is also expressed by innate immune populations, including NK and DC. Dual blocking PD-1 and TIM-3 not only on T cells but also on DC, NK cells may achieve better clinical benefit for patients who are resistant to or refractory from ICI treatment. Method A bivalent TIM-3 and PD-1 bispecific antibody (Bis5) was developed, a series of in vitro and in vivo efficacy, preclinical pharmacokinetic and toxicity studies were conducted. A Phase I, multicenter, open-label study to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity and preliminary efficacy of Bis5 in patients with advanced and/or metastatic solid tumors is ongoing in China. Results Bis5 showed affinity of 5-8 nM to both TIM-3 and PD-1, with better cell activity than TIM-3 and PD-1 mAb combination to activated T cell as well as NK and DC, over the other clinical stage reference BsAb. In huPD-1/TIM-3 double knock in mice-CT26 tumor model, Bis5 showed significant tumor inhibition activity and doubled the survival rate, while neither PD-1 mAb, TIM-3 mAb nor PD-1 and TIM-3 antibody combination showed activity. The highest non-severe toxicity dose (HNSTD) was 200mg/kg in monkeys. Nine cohorts (0.001-15 mg/kg) are planned to be enrolled sequentially in the dose escalation part in the Phase I study, as of April 2023, seven cohorts enrollment have completed. No dose limiting toxicity was observed, and the optimal effective dose was not reached. No TRAE higher than grade 2 was observed. The TRAE with ≥10% Incidence was anemia. SD >4 or 2 months were shown in the suboptimal dose levels in NSCLC and CRC (0.3mg/kg, 1mg/kg). The Part 2 dose expansion will further characterize the safety profile and preliminary tumor response in several cohorts including NSCLC, CRC, ESCC etc. Conclusion Bis5 showed good preclinical efficacy and safety profile, its clinical performance is expected. Clinical trial information: NCT05357651.
{"title":"A BIVALENT TIM-3/PD-1 BISPECIFIC ANTIBODY FOR THE TREATMENT OF PD-1 ANTIBODY RESISTANT OR REFRACTORY SOLID TUMORS","authors":"Yansong Luan, Hong-Ying Deng, Fengpo Wang, Cuihui Wang, Zhen Zhang, Xun Liu, K. Abuduwaili, Jiajian Liu","doi":"10.1093/abt/tbad014.002","DOIUrl":"https://doi.org/10.1093/abt/tbad014.002","url":null,"abstract":"Abstract Background Immune checkpoint inhibitors (ICI) PD-1/PD-L1 antibody are key drugs for the treatment of cancer. Bispecific antibody is one of the strategies aimed to meet the clinical needs for cancer patients who are resistant to or refractory from ICI treatment. TIM-3, one of the next generation of ICI targets, co-expressed on exhausted T cells with PD-1. It is also expressed by innate immune populations, including NK and DC. Dual blocking PD-1 and TIM-3 not only on T cells but also on DC, NK cells may achieve better clinical benefit for patients who are resistant to or refractory from ICI treatment. Method A bivalent TIM-3 and PD-1 bispecific antibody (Bis5) was developed, a series of in vitro and in vivo efficacy, preclinical pharmacokinetic and toxicity studies were conducted. A Phase I, multicenter, open-label study to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity and preliminary efficacy of Bis5 in patients with advanced and/or metastatic solid tumors is ongoing in China. Results Bis5 showed affinity of 5-8 nM to both TIM-3 and PD-1, with better cell activity than TIM-3 and PD-1 mAb combination to activated T cell as well as NK and DC, over the other clinical stage reference BsAb. In huPD-1/TIM-3 double knock in mice-CT26 tumor model, Bis5 showed significant tumor inhibition activity and doubled the survival rate, while neither PD-1 mAb, TIM-3 mAb nor PD-1 and TIM-3 antibody combination showed activity. The highest non-severe toxicity dose (HNSTD) was 200mg/kg in monkeys. Nine cohorts (0.001-15 mg/kg) are planned to be enrolled sequentially in the dose escalation part in the Phase I study, as of April 2023, seven cohorts enrollment have completed. No dose limiting toxicity was observed, and the optimal effective dose was not reached. No TRAE higher than grade 2 was observed. The TRAE with ≥10% Incidence was anemia. SD >4 or 2 months were shown in the suboptimal dose levels in NSCLC and CRC (0.3mg/kg, 1mg/kg). The Part 2 dose expansion will further characterize the safety profile and preliminary tumor response in several cohorts including NSCLC, CRC, ESCC etc. Conclusion Bis5 showed good preclinical efficacy and safety profile, its clinical performance is expected. Clinical trial information: NCT05357651.","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":"45359788","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}
