T. Akuta, T. Maruyama, Chiaki Sakuma, Masataka Nakagawa, Yui Tomioka, Kevin C Entzminger, J. Fleming, Ryo Sato, Takashi Shibata, Y. Kurosawa, C. Okumura, T. Arakawa
In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein.
{"title":"A New Method to Characterize Conformation-Specific Antibody by a Combination of Agarose Native Gel Electrophoresis and Contact Blotting","authors":"T. Akuta, T. Maruyama, Chiaki Sakuma, Masataka Nakagawa, Yui Tomioka, Kevin C Entzminger, J. Fleming, Ryo Sato, Takashi Shibata, Y. Kurosawa, C. Okumura, T. Arakawa","doi":"10.3390/antib11020036","DOIUrl":"https://doi.org/10.3390/antib11020036","url":null,"abstract":"In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47578830","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}
Janine Dilchert, M. Hofmann, F. Unverdorben, R. Kontermann, S. Bunk
Bispecific T cell receptor (TCR)-based molecules capable of redirecting and activating T cells towards tumor cells represent a novel and promising class of biotherapeutics for the treatment of cancer. Usage of TCRs allows for targeting of intracellularly expressed and highly selective cancer antigens, but also requires a complex maturation process to increase the naturally low affinity and stability of TCRs. Even though TCR domains can be matured via phage and yeast display, these techniques share the disadvantages of non-human glycosylation patterns and the need for a later reformatting into the final bispecific format. Here, we describe the development and application of a Chinese Hamster Ovary (CHO) display for affinity engineering of TCRs in the context of the final bispecific TCR format. The recombinase-mediated cassette exchange (RCME)-based system allows for stable, single-copy integration of bispecific TCR molecules with high efficiency into a defined genetic locus of CHO cells. We used the system to isolate affinity-increased variants of bispecific T cell engaging receptor (TCER) molecules from a library encoding different CDR variants of a model TCR targeting preferentially expressed antigen in melanoma (PRAME). When expressed as a soluble protein, the selected TCER molecules exhibited strong reactivity against PRAME-positive tumor cells associated with a pronounced cytokine release from activated T cells. The obtained data support the usage of the CHO display-based maturation system for TCR affinity maturation in the context of the final bispecific TCER format.
{"title":"Mammalian Display Platform for the Maturation of Bispecific TCR-Based Molecules","authors":"Janine Dilchert, M. Hofmann, F. Unverdorben, R. Kontermann, S. Bunk","doi":"10.3390/antib11020034","DOIUrl":"https://doi.org/10.3390/antib11020034","url":null,"abstract":"Bispecific T cell receptor (TCR)-based molecules capable of redirecting and activating T cells towards tumor cells represent a novel and promising class of biotherapeutics for the treatment of cancer. Usage of TCRs allows for targeting of intracellularly expressed and highly selective cancer antigens, but also requires a complex maturation process to increase the naturally low affinity and stability of TCRs. Even though TCR domains can be matured via phage and yeast display, these techniques share the disadvantages of non-human glycosylation patterns and the need for a later reformatting into the final bispecific format. Here, we describe the development and application of a Chinese Hamster Ovary (CHO) display for affinity engineering of TCRs in the context of the final bispecific TCR format. The recombinase-mediated cassette exchange (RCME)-based system allows for stable, single-copy integration of bispecific TCR molecules with high efficiency into a defined genetic locus of CHO cells. We used the system to isolate affinity-increased variants of bispecific T cell engaging receptor (TCER) molecules from a library encoding different CDR variants of a model TCR targeting preferentially expressed antigen in melanoma (PRAME). When expressed as a soluble protein, the selected TCER molecules exhibited strong reactivity against PRAME-positive tumor cells associated with a pronounced cytokine release from activated T cells. The obtained data support the usage of the CHO display-based maturation system for TCR affinity maturation in the context of the final bispecific TCER format.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48206817","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}
Adoptive cell therapy holds great promise for treating a myriad of diseases, especially cancer. Within the last decade, immunotherapy has provided a significant leap in the successful treatment of leukemia. The research conducted throughout this period to understand the interrelationships between cancer cells and infiltrating immune cells winds up having one very common feature, bioenergetics. Cancer cells and immune cells both need ATP to perform their individual functions and cancer cells have adopted means to limit immune cell activity via changes in immune cell bioenergetics that redirect immune cell behavior to encourage tumor growth. Current leading strategies for cancer treatment super-charge an individual’s own immune cells against cancer. Successful Chimeric Antigen Receptor T Cells (CAR T) target pathways that ultimately influence bioenergetics. In the last decade, scientists identified that mitochondria play a crucial role in T cell physiology. When modifying T cells to create chimeras, a unique mitochondrial fitness emerges that establishes stemness and persistence. This review highlights many of the key findings leading to this generation’s CAR T treatments and the work currently being done to advance immunotherapy, to empower not just T cells but other immune cells as well against a variety of cancers.
