Pub Date : 2024-01-01Epub Date: 2024-09-22DOI: 10.1080/19420862.2024.2406539
Geoff Hale, Alastair Douglas Davy, Ian Wilkinson
A critical attribute of therapeutic antibodies is their ability to engage with humoral or cellular effector mechanisms, and this depends on the ability of the Fc region to bind to complement (C1q) or Fc receptors. Investigators have sought to optimize these effects by engineering the Fc region to bind to a greater or lesser extent to individual receptors. Different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies representing a range of variants and compared their activity in cell-based assays for complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent phagocytosis using a range of individual Fc receptors. We have also compared the thermal stability of the variants by differential scanning fluorimetry (DSF). The results reveal a spectrum of activities which may be appropriate for different applications.
治疗性抗体的一个关键属性是它们与体液或细胞效应机制结合的能力,而这取决于Fc区与补体(C1q)或Fc受体结合的能力。研究人员试图通过对 Fc 区进行工程设计,使其在更大或更小的程度上与个别受体结合,从而优化这些效应。不同的方法已用于临床,但还没有对它们进行系统的比较。现在,我们已经制备出了一组匹配的抗 CD20 抗体,代表了一系列变体,并比较了它们在基于细胞的补体依赖性细胞毒性、抗体依赖性细胞介导的细胞毒性和抗体依赖性吞噬作用试验中使用一系列单个 Fc 受体的活性。我们还通过差示扫描荧光测定法(DSF)比较了变体的热稳定性。结果表明,这些变体具有不同的活性,可能适用于不同的应用领域。
{"title":"Systematic analysis of Fc mutations designed to enhance binding to Fc-gamma receptors.","authors":"Geoff Hale, Alastair Douglas Davy, Ian Wilkinson","doi":"10.1080/19420862.2024.2406539","DOIUrl":"10.1080/19420862.2024.2406539","url":null,"abstract":"<p><p>A critical attribute of therapeutic antibodies is their ability to engage with humoral or cellular effector mechanisms, and this depends on the ability of the Fc region to bind to complement (C1q) or Fc receptors. Investigators have sought to optimize these effects by engineering the Fc region to bind to a greater or lesser extent to individual receptors. Different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies representing a range of variants and compared their activity in cell-based assays for complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent phagocytosis using a range of individual Fc receptors. We have also compared the thermal stability of the variants by differential scanning fluorimetry (DSF). The results reveal a spectrum of activities which may be appropriate for different applications.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142290483","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01Epub Date: 2024-03-13DOI: 10.1080/19420862.2024.2322533
Chu'nan Liu, Lilian M Denzler, Oliver E C Hood, Andrew C R Martin
Antibodies have increasingly been developed as drugs with over 100 now licensed in the US or EU. During development, it is often necessary to increase or reduce the affinity of an antibody and rational attempts to do so rely on having a structure of the antibody-antigen complex often obtained by modeling. The antigen-binding site consists primarily of six loops known as complementarity-determining regions (CDRs), and an open question has been whether these loops change their conformation when they bind to an antigen. Existing surveys of antibody-antigen complex structures have only examined CDR conformational change in case studies or small-scale surveys. With an increasing number of antibodies where both free and complexed structures have been deposited in the Protein Data Bank, a large-scale survey of CDR conformational change during binding is now possible. To this end, we built a dataset, AbAgDb, that currently includes 177 antibodies with high-quality CDRs, each of which has at least one bound and one unbound structure. We analyzed the conformational change of the Cα backbone of each CDR upon binding and found that, in most cases, the CDRs (other than CDR-H3) show minimal movement, while 70.6% and 87% of CDR-H3s showed global Cα RMSD ≤ 1.0Å and ≤ 2.0Å, respectively. We also compared bound CDR conformations with the conformational space of unbound CDRs and found most of the bound conformations are included in the unbound conformational space. In future, our results will contribute to developing insights into antibodies and new methods for modeling and docking.