Abstract Background Antibody–drug conjugates (ADC) typically consist of a monoclonal antibody (mAbs) attached to a cytotoxic payload via a chemical linker. ADC development requires careful consideration of each of its key components and development strategy since each element has the potential to affect the final therapeutic efficacy and safety. One important characteristic of an ADC drug is the drug-to-antibody ratio (DAR). This ratio elucidates the number of drug molecules bound onto a single antibody. Based on the conjugation strategy, the number of drug molecules that are bound to a single antibody varies. Low-drug loading reduces the overall potency, whereas high-drug loading can have higher cytotoxic effects but increased side-effects and altered pharmacokinetics (PK). As such, appropriate selection of conjugation strategy can affect the homogeneity of ADCs and resulting effectiveness. To increase the efficacy of ADCs, site-specific conjugation technologies, including engineered cysteine residues, unnatural amino acids, or enzymatic conjugation through glycosyltransferases, have been applied to obtain more homogeneous ADCs. This has proven to be clinically effective by improving ADC pharmacokinetics and therapeutic index. Furthermore, the increased control over conjugation site reduces the overall hydrophobicity of the linker–payload, preventing unintended payload release in blood. AGLink ADC site-specific conjugation kits were used to perform site-specific conjugation. The AGLink technology utilizes an enzymatic modification method (one-pot process) to reduce antibody N-glycans by fucosylation and enable site-specific and controllable conjugation. After conjugation, the resulting ADCs were evaluated for ADC homogeneity, immunoreactivity, and cytotoxicity. Methods and Results AGLink ADC site-specific conjugation were used and based on the conjugation platform YTConju™. To characterize the efficacy of AGLink, Trastuzumab and MMAE were used. N-glycans were identified to be at the asparagine 297 (N297) position of the CH2 domain on each heavy chain Fc fragment. The N-glycans were reduced to form reactive sites linked with payloads through glycosylation. The resulting glycosylation is predominantly composed of varied amounts of N-acetylglucosamine, fucose, galactose, mannose and N-acetylneuraminic acid (sialic acid) residues, which are assembled in different complex-type biantennary structures. The resulting ADCs (Trastuzumab-MMAE) with different DARs (2 or 4) were developed and characterized through varying studies. Conclusion Site-specific modifications are beginning to be used more frequently to meet the rapidly evolving applications of ADCs. Of these modification methods, we see that glycoengineering has been demonstrated as a useful approach for site-specific antibody conjugation methods. The AGLink site-specific conjugation kit utilizes glycoengineering by performing an enzymatic modification method of IgG Fc glycans to perform conjugatio
{"title":"ACCELERATING THE DEVELOPMENT OF NOVEL ANTIBODY-DRUG CONJUGATES THROUGH SITE-SPECIFIC CONJUGATION METHODS","authors":"A. Ouyang, Spencer Chiang, Chao Wang","doi":"10.1093/abt/tbad014.021","DOIUrl":"https://doi.org/10.1093/abt/tbad014.021","url":null,"abstract":"Abstract Background Antibody–drug conjugates (ADC) typically consist of a monoclonal antibody (mAbs) attached to a cytotoxic payload via a chemical linker. ADC development requires careful consideration of each of its key components and development strategy since each element has the potential to affect the final therapeutic efficacy and safety. One important characteristic of an ADC drug is the drug-to-antibody ratio (DAR). This ratio elucidates the number of drug molecules bound onto a single antibody. Based on the conjugation strategy, the number of drug molecules that are bound to a single antibody varies. Low-drug loading reduces the overall potency, whereas high-drug loading can have higher cytotoxic effects but increased side-effects and altered pharmacokinetics (PK). As such, appropriate selection of conjugation strategy can affect the homogeneity of ADCs and resulting effectiveness. To increase the efficacy of ADCs, site-specific conjugation technologies, including engineered cysteine residues, unnatural amino acids, or enzymatic conjugation through glycosyltransferases, have been applied to obtain more homogeneous ADCs. This has proven to be clinically effective by improving ADC pharmacokinetics and therapeutic index. Furthermore, the increased control over conjugation site reduces the overall hydrophobicity of the linker–payload, preventing unintended payload release in blood. AGLink ADC site-specific conjugation kits were used to perform site-specific conjugation. The AGLink technology utilizes an enzymatic modification method (one-pot process) to reduce antibody N-glycans by fucosylation and enable site-specific and controllable conjugation. After conjugation, the resulting ADCs were evaluated for ADC homogeneity, immunoreactivity, and cytotoxicity. Methods and Results AGLink ADC site-specific conjugation were used and based on the conjugation platform YTConju™. To characterize the efficacy of AGLink, Trastuzumab and MMAE were used. N-glycans were identified to be at the asparagine 297 (N297) position of the CH2 domain on each heavy chain Fc fragment. The N-glycans were reduced to form reactive sites linked with payloads through glycosylation. The resulting glycosylation is predominantly composed of varied amounts of N-acetylglucosamine, fucose, galactose, mannose and N-acetylneuraminic acid (sialic acid) residues, which are assembled in different complex-type biantennary structures. The resulting ADCs (Trastuzumab-MMAE) with different DARs (2 or 4) were developed and characterized through varying studies. Conclusion Site-specific modifications are beginning to be used more frequently to meet the rapidly evolving applications of ADCs. Of these modification methods, we see that glycoengineering has been demonstrated as a useful approach for site-specific antibody conjugation methods. The AGLink site-specific conjugation kit utilizes glycoengineering by performing an enzymatic modification method of IgG Fc glycans to perform conjugatio","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":"44203963","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}