{"title":"Immune Cell Metabolic Fitness for Life","authors":"K. Bittman","doi":"10.3390/antib11020032","DOIUrl":"https://doi.org/10.3390/antib11020032","url":null,"abstract":"Adoptive cell therapy holds great promise for treating a myriad of diseases, especially cancer. Within the last decade, immunotherapy has provided a significant leap in the successful treatment of leukemia. The research conducted throughout this period to understand the interrelationships between cancer cells and infiltrating immune cells winds up having one very common feature, bioenergetics. Cancer cells and immune cells both need ATP to perform their individual functions and cancer cells have adopted means to limit immune cell activity via changes in immune cell bioenergetics that redirect immune cell behavior to encourage tumor growth. Current leading strategies for cancer treatment super-charge an individual’s own immune cells against cancer. Successful Chimeric Antigen Receptor T Cells (CAR T) target pathways that ultimately influence bioenergetics. In the last decade, scientists identified that mitochondria play a crucial role in T cell physiology. When modifying T cells to create chimeras, a unique mitochondrial fitness emerges that establishes stemness and persistence. This review highlights many of the key findings leading to this generation’s CAR T treatments and the work currently being done to advance immunotherapy, to empower not just T cells but other immune cells as well against a variety of cancers.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41862427","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}
Immunotherapy has revolutionized the care of cancer patients. A diverse set of strategies to overcome cancer immunosuppression and enhance the tumor-directed immune response are in clinical use, but have not achieved transformative benefits for brain tumor patients. Adoptive cell therapies, which employ a patient’s own immune cells to generate directed anti-tumor activity, are emerging technologies that hold promise to improve the treatment of primary brain tumors in children and adults. Here, we review recent advances in chimeric antigen receptor (CAR) T-cell therapies for the treatment of aggressive primary brain tumors, including glioblastoma and diffuse midline glioma, H3 K27M-mutant. We highlight current approaches, discuss encouraging investigational data, and describe key challenges in the development and implementation of these types of therapies in the neuro-oncology setting.
{"title":"Advances in Chimeric Antigen Receptor (CAR) T-Cell Therapies for the Treatment of Primary Brain Tumors","authors":"Christopher W. Mount, L. G. Gonzalez Castro","doi":"10.3390/antib11020031","DOIUrl":"https://doi.org/10.3390/antib11020031","url":null,"abstract":"Immunotherapy has revolutionized the care of cancer patients. A diverse set of strategies to overcome cancer immunosuppression and enhance the tumor-directed immune response are in clinical use, but have not achieved transformative benefits for brain tumor patients. Adoptive cell therapies, which employ a patient’s own immune cells to generate directed anti-tumor activity, are emerging technologies that hold promise to improve the treatment of primary brain tumors in children and adults. Here, we review recent advances in chimeric antigen receptor (CAR) T-cell therapies for the treatment of aggressive primary brain tumors, including glioblastoma and diffuse midline glioma, H3 K27M-mutant. We highlight current approaches, discuss encouraging investigational data, and describe key challenges in the development and implementation of these types of therapies in the neuro-oncology setting.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43119730","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}
An elevated IgA level obtained in a 10-year-old male a year after an episode of pneumococcal sepsis led to the discovery of a broad-based IgG-specific antibody deficiency syndrome. The specifics of the case and pertinent literature are presented, including a discussion of the hyper-IgD syndrome. An elevated IgA, greater than two standard deviations above the expected age range should prompt a complete workup for selective antibody deficiency syndrome and adds an additional associated marker of an indolent hyper-IgD syndrome in a different clinical circumstance, although the lack of antibody response to vaccines is atypical of the hyper-IgD syndrome.