{"title":"Do antibody CDR loops change conformation upon binding?","authors":"Chu'nan Liu, Lilian M Denzler, Oliver E C Hood, Andrew C R Martin","doi":"10.1080/19420862.2024.2322533","DOIUrl":"10.1080/19420862.2024.2322533","url":null,"abstract":"<p><p>Antibodies have increasingly been developed as drugs with over 100 now licensed in the US or EU. During development, it is often necessary to increase or reduce the affinity of an antibody and rational attempts to do so rely on having a structure of the antibody-antigen complex often obtained by modeling. The antigen-binding site consists primarily of six loops known as complementarity-determining regions (CDRs), and an open question has been whether these loops change their conformation when they bind to an antigen. Existing surveys of antibody-antigen complex structures have only examined CDR conformational change in case studies or small-scale surveys. With an increasing number of antibodies where both free and complexed structures have been deposited in the Protein Data Bank, a large-scale survey of CDR conformational change during binding is now possible. To this end, we built a dataset, AbAgDb, that currently includes 177 antibodies with high-quality CDRs, each of which has at least one bound and one unbound structure. We analyzed the conformational change of the C<i>α</i> backbone of each CDR upon binding and found that, in most cases, the CDRs (other than CDR-H3) show minimal movement, while 70.6% and 87% of CDR-H3s showed global C<i>α</i> RMSD ≤ 1.0Å and ≤ 2.0Å, respectively. We also compared bound CDR conformations with the conformational space of unbound CDRs and found most of the bound conformations are included in the unbound conformational space. In future, our results will contribute to developing insights into antibodies and new methods for modeling and docking.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10939163/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140110600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01Epub Date: 2024-07-04DOI: 10.1080/19420862.2024.2373325
Amelia C McCue, Stephen J Demarest, Karen J Froning, Michael J Hickey, Stephen Antonysamy, Brian Kuhlman
T-cell engaging (TCE) bispecific antibodies are potent drugs that trigger the immune system to eliminate cancer cells, but administration can be accompanied by toxic side effects that limit dosing. TCEs function by binding to cell surface receptors on T cells, frequently CD3, with one arm of the bispecific antibody while the other arm binds to cell surface antigens on cancer cells. On-target, off-tumor toxicity can arise when the target antigen is also present on healthy cells. The toxicity of TCEs may be ameliorated through the use of pro-drug forms of the TCE, which are not fully functional until recruited to the tumor microenvironment. This can be accomplished by masking the anti-CD3 arm of the TCE with an autoinhibitory motif that is released by tumor-enriched proteases. Here, we solve the crystal structure of the antigen-binding fragment of a novel anti-CD3 antibody, E10, in complex with its epitope from CD3 and use this information to engineer a masked form of the antibody that can activate by the tumor-enriched protease matrix metalloproteinase 2 (MMP-2). We demonstrate with binding experiments and in vitro T-cell activation and killing assays that our designed prodrug TCE is capable of tumor-selective T-cell activity that is dependent upon MMP-2. Furthermore, we demonstrate that a similar masking strategy can be used to create a pro-drug form of the frequently used anti-CD3 antibody SP34. This study showcases an approach to developing immune-modulating therapeutics that prioritizes safety and has the potential to advance cancer immunotherapy treatment strategies.