Mariya B Shapiro, Jacqueline Boucher, Anna Brousseau, Amin Dehkharghani, Justin Gabriel, Vishal Kamat, Ketan Patil, Feng Gao, Jennifer Walker, Ryan Kelly, Colby A Souders
In vivo VHH discovery approaches have been limited by the lack of methodologies for camelid B cell interrogation. Here, we report a novel application of the Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening alpaca B cells. Methods for alpaca B cell enrichment, culture, IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH from an alpaca immunized with prostate-specific membrane antigen (PSMA) or a second target. PSMA-specific hits were expressed as VHH-Fc and characterized using label-free techniques. Anti-PSMA IgG2/3 titer plateaued on day 153, when on-Beacon IgG2/3 secretion and target binding rates peaked. Of 13 recombinantly expressed VHH-Fc, all but one bound with nanomolar affinity, and five were successfully humanized. Repertoire sequencing uncovered additional variants within the clonal lineages of the validated hits. The establishment of this workflow extends the powerful Beacon technology to enable rapid VHH discovery directly from natural camelid immune repertoires.
{"title":"Alpaca single B cell interrogation and heavy-chain-only antibody discovery on an optofluidic platform.","authors":"Mariya B Shapiro, Jacqueline Boucher, Anna Brousseau, Amin Dehkharghani, Justin Gabriel, Vishal Kamat, Ketan Patil, Feng Gao, Jennifer Walker, Ryan Kelly, Colby A Souders","doi":"10.1093/abt/tbad018","DOIUrl":"https://doi.org/10.1093/abt/tbad018","url":null,"abstract":"<p><p><i>In vivo</i> VHH discovery approaches have been limited by the lack of methodologies for camelid B cell interrogation. Here, we report a novel application of the Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening alpaca B cells. Methods for alpaca B cell enrichment, culture, IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH from an alpaca immunized with prostate-specific membrane antigen (PSMA) or a second target. PSMA-specific hits were expressed as VHH-Fc and characterized using label-free techniques. Anti-PSMA IgG2/3 titer plateaued on day 153, when on-Beacon IgG2/3 secretion and target binding rates peaked. Of 13 recombinantly expressed VHH-Fc, all but one bound with nanomolar affinity, and five were successfully humanized. Repertoire sequencing uncovered additional variants within the clonal lineages of the validated hits. The establishment of this workflow extends the powerful Beacon technology to enable rapid VHH discovery directly from natural camelid immune repertoires.</p>","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":"6 3","pages":"211-223"},"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/7f/ef/tbad018.PMC10481890.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10177234","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}
Yunying Chen, Xin Lin, Yi Qin, Donghui Wu, Jijie Gu, Siwei Nie
Abstract Background The serum half-life of endogenous albumin is approximately 19 days in humans and 1.5-2.5 days in rodents. This extended half-life in vivo is primarily due to effective recycling upon internalization mediated by the neonatal Fc receptor (FcRn). Many protein therapeutics smaller than 60 kD have short serum half-life, which can be extended by fusing the proteins of interest with an anti-albumin antibody. The fusion proteins then take advantage of FcRn-mediated recycling. Single domain antibody (VHH) molecules (around 15 kD), derived from camelid heavy-chain-only IgG, are attracting increased attention globally in the field of antibody-based therapies due to several features including small size, high stability, low immunogenicity, good tissue penetration and cost effectiveness in manufacturing. Methods an anti-human/cynomolgus monkey/murine/canine/feline serum albumin VHH lead was discovered from a proprietary, large native phage-displayed VHH library, which was then humanized, and affinity matured. The optimized VHH was fused to bispecific and trispecific antibodies for in vivo PK studies. Results An anti-human serum albumin VHH Ab was discovered, and it also bound to cyno and mouse serum albumin with high affinity without affecting the interaction of HSA with FcRn. The VHH showed good developability and, once fused to protein therapeutics, long half-life and anti-tumor activity in rodents. Conclusions a novel VHH against serum albumin of different species was discovered from native VHH libraries, and it can be easily assembled into bispecific Abs and multispecific Abs to prolong the molecules’ PK profile.