{"title":"Persistent Hyper IgA as a Marker of Immune Deficiency: A Case Report","authors":"R. Hopp, H. Niebur","doi":"10.3390/antib11020030","DOIUrl":"https://doi.org/10.3390/antib11020030","url":null,"abstract":"An elevated IgA level obtained in a 10-year-old male a year after an episode of pneumococcal sepsis led to the discovery of a broad-based IgG-specific antibody deficiency syndrome. The specifics of the case and pertinent literature are presented, including a discussion of the hyper-IgD syndrome. An elevated IgA, greater than two standard deviations above the expected age range should prompt a complete workup for selective antibody deficiency syndrome and adds an additional associated marker of an indolent hyper-IgD syndrome in a different clinical circumstance, although the lack of antibody response to vaccines is atypical of the hyper-IgD syndrome.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48882055","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}
Immune checkpoint blockades prescribed in the neoadjuvant setting are now under active investigation for many types of tumors, and many have shown early success. The primary tumor (PT) and tumor-draining lymph node (TDLN) immune factors, along with adequate therapeutic antibody distributions to the PT and TDLN, are critical for optimal immune activation and anti-tumor efficacy in neoadjuvant immunotherapy. However, it remains largely unknown how much of the antibody can be distributed into the PT-TDLN axis at different clinical scenarios. The goal of the current work is to build a physiologically based pharmacokinetic (PBPK) model framework capable of characterizing antibody distribution gradients in the PT-TDLN axis across various clinical and pathophysiological scenarios. The model was calibrated using clinical data from immuno-PET antibody-imaging studies quantifying antibody pharmacokinetics (PK) in the blood, PTs, and TDLNs. The effects of metastatic lesion location, tumor-induced compression, and inflammation, as well as surgery, on antibody concentration gradients in the PT-TDLN axis were characterized. The PBPK model serves as a valuable tool to predict antibody exposures in various types of tumors, metastases, and the associated lymph node, supporting effective immunotherapy.
{"title":"A Physiologically Based Pharmacokinetic Framework for Quantifying Antibody Distribution Gradients from Tumors to Tumor-Draining Lymph Nodes","authors":"Eric Salgado, Yanguang Cao","doi":"10.3390/antib11020028","DOIUrl":"https://doi.org/10.3390/antib11020028","url":null,"abstract":"Immune checkpoint blockades prescribed in the neoadjuvant setting are now under active investigation for many types of tumors, and many have shown early success. The primary tumor (PT) and tumor-draining lymph node (TDLN) immune factors, along with adequate therapeutic antibody distributions to the PT and TDLN, are critical for optimal immune activation and anti-tumor efficacy in neoadjuvant immunotherapy. However, it remains largely unknown how much of the antibody can be distributed into the PT-TDLN axis at different clinical scenarios. The goal of the current work is to build a physiologically based pharmacokinetic (PBPK) model framework capable of characterizing antibody distribution gradients in the PT-TDLN axis across various clinical and pathophysiological scenarios. The model was calibrated using clinical data from immuno-PET antibody-imaging studies quantifying antibody pharmacokinetics (PK) in the blood, PTs, and TDLNs. The effects of metastatic lesion location, tumor-induced compression, and inflammation, as well as surgery, on antibody concentration gradients in the PT-TDLN axis were characterized. The PBPK model serves as a valuable tool to predict antibody exposures in various types of tumors, metastases, and the associated lymph node, supporting effective immunotherapy.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49467148","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}
Georg Tscheuschner, M. Kaiser, J. Lisec, Denis Beslic, T. Muth, Maren Krüger, H. Mages, B. Dorner, Julia Knospe, J. Schenk, F. Sellrie, M. Weller
During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.