{"title":"Engineering a tumor-selective prodrug T-cell engager bispecific antibody for safer immunotherapy.","authors":"Amelia C McCue, Stephen J Demarest, Karen J Froning, Michael J Hickey, Stephen Antonysamy, Brian Kuhlman","doi":"10.1080/19420862.2024.2373325","DOIUrl":"10.1080/19420862.2024.2373325","url":null,"abstract":"<p><p>T-cell engaging (TCE) bispecific antibodies are potent drugs that trigger the immune system to eliminate cancer cells, but administration can be accompanied by toxic side effects that limit dosing. TCEs function by binding to cell surface receptors on T cells, frequently CD3, with one arm of the bispecific antibody while the other arm binds to cell surface antigens on cancer cells. On-target, off-tumor toxicity can arise when the target antigen is also present on healthy cells. The toxicity of TCEs may be ameliorated through the use of pro-drug forms of the TCE, which are not fully functional until recruited to the tumor microenvironment. This can be accomplished by masking the anti-CD3 arm of the TCE with an autoinhibitory motif that is released by tumor-enriched proteases. Here, we solve the crystal structure of the antigen-binding fragment of a novel anti-CD3 antibody, E10, in complex with its epitope from CD3 and use this information to engineer a masked form of the antibody that can activate by the tumor-enriched protease matrix metalloproteinase 2 (MMP-2). We demonstrate with binding experiments and <i>in vitro</i> T-cell activation and killing assays that our designed prodrug TCE is capable of tumor-selective T-cell activity that is dependent upon MMP-2. Furthermore, we demonstrate that a similar masking strategy can be used to create a pro-drug form of the frequently used anti-CD3 antibody SP34. This study showcases an approach to developing immune-modulating therapeutics that prioritizes safety and has the potential to advance cancer immunotherapy treatment strategies.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11225918/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498394","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01Epub Date: 2024-06-06DOI: 10.1080/19420862.2024.2362789
Wen-Ting K Tsai, Yinyin Li, Zhaojun Yin, Peter Tran, Qui Phung, Zhenru Zhou, Kun Peng, Dan Qin, Sien Tam, Christoph Spiess, Jochen Brumm, Manda Wong, Zhengmao Ye, Patrick Wu, Sivan Cohen, Paul J Carter
Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.
包括双特异性 IgG 在内的双特异性抗体正在成为一类重要的新型抗体疗法。因此,我们和其他公司一起开发了工程策略,旨在促进双特异性 IgG 的高效生产,以用于临床开发。例如,我们广泛使用 "knobs-into-holes"(KIH)突变来促进抗体重链的异源二聚化,最近又使用 Fab 突变来促进同源重链/轻链配对,从而在单个宿主细胞中高效地在体内组装双特异性 IgG。我们构建了一个相关的单特异性和双特异性 IgG1 抗体面板,并通过与已知抗药抗体临床发生率低(阿瓦斯汀和赫赛汀)或高(博西珠单抗和 ATR-107)的基准抗体进行比较,评估免疫原性风险。使用的检测方法包括树突状细胞内化、T 细胞增殖、通过硅预测和 MHC 相关肽蛋白质组学鉴定 T 细胞表位。每种方法的数据都是独立考虑的,然后一起进行整体综合免疫原性风险评估。总之,这些数据表明,KIH 突变和体外组装半抗并不构成双特异性 IgG1 免疫原性的主要风险,用于在体内单宿主细胞中高效组装双特异性抗体的 Fab 突变也不构成主要风险。研究级制剂曲妥珠单抗和贝伐珠单抗的免疫原性风险评估数据分别与赫赛汀和阿瓦斯汀相当或略高。这些数据为使用研究级 IgG1 制剂作为替代物对相应药物进行免疫原性风险评估的普遍做法提供了实验支持。
{"title":"Nonclinical immunogenicity risk assessment for knobs-into-holes bispecific IgG<sub>1</sub> antibodies.","authors":"Wen-Ting K Tsai, Yinyin Li, Zhaojun Yin, Peter Tran, Qui Phung, Zhenru Zhou, Kun Peng, Dan Qin, Sien Tam, Christoph Spiess, Jochen Brumm, Manda Wong, Zhengmao Ye, Patrick Wu, Sivan Cohen, Paul J Carter","doi":"10.1080/19420862.2024.2362789","DOIUrl":"10.1080/19420862.2024.2362789","url":null,"abstract":"<p><p>Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient <i>in vivo</i> assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG<sub>1</sub> antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by <i>in silico</i> prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. <i>In toto</i>, these data suggest that the KIH mutations and <i>in vitro</i> assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG<sub>1</sub>, nor do the Fab mutations used for efficient <i>in vivo</i> assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG<sub>1</sub> as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11164226/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141284152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01Epub Date: 2024-02-13DOI: 10.1080/19420862.2024.2310248