{"title":"DISCOVERY OF NOVEL ANTI-SERUM ALBUMIN VHH AS A BUILDING BLOCK FOR PK PROLONGATION","authors":"Yunying Chen, Xin Lin, Yi Qin, Donghui Wu, Jijie Gu, Siwei Nie","doi":"10.1093/abt/tbad014.011","DOIUrl":"https://doi.org/10.1093/abt/tbad014.011","url":null,"abstract":"Abstract Background The serum half-life of endogenous albumin is approximately 19 days in humans and 1.5-2.5 days in rodents. This extended half-life in vivo is primarily due to effective recycling upon internalization mediated by the neonatal Fc receptor (FcRn). Many protein therapeutics smaller than 60 kD have short serum half-life, which can be extended by fusing the proteins of interest with an anti-albumin antibody. The fusion proteins then take advantage of FcRn-mediated recycling. Single domain antibody (VHH) molecules (around 15 kD), derived from camelid heavy-chain-only IgG, are attracting increased attention globally in the field of antibody-based therapies due to several features including small size, high stability, low immunogenicity, good tissue penetration and cost effectiveness in manufacturing. Methods an anti-human/cynomolgus monkey/murine/canine/feline serum albumin VHH lead was discovered from a proprietary, large native phage-displayed VHH library, which was then humanized, and affinity matured. The optimized VHH was fused to bispecific and trispecific antibodies for in vivo PK studies. Results An anti-human serum albumin VHH Ab was discovered, and it also bound to cyno and mouse serum albumin with high affinity without affecting the interaction of HSA with FcRn. The VHH showed good developability and, once fused to protein therapeutics, long half-life and anti-tumor activity in rodents. Conclusions a novel VHH against serum albumin of different species was discovered from native VHH libraries, and it can be easily assembled into bispecific Abs and multispecific Abs to prolong the molecules’ PK profile.","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":"48158167","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}
Abstract Background and significance The presence of adenosine at abnormally high concentrations in tumor microenvironment leads to the activation of adenosine receptor 2A (A2aR) and represents one of the mechanisms for cancer patients to be resistance to immune checkpoint blockade therapies. CD39 and CD73 play an important role in converting ATP to adenosine. Currently antibodies or small molecule inhibitors (SMIs) targeting CD39 and CD73, as well as SMIs targeting adenosine receptors, are under preclinical investigation and early clinical development. However, effective biologics targeting adenosine receptors has not been reported. Here we demonstrated that humanized anti-A2aR antibodies represent promising novel therapeutic candidates for developing immunotherapy to restore anti-tumor responses in solid tumors that only partially respond or are completely resistance to ICB therapies. Methods Humanized anti-human A2aR monoclonal antibodies (mAbs) were generated from mouse hybridoma antibody via classic CDR grafting method. The A2aR binding and non-human primate cross-reactivity were measured by FACS using human A2aR-expressing HEK293 cells as well as human and cynomolgus monkey PBMCs. Internalizing properties were determined by FACS and immunofluorescent method. The cellular potency of these mAbs was assessed by their capability of inhibiting cAMP production induced by A2aR agonist adenosine and NECA in hA2aR-overexpressing HEK293 cells. The ability of lead candidates to reverse adenosine-mediated suppression of human effector cell function was determined using standard CD3/CD28 activation conditions in the presence of A2aR agonist NECA. Results and Conclusion We successfully humanized anti-A2aR antibody discovered via hybridoma technology. Humanized anti-A2aR antibodies maintain binding, cross-reactivity to human and cynomolgus monkey A2aR, as well as internalizing property and A2aR-antagonizing potency, in comparison with parental antibody. Humanized antibodies are 5~10 fold more potent than clinical stage small molecule inhibitors CPI-444 and AB928 in inhibiting cAMP production. Blockade of A2aR with humanized antibody can restore activation and cytokine production of human T and NK cells suppressed by A2aR agonist adenosine or NECA. Thus, antibody-mediated blockade of A2aR pathway represents a novel strategy to mitigate adenosine-mediated tumor resistance to immune checkpoint blockade therapies.
