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Oosterhoff JJ, Linty F, Visser R, de Vos T, Hofstede-van Egmond S, van de Weerd M, Porcelijn L, de Haas M, van der Schoot E, Vidarsson G. Generation of human antibodies targeting human platelet antigen (HPA)-1a. Transfusion 2024; 64:893-905. [PMID: 38400657 DOI: 10.1111/trf.17758] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Revised: 01/26/2024] [Accepted: 01/27/2024] [Indexed: 02/25/2024]
Abstract
BACKGROUND Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. STUDY DESIGN AND METHODS This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. RESULTS Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. DISCUSSION This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.
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Affiliation(s)
- Janita J Oosterhoff
- Department of Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands
- Department of Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands
| | - Federica Linty
- Department of Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands
- Department of Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands
| | - Remco Visser
- Department of Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands
- Department of Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands
| | - Thijs de Vos
- Department of Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands
- Department of Pediatrics, Division of Neonatology, Leiden University Medical Centre, Leiden, The Netherlands
| | | | - Miranda van de Weerd
- Department of Immunogenetics, Sanquin Diagnostic Services, Amsterdam, The Netherlands
| | - Leendert Porcelijn
- Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, The Netherlands
| | - Masja de Haas
- Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, The Netherlands
- Department of Haematology, Leiden University Medical Centre, Leiden, The Netherlands
| | - Ellen van der Schoot
- Department of Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands
- Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
| | - Gestur Vidarsson
- Department of Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands
- Department of Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands
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Eksteen M, Tiller H, Averina M, Heide G, Kjaer M, Ghevaert C, Michaelsen TE, Ihle Ø, Husebekk A, Skogen B, Stuge TB. Characterization of a human platelet antigen-1a-specific monoclonal antibody derived from a B cell from a woman alloimmunized in pregnancy. THE JOURNAL OF IMMUNOLOGY 2015; 194:5751-60. [PMID: 25972474 DOI: 10.4049/jimmunol.1401599] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/24/2014] [Accepted: 04/09/2015] [Indexed: 11/19/2022]
Abstract
Human platelet Ag (HPA)-1a, located on integrin β3, is the main target for alloantibodies responsible for fetal and neonatal alloimmune thrombocytopenia (FNAIT) in the white population. There are ongoing efforts to develop an Ab prophylaxis and therapy to prevent or treat FNAIT. In this study, an mAb specific for HPA-1a, named 26.4, was derived from an immortalized B cell from an alloimmunized woman who had an infant affected by FNAIT. It is the only HPA-