Dan Zhu, Haralambos Hadjivassiliou, Catherine Jennings, David Mikolon, Massimo Ammirante, Sharmistha Acharya, Jon Lloyd, Mahan Abbasian, Rama Krishna Narla, Joseph R Piccotti, Katie Stamp, Ho Cho, Kandasamy Hariharan
Cluster of differentiation 47 (CD47) is a transmembrane protein highly expressed in tumor cells that interacts with signal regulatory protein alpha (SIRPα) and triggers a "don't eat me" signal to the macrophage, inhibiting phagocytosis and enabling tumor escape from immunosurveillance. The CD47-SIRPα axis has become an important target for cancer immunotherapy. To date, the advancement of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hematologic toxicity including anemia. To overcome those challenges a bispecific approach was taken. CC-96673, a humanized IgG1 bispecific antibody co-targeting CD47 and CD20, is designed to bind CD20 with high affinity and CD47 with optimally lowered affinity. As a result of the detuned CD47 affinity, CC-96673 selectively binds to CD20-expressing cells, blocking the interaction of CD47 with SIRPα. This increased selectivity of CC-96673 over monospecific anti-CD47 approaches allows for the use of wild-type IgG1 Fc, which engages activating crystallizable fragment gamma receptors (FcγRs) to fully potentiate macrophages to engulf and destroy CD20+ cells, while sparing CD47+CD20- normal cells. The combined targeting of anti-CD20 and anti-CD47 results in enhanced anti- tumor activity compared to anti-CD20 targeting antibodies alone. Furthermore, preclinical studies have demonstrated that CC-96673 exhibits acceptable pharmacokinetic properties with a favorable toxicity profile in non-human primates. Collectively, these findings define CC-96673 as a promising CD47 × CD20 bispecific antibody that selectively destroys CD20+ cancer cells via enhanced phagocytosis and other effector functions.
{"title":"CC-96673 (BMS-986358), an affinity-tuned anti-CD47 and CD20 bispecific antibody with fully functional fc, selectively targets and depletes non-Hodgkin's lymphoma.","authors":"Dan Zhu, Haralambos Hadjivassiliou, Catherine Jennings, David Mikolon, Massimo Ammirante, Sharmistha Acharya, Jon Lloyd, Mahan Abbasian, Rama Krishna Narla, Joseph R Piccotti, Katie Stamp, Ho Cho, Kandasamy Hariharan","doi":"10.1080/19420862.2024.2310248","DOIUrl":"10.1080/19420862.2024.2310248","url":null,"abstract":"<p><p>Cluster of differentiation 47 (CD47) is a transmembrane protein highly expressed in tumor cells that interacts with signal regulatory protein alpha (SIRPα) and triggers a \"don't eat me\" signal to the macrophage, inhibiting phagocytosis and enabling tumor escape from immunosurveillance. The CD47-SIRPα axis has become an important target for cancer immunotherapy. To date, the advancement of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hematologic toxicity including anemia. To overcome those challenges a bispecific approach was taken. CC-96673, a humanized IgG1 bispecific antibody co-targeting CD47 and CD20, is designed to bind CD20 with high affinity and CD47 with optimally lowered affinity. As a result of the detuned CD47 affinity, CC-96673 selectively binds to CD20-expressing cells, blocking the interaction of CD47 with SIRPα. This increased selectivity of CC-96673 over monospecific anti-CD47 approaches allows for the use of wild-type IgG1 Fc, which engages activating crystallizable fragment gamma receptors (FcγRs) to fully potentiate macrophages to engulf and destroy CD20<sup>+</sup> cells, while sparing CD47<sup>+</sup>CD20<sup>-</sup> normal cells. The combined targeting of anti-CD20 and anti-CD47 results in enhanced anti- tumor activity compared to anti-CD20 targeting antibodies alone. Furthermore, preclinical studies have demonstrated that CC-96673 exhibits acceptable pharmacokinetic properties with a favorable toxicity profile in non-human primates. Collectively, these findings define CC-96673 as a promising CD47 × CD20 bispecific antibody that selectively destroys CD20<sup>+</sup> cancer cells via enhanced phagocytosis and other effector functions.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10865928/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139723151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01Epub Date: 2024-07-25DOI: 10.1080/19420862.2024.2383013