{"title":"POTENT AND SELECTIVE A2AR MONOCLONAL ANTIBODY ANTAGONIST FOR THE TREATMENT OF CANCER","authors":"Changyun Hu, Yanyan Wang, Xiqian Lan, Jiandong Zhang, Xinyan Zhao","doi":"10.1093/abt/tbad014.010","DOIUrl":"https://doi.org/10.1093/abt/tbad014.010","url":null,"abstract":"Abstract Background and significance The presence of adenosine at abnormally high concentrations in tumor microenvironment leads to the activation of adenosine receptor 2A (A2aR) and represents one of the mechanisms for cancer patients to be resistance to immune checkpoint blockade therapies. CD39 and CD73 play an important role in converting ATP to adenosine. Currently antibodies or small molecule inhibitors (SMIs) targeting CD39 and CD73, as well as SMIs targeting adenosine receptors, are under preclinical investigation and early clinical development. However, effective biologics targeting adenosine receptors has not been reported. Here we demonstrated that humanized anti-A2aR antibodies represent promising novel therapeutic candidates for developing immunotherapy to restore anti-tumor responses in solid tumors that only partially respond or are completely resistance to ICB therapies. Methods Humanized anti-human A2aR monoclonal antibodies (mAbs) were generated from mouse hybridoma antibody via classic CDR grafting method. The A2aR binding and non-human primate cross-reactivity were measured by FACS using human A2aR-expressing HEK293 cells as well as human and cynomolgus monkey PBMCs. Internalizing properties were determined by FACS and immunofluorescent method. The cellular potency of these mAbs was assessed by their capability of inhibiting cAMP production induced by A2aR agonist adenosine and NECA in hA2aR-overexpressing HEK293 cells. The ability of lead candidates to reverse adenosine-mediated suppression of human effector cell function was determined using standard CD3/CD28 activation conditions in the presence of A2aR agonist NECA. Results and Conclusion We successfully humanized anti-A2aR antibody discovered via hybridoma technology. Humanized anti-A2aR antibodies maintain binding, cross-reactivity to human and cynomolgus monkey A2aR, as well as internalizing property and A2aR-antagonizing potency, in comparison with parental antibody. Humanized antibodies are 5~10 fold more potent than clinical stage small molecule inhibitors CPI-444 and AB928 in inhibiting cAMP production. Blockade of A2aR with humanized antibody can restore activation and cytokine production of human T and NK cells suppressed by A2aR agonist adenosine or NECA. Thus, antibody-mediated blockade of A2aR pathway represents a novel strategy to mitigate adenosine-mediated tumor resistance to immune checkpoint blockade therapies.","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":"43810777","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}
T. Xu, Yuan-Fei Lv, Jie Sun, Jianjian Peng, Jingqiu Li, Xionghui Li, B. Guo
Abstract Background KN026 is a novel bispecific antibody and simultaneously binds to two distinct HER2 epitopes which are the same domains as trastuzumab (ECD4) and pertuzumab (ECD2). It showed higher maximal binding than monospecific HER2 antibodies and favors that crosslinking of HER2 receptors could enhance the receptors internalization in preclinical studies. KN026 significantly inhibited the growth of human cancer cell lines and demonstrated obviously antitumor activity against different xenografts models. The encouraging efficacy and manageable safety of KN026 were reported in Chinese cancer patients (pts) with HER2 expression and ph3 study is ongoing in gastric cancer (NCT05427383). Methods The late line solid tumor pts with HER2 expression who were refractory to or ineligible for standard therapy were recruited in this phase I dose-escalation study in US (NCT03847168). All pts intravenously received KN026 monotherapy at ascending dose of 10 mg/kg (QW), 20 mg/kg (Q2W) or 30 mg/kg (Q3W). Dose escalation followed standard “3 + 3” design. Expansion was determined by Safety Monitoring Committee (SMC). The primary objectives were to evaluate safety and tolerability of KN026 and determine the MTD and/or RP2D. Safety was evaluated according to CTCAE v 5.0, and efficacy was assessed by RECIST 1.1. Results A total of 22 pts with median 4 lines prior treatment were enrolled, including 5 breast cancer pts, 3 gastric or gastroesophageal junction cancer pts and 14 other solid tumor pts. 21 pts were included in the efficacy analysis. The median age was 57.0 (47-79), with 8 males and 14 females and most of pts were Caucasian (72.7%). 