{"title":"MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour","authors":"Georg Tscheuschner, M. Kaiser, J. Lisec, Denis Beslic, T. Muth, Maren Krüger, H. Mages, B. Dorner, Julia Knospe, J. Schenk, F. Sellrie, M. Weller","doi":"10.3390/antib11020027","DOIUrl":"https://doi.org/10.3390/antib11020027","url":null,"abstract":"During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43059075","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}
P. Dimou, S. Trivedi, Maria Liousia, Reena R D'Souza, A. Klampatsa
Precision-cut tumor slices (PCTS) have recently emerged as important ex vivo human tumor models, offering the opportunity to study individual patient responses to targeted immunotherapies, including CAR-T cell therapies. In this review, an outline of different human tumor models available in laboratory settings is provided, with a focus on the unique characteristics of PCTS. Standard PCTS generation and maintenance procedures are outlined, followed by an in-depth overview of PCTS utilization in preclinical research aiming to better understand the unique functional characteristics of cytotoxic T cells within human tumors. Furthermore, recent studies using PCTS as an ex vivo model for predicting patient responses to immunotherapies and other targeted therapies against solid tumors are thoroughly presented. Finally, the advantages and limitations of the PCTS models are discussed. PCTS are expected to gain momentum and be fully utilized as a significant tool towards better patient stratification and personalized medicine.
{"title":"Precision-Cut Tumor Slices (PCTS) as an Ex Vivo Model in Immunotherapy Research","authors":"P. Dimou, S. Trivedi, Maria Liousia, Reena R D'Souza, A. Klampatsa","doi":"10.3390/antib11020026","DOIUrl":"https://doi.org/10.3390/antib11020026","url":null,"abstract":"Precision-cut tumor slices (PCTS) have recently emerged as important ex vivo human tumor models, offering the opportunity to study individual patient responses to targeted immunotherapies, including CAR-T cell therapies. In this review, an outline of different human tumor models available in laboratory settings is provided, with a focus on the unique characteristics of PCTS. Standard PCTS generation and maintenance procedures are outlined, followed by an in-depth overview of PCTS utilization in preclinical research aiming to better understand the unique functional characteristics of cytotoxic T cells within human tumors. Furthermore, recent studies using PCTS as an ex vivo model for predicting patient responses to immunotherapies and other targeted therapies against solid tumors are thoroughly presented. Finally, the advantages and limitations of the PCTS models are discussed. PCTS are expected to gain momentum and be fully utilized as a significant tool towards better patient stratification and personalized medicine.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42840223","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}
A. Xu, Nicholas J. Clark, Joey Pollastrini, Maribel Espinoza, Hyo-Jin Kim, Sekhar R. Kanapuram, B. Kerwin, M. Treuheit, S. Krueger, A. Mcauley, J. E. Curtis
In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (CNaCl) and monoclonal antibody (mAb) concentration (CmAb) were assessed using the diffusion interaction parameter kD and second virial coefficient B22 measured from solutions with low to moderate CmAb. The effective structure factor S(q)eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with CNaCl, but also with increasing CmAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including kD and S(q)eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low CmAb.
{"title":"Effects of Monovalent Salt on Protein-Protein Interactions of Dilute and Concentrated Monoclonal Antibody Formulations","authors":"A. Xu, Nicholas J. Clark, Joey Pollastrini, Maribel Espinoza, Hyo-Jin Kim, Sekhar R. Kanapuram, B. Kerwin, M. Treuheit, S. Krueger, A. Mcauley, J. E. Curtis","doi":"10.3390/antib11020024","DOIUrl":"https://doi.org/10.3390/antib11020024","url":null,"abstract":"In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (CNaCl) and monoclonal antibody (mAb) concentration (CmAb) were assessed using the diffusion interaction parameter kD and second virial coefficient B22 measured from solutions with low to moderate CmAb. The effective structure factor S(q)eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with CNaCl, but also with increasing CmAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including kD and S(q)eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low CmAb.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46281099","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}