Siva Charan Devanaboyina, Peng Li, Edward L LaGory, Carrie Poon-Andersen, Kevin D Cook, Marcus Soto, Zhe Wang, Khue Dang, Craig Uyeda, Ryan B Case, Veena A Thomas, Ronya Primack, Manuel Ponce, Mei Di, Brian Ouyang, Joelle Kaner, Sheung Kwan Lam, Mina Mostafavi
Targeting antigens with antibodies exhibiting pH/Ca2+-dependent binding against an antigen is an attractive strategy to mitigate target-mediated disposition and antigen buffering. Studies have reported improved serum exposure of antibodies exhibiting pH/Ca2+-binding against membrane-bound receptors. Asialoglycoprotein receptor 1 (ASGR1) is a membrane-bound receptor primarily localized in hepatocytes. With a high expression level of approximately one million receptors per cell, high turnover, and rapid recycling, targeting this receptor with a conventional antibody is a challenge. In this study, we identified an antibody exhibiting pH/Ca2+-dependent binding to ASGR1 and generated antibody variants with increased binding to neonatal crystallizable fragment receptor (FcRn). Serum exposures of the generated anti-ASGR1 antibodies were analyzed in transgenic mice expressing human FcRn. Contrary to published reports of increased serum exposure of pH/Ca2+-dependent antibodies, the pH/Ca2+-dependent anti-ASGR1 antibody had rapid serum clearance in comparison to a conventional anti-ASGR1 antibody. We conducted sub-cellular trafficking studies of the anti-ASGR1 antibodies along with receptor quantification analysis for mechanistic understanding of the rapid serum clearance of pH/Ca2+-dependent anti-ASGR1 antibody. The findings from our study provide valuable insights in identifying the antigens, especially membrane bound, that may benefit from targeting with pH/Ca2+-dependent antibodies to obtain increased serum exposure.
{"title":"Rapid depletion of \"catch-and-release\" anti-ASGR1 antibody in vivo.","authors":"Siva Charan Devanaboyina, Peng Li, Edward L LaGory, Carrie Poon-Andersen, Kevin D Cook, Marcus Soto, Zhe Wang, Khue Dang, Craig Uyeda, Ryan B Case, Veena A Thomas, Ronya Primack, Manuel Ponce, Mei Di, Brian Ouyang, Joelle Kaner, Sheung Kwan Lam, Mina Mostafavi","doi":"10.1080/19420862.2024.2383013","DOIUrl":"10.1080/19420862.2024.2383013","url":null,"abstract":"<p><p>Targeting antigens with antibodies exhibiting pH/Ca<sup>2+</sup>-dependent binding against an antigen is an attractive strategy to mitigate target-mediated disposition and antigen buffering. Studies have reported improved serum exposure of antibodies exhibiting pH/Ca<sup>2+</sup>-binding against membrane-bound receptors. Asialoglycoprotein receptor 1 (ASGR1) is a membrane-bound receptor primarily localized in hepatocytes. With a high expression level of approximately one million receptors per cell, high turnover, and rapid recycling, targeting this receptor with a conventional antibody is a challenge. In this study, we identified an antibody exhibiting pH/Ca<sup>2+</sup>-dependent binding to ASGR1 and generated antibody variants with increased binding to neonatal crystallizable fragment receptor (FcRn). Serum exposures of the generated anti-ASGR1 antibodies were analyzed in transgenic mice expressing human FcRn. Contrary to published reports of increased serum exposure of pH/Ca<sup>2+</sup>-dependent antibodies, the pH/Ca<sup>2+</sup>-dependent anti-ASGR1 antibody had rapid serum clearance in comparison to a conventional anti-ASGR1 antibody. We conducted sub-cellular trafficking studies of the anti-ASGR1 antibodies along with receptor quantification analysis for mechanistic understanding of the rapid serum clearance of pH/Ca<sup>2+</sup>-dependent anti-ASGR1 antibody. The findings from our study provide valuable insights in identifying the antigens, especially membrane bound, that may benefit from targeting with pH/Ca<sup>2+</sup>-dependent antibodies to obtain increased serum exposure.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11275528/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141759614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01Epub Date: 2024-01-03DOI: 10.1080/19420862.2023.2297451
Jennifer L Schwedler, Maxwell A Stefan, Christine E Thatcher, Peter R McIlroy, Anupama Sinha, Ashlee M Phillips, Christopher A Sumner, Colleen M Courtney, Christina Y Kim, Dina R Weilhammer, Brooke Harmon
The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.