3, 10 (7 at expansion phase), and 9 (6 at expansion phase) pts were treated at 10mg/kg, 20mg/kg, 30mg/kg, respectively. No dose-limiting toxicities (DLTs) were observed and MTD wasn’t reached. 20 pts (90.9%) occurred TRAEs and the common (≥20%) TRAEs were diarrhea (45.5%), fatigue (40.9%), chills (36.4%) and nausea (27.3%). Most of TRAE were Gr 1-2, and only 3 pts (13.6%) experienced ≥ Gr3 TRAE (anemia, fatigue and pneumonitis) without treatment discontinuation and death caused by KN026. 4 of 21 pts (1 at 10mg/kg, 2 at 20 mg/kg, 1 at 30mg/kg) achieved objective response (ORR 19.0%, 95% CI: 5.5, 41.9), and DCR was 61.9% (95% CI: 38.4, 81.9). The exposure of KN026 and T1/2 increased with dose escalation. Accumulation ratio of Cmax and AUC was 1.18-1.42 and 1.14-1.71. The concentration of Ctrough was over 20μM at all dose levels. There was only one positive ADA sample in 20 mg/kg group in C1D1. And the RP2D was 20mg/kg Q2W or 30mg/kg Q3W. Conclusion KN026 was well tolerated and showed the promising anti-tumor activity in US pts with HER2 expression. Besides, KN026 has well pharmacokinetic profile and favorable immunogenicity.
{"title":"PRELIMINARY SAFETY, EFFICACY AND PHARMACOKINETICS (PK) RESULTS OF KN026 (A HER2 BISPECIFIC ANTIBODY) MONOTHERAPY IN ADVANCED SOLID TUMOR PATIENTS WITH HER2 EXPRESSION","authors":"T. Xu, Yuan-Fei Lv, Jie Sun, Jianjian Peng, Jingqiu Li, Xionghui Li, B. Guo","doi":"10.1093/abt/tbad014.006","DOIUrl":"https://doi.org/10.1093/abt/tbad014.006","url":null,"abstract":"Abstract Background KN026 is a novel bispecific antibody and simultaneously binds to two distinct HER2 epitopes which are the same domains as trastuzumab (ECD4) and pertuzumab (ECD2). It showed higher maximal binding than monospecific HER2 antibodies and favors that crosslinking of HER2 receptors could enhance the receptors internalization in preclinical studies. KN026 significantly inhibited the growth of human cancer cell lines and demonstrated obviously antitumor activity against different xenografts models. The encouraging efficacy and manageable safety of KN026 were reported in Chinese cancer patients (pts) with HER2 expression and ph3 study is ongoing in gastric cancer (NCT05427383). Methods The late line solid tumor pts with HER2 expression who were refractory to or ineligible for standard therapy were recruited in this phase I dose-escalation study in US (NCT03847168). All pts intravenously received KN026 monotherapy at ascending dose of 10 mg/kg (QW), 20 mg/kg (Q2W) or 30 mg/kg (Q3W). Dose escalation followed standard “3 + 3” design. Expansion was determined by Safety Monitoring Committee (SMC). The primary objectives were to evaluate safety and tolerability of KN026 and determine the MTD and/or RP2D. Safety was evaluated according to CTCAE v 5.0, and efficacy was assessed by RECIST 1.1. Results A total of 22 pts with median 4 lines prior treatment were enrolled, including 5 breast cancer pts, 3 gastric or gastroesophageal junction cancer pts and 14 other solid tumor pts. 21 pts were included in the efficacy analysis. The median age was 57.0 (47-79), with 8 males and 14 females and most of pts were Caucasian (72.7%). 3, 10 (7 at expansion phase), and 9 (6 at expansion phase) pts were treated at 10mg/kg, 20mg/kg, 30mg/kg, respectively. No dose-limiting toxicities (DLTs) were observed and MTD wasn’t reached. 20 pts (90.9%) occurred TRAEs and the common (≥20%) TRAEs were diarrhea (45.5%), fatigue (40.9%), chills (36.4%) and nausea (27.3%). Most of TRAE were Gr 1-2, and only 3 pts (13.6%) experienced ≥ Gr3 TRAE (anemia, fatigue and pneumonitis) without treatment discontinuation and death caused by KN026. 