This study aimed to investigate PD-L1 and PD-1 expression in circulating CD20+ cells in diffuse larger B-cell lymphoma (DLBCL) and to evaluate the predictive and diagnostic performance of PD-L1 versus PD-1 expression in circulating CD20+ cells in DLBCL. Percentages of CD20+, PD-L1+CD20+, and PD-1+CD20+ cells were measured by flow cytometry in 40 DLBCL blood samples and 19 healthy controls. The DLBCL patient group was subdivided into 20 newly diagnosed patients with no treatment yet and 20 patients that had finished six cycles of CHOP therapy. Percentages of PD-L1+CD20+ and PD-1+CD20+ cells were highly significantly increased in pre-therapy patients in comparison to healthy volunteers (p < 0.001). Meanwhile, a significant decrease in percentages of PD-L1+CD20+ and PD-1+CD20+ was observed in post-CHOP therapy patients in comparison to pre-therapy patients (p < 0.001). PD-L1+CD20+ cells were significantly decreased in post-therapy patients when compared to normal controls (p < 0.001), while not for PD-1+CD20+ cells. A strong significant positive correlation between percentages of PD-L1+CD20+ and PD-1+CD20+ was detected in DLBCL patients (p < 0.001). In the pre-therapy group, high PD-L1+CD20+ and PD-1+CD20+ percentages were correlated with serum LDH levels (p = 0.021, p < 0.001). High percentages of PD-1+CD20+ were found in DLBCL patients with splenomegaly (p = 0.027). The results revealed that patients with advanced tumor stages, poor ECOG performance, and non-GCB DLBCL type had increased percentages of PD-L1+CD20+ and PD-1+CD20+ cells. Moreover, PD-L1+CD20+ % and PD-1+CD20+ % were significantly increased in DLBCL patients with bone marrow involvement or B symptoms. The superiority of PD-L1+CD20+ over PD-1+CD20+ was more profound in DLBCL prediction [AUC: 1.0] and in discriminating newly diagnosed patients [AUC: 1.0]. The findings suggest that increased PD-L1/PD-1 expression in peripheral CD20 cells may serve as a companion diagnostic marker for DLBCL. Moreover, percentages of PD-L1+CD20+ cells have better diagnostic performance with higher sensitivity and specificity than PD-1+CD20+ %.
{"title":"Diagnostic Performance of PD-L1 versus PD-1 Expression in Circulating CD20 Cells in Diffuse Large B-Cell Lymphoma","authors":"M. Saber","doi":"10.3390/antib11010015","DOIUrl":"https://doi.org/10.3390/antib11010015","url":null,"abstract":"This study aimed to investigate PD-L1 and PD-1 expression in circulating CD20+ cells in diffuse larger B-cell lymphoma (DLBCL) and to evaluate the predictive and diagnostic performance of PD-L1 versus PD-1 expression in circulating CD20+ cells in DLBCL. Percentages of CD20+, PD-L1+CD20+, and PD-1+CD20+ cells were measured by flow cytometry in 40 DLBCL blood samples and 19 healthy controls. The DLBCL patient group was subdivided into 20 newly diagnosed patients with no treatment yet and 20 patients that had finished six cycles of CHOP therapy. Percentages of PD-L1+CD20+ and PD-1+CD20+ cells were highly significantly increased in pre-therapy patients in comparison to healthy volunteers (p < 0.001). Meanwhile, a significant decrease in percentages of PD-L1+CD20+ and PD-1+CD20+ was observed in post-CHOP therapy patients in comparison to pre-therapy patients (p < 0.001). PD-L1+CD20+ cells were significantly decreased in post-therapy patients when compared to normal controls (p < 0.001), while not for PD-1+CD20+ cells. A strong significant positive correlation between percentages of PD-L1+CD20+ and PD-1+CD20+ was detected in DLBCL patients (p < 0.001). In the pre-therapy group, high PD-L1+CD20+ and PD-1+CD20+ percentages were correlated with serum LDH levels (p = 0.021, p < 0.001). High percentages of PD-1+CD20+ were found in DLBCL patients with splenomegaly (p = 0.027). The results revealed that patients with advanced tumor stages, poor ECOG performance, and non-GCB DLBCL type had increased percentages of PD-L1+CD20+ and PD-1+CD20+ cells. Moreover, PD-L1+CD20+ % and PD-1+CD20+ % were significantly increased in DLBCL patients with bone marrow involvement or B symptoms. The superiority of PD-L1+CD20+ over PD-1+CD20+ was more profound in DLBCL prediction [AUC: 1.0] and in discriminating newly diagnosed patients [AUC: 1.0]. The findings suggest that increased PD-L1/PD-1 expression in peripheral CD20 cells may serve as a companion diagnostic marker for DLBCL. Moreover, percentages of PD-L1+CD20+ cells have better diagnostic performance with higher sensitivity and specificity than PD-1+CD20+ %.","PeriodicalId":8188,"journal":{"name":"Antibodies","volume":" ","pages":""},"PeriodicalIF":4.7,"publicationDate":"2022-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42927950","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}
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