特异、安全和强效单克隆抗体(Abs)的开发为传染病带来了新的治疗选择。除了通过中和防止病毒感染外,Abs 还能清除受感染的细胞,并通过其可结晶片段(Fc)与补体蛋白和免疫细胞上的 Fc 受体的结合诱导免疫调节功能。人们对中和Abs的Fc效应功能在脑炎病毒感染中的作用知之甚少。为了确定 Fc 效应器功能在抗委内瑞拉马脑炎病毒(VEEV)疗效中的作用,我们比较了在 VEEV 感染情况下具有完整 Fc 功能(hF5-WT)或含有功能缺失 Fc 突变 L234A 和 L235A(hF5-LALA)的强效中和抗 VEEV 人 IgG F5(hF5)抗体。我们观察到,与 hF5-WT 相比,hF5-LALA 与补体和 Fc 受体的结合明显减少,Fc 介导的体外细胞毒性动力学也有所不同。在感染后-24小时和+24小时,hF5-LALA的体内疗效与hF5-WT相当,两种Abs都能提供高水平的保护。然而,当感染后+ 48小时注射hF5-WT和hF5-LALA时,hF5-LALA的疗效显著下降。这些结果共同表明,针对 VEEV(可能还包括其他脑炎α-病毒)的最佳治疗性抗体疗法需要通过 Fc 区域中和与免疫效应因子的结合。
{"title":"Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function.","authors":"Jennifer L Schwedler, Maxwell A Stefan, Christine E Thatcher, Peter R McIlroy, Anupama Sinha, Ashlee M Phillips, Christopher A Sumner, Colleen M Courtney, Christina Y Kim, Dina R Weilhammer, Brooke Harmon","doi":"10.1080/19420862.2023.2297451","DOIUrl":"10.1080/19420862.2023.2297451","url":null,"abstract":"<p><p>The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential <i>in vitro</i> kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The <i>in vivo</i> efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10766394/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139087446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01Epub Date: 2024-07-30DOI: 10.1080/19420862.2024.2379903
Linlin Dong, Susan Chen, Konstantin Piatkov, Dong Wei, Mark G Qian
A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.