4 of 21 pts (1 at 10mg/kg, 2 at 20 mg/kg, 1 at 30mg/kg) achieved objective response (ORR 19.0%, 95% CI: 5.5, 41.9), and DCR was 61.9% (95% CI: 38.4, 81.9). The exposure of KN026 and T1/2 increased with dose escalation. Accumulation ratio of Cmax and AUC was 1.18-1.42 and 1.14-1.71. The concentration of Ctrough was over 20μM at all dose levels. There was only one positive ADA sample in 20 mg/kg group in C1D1. And the RP2D was 20mg/kg Q2W or 30mg/kg Q3W. Conclusion KN026 was well tolerated and showed the promising anti-tumor activity in US pts with HER2 expression. Besides, KN026 has well pharmacokinetic profile and favorable immunogenicity.","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":"42160602","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}
Pub Date : 2023-05-26eCollection Date: 2023-07-01DOI: 10.1093/abt/tbad009
Jing Chang, Christoph Rader, Haiyong Peng
In vitro display technologies have been successfully utilized for the discovery and evolution of monoclonal antibodies (mAbs) for diagnostic and therapeutic applications, with phage display and yeast display being the most commonly used platforms due to their simplicity and high efficiency. As their prokaryotic or lower eukaryotic host organisms typically have no or different post-translational modifications, several mammalian cell-based display and screening technologies for isolation and optimization of mAbs have emerged and are being developed. We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in a single-chain Fab (scFab) format on the surface of HEK293F cells. Immune rabbit antibody libraries encompassing ~7 × 107 independent clones were generated in an all-in-one transposon vector, stably delivered into HEK293F cells and displayed as an scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting and two rounds of fluorescence activated cell sorting, mAbs with high affinity in the subnanomolar range and cross-reactivity to the corresponding human and mouse antigens were identified, demonstrating the power of this platform for antibody discovery. We developed a highly efficient mammalian cell display platform based on the PiggyBac transposon system for antibody discovery, which could be further utilized for humanization as well as affinity and specificity maturation.
{"title":"A mammalian cell display platform based on scFab transposition.","authors":"Jing Chang, Christoph Rader, Haiyong Peng","doi":"10.1093/abt/tbad009","DOIUrl":"10.1093/abt/tbad009","url":null,"abstract":"<p><p><i>In vitro</i> display technologies have been successfully utilized for the discovery and evolution of monoclonal antibodies (mAbs) for diagnostic and therapeutic applications, with phage display and yeast display being the most commonly used platforms due to their simplicity and high efficiency. As their prokaryotic or lower eukaryotic host organisms typically have no or different post-translational modifications, several mammalian cell-based display and screening technologies for isolation and optimization of mAbs have emerged and are being developed. We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in a single-chain Fab (scFab) format on the surface of HEK293F cells. Immune rabbit antibody libraries encompassing ~7 × 10<sup>7</sup> independent clones were generated in an all-in-one transposon vector, stably delivered into HEK293F cells and displayed as an scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting and two rounds of fluorescence activated cell sorting, mAbs with high affinity in the subnanomolar range and cross-reactivity to the corresponding human and mouse antigens were identified, demonstrating the power of this platform for antibody discovery. We developed a highly efficient mammalian cell display platform based on the PiggyBac transposon system for antibody discovery, which could be further utilized for humanization as well as affinity and specificity maturation.</p>","PeriodicalId":36655,"journal":{"name":"Antibody Therapeutics","volume":"6 3","pages":"157-169"},"PeriodicalIF":0.0,"publicationDate":"2023-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10365156/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9878127","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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