我们需要一种灵敏而特异的生物分析方法来测量小鼠血浆中 LAGA 突变代用品小鼠 IgG2a 单克隆抗体的暴露量,但由于缺乏针对 LAGA 突变体的高度特异性试剂,配体结合检测方法的开发受到了阻碍。同样存在问题的是,在 mIgG2a 互补性决定区无法鉴定出适用于定量质谱分析的敏感的独特胰蛋白酶肽。为了克服这些难题,我们系统地研究了一种胰蛋白酶替代品胃蛋白酶(一种天冬氨酸蛋白酶),研究它在消化变异 mIgG2a 抗体时的用途,以便生成用于生物分析定量目的的特征肽。经过一系列评估后,针对突变的 Fc 骨架建立了一小时快速胃蛋白酶消化方案。因此,成功开发了一种基于胃蛋白酶消化的液相色谱-串联质谱(LC/MS/MS)新方法,以支持小鼠药代动力学(PK)样本分析。简而言之,在pH≤2和37°C条件下,对突变mIgG2a双突变位点附近的亮氨酸和苯丙氨酸进行了稳健且可重复的C端裂解。结合市售的大鼠抗 mIgG2a 重链抗体,所建立的免疫亲和 LC/MS/MS 检测方法在相关动态范围内的定量限为 20 ng/mL,检测精度和准确度令人满意。在发现 PK 研究中成功实施这种新方法后,就不再需要为 LAGA 突变体制作繁琐而昂贵的特异性免疫捕获试剂了。这种方法应广泛适用于开发基于 LAGA 突变体的流行生物疗法。
{"title":"Quantifying LAGA mutated mouse IgG2a monoclonal antibody with a rapid pepsin digestion enabled immunoaffinity LC/MS/MS assay.","authors":"Linlin Dong, Susan Chen, Konstantin Piatkov, Dong Wei, Mark G Qian","doi":"10.1080/19420862.2024.2379903","DOIUrl":"10.1080/19420862.2024.2379903","url":null,"abstract":"<p><p>A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.6,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11290748/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01Epub Date: 2024-03-21DOI: 10.1080/19420862.2024.2324836
Ruoxuan Sun, Mark G Qian, Xiaobin Zhang
The surge in the clinical use of therapeutic antibodies has reshaped the landscape of pharmaceutical therapy for many diseases, including rare and challenging conditions. However, the administration of exogenous biologics could potentially trigger unwanted immune responses such as generation of anti-drug antibodies (ADAs). Real-world experiences have illuminated the clear correlation between the ADA occurrence and unsatisfactory therapeutic outcomes as well as immune-related adverse events. By retrospectively examining research involving immunogenicity analysis, we noticed the growing emphasis on elucidating the immunogenic epitope profiles of antibody-based therapeutics aiming for mechanistic understanding the immunogenicity generation and, ideally, mitigating the risks. As such, we have comprehensively summarized here the progress in both experimental and computational methodologies for the characterization of T and B cell epitopes of therapeutics. Furthermore, the successful practice of epitope-driven deimmunization of biotherapeutics is exceptionally highlighted in this article.
治疗性抗体的临床应用激增,重塑了许多疾病的药物治疗格局,包括罕见和具有挑战性的疾病。然而,施用外源性生物制剂有可能引发不必要的免疫反应,如产生抗药抗体(ADA)。现实世界的经验表明,ADA 的产生与不理想的治疗效果以及与免疫相关的不良事件之间存在明显的相关性。通过对涉及免疫原性分析的研究进行回顾性研究,我们注意到人们越来越重视阐明抗体疗法的免疫原性表位谱,目的是从机理上理解免疫原性的产生,并在理想情况下降低风险。因此,我们在此全面总结了用于表征治疗药物 T 细胞和 B 细胞表位的实验和计算方法的进展。此外,本文还特别强调了表位驱动的生物治疗药物去免疫的成功实践。
{"title":"T and B cell epitope analysis for the immunogenicity evaluation and mitigation of antibody-based therapeutics.","authors":"Ruoxuan Sun, Mark G Qian, Xiaobin Zhang","doi":"10.1080/19420862.2024.2324836","DOIUrl":"10.1080/19420862.2024.2324836","url":null,"abstract":"<p><p>The surge in the clinical use of therapeutic antibodies has reshaped the landscape of pharmaceutical therapy for many diseases, including rare and challenging conditions. However, the administration of exogenous biologics could potentially trigger unwanted immune responses such as generation of anti-drug antibodies (ADAs). Real-world experiences have illuminated the clear correlation between the ADA occurrence and unsatisfactory therapeutic outcomes as well as immune-related adverse events. By retrospectively examining research involving immunogenicity analysis, we noticed the growing emphasis on elucidating the immunogenic epitope profiles of antibody-based therapeutics aiming for mechanistic understanding the immunogenicity generation and, ideally, mitigating the risks. As such, we have comprehensively summarized here the progress in both experimental and computational methodologies for the characterization of T and B cell epitopes of therapeutics. Furthermore, the successful practice of epitope-driven deimmunization of biotherapeutics is exceptionally highlighted in this article.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":null,"pages":null},"PeriodicalIF":5.3,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10962608/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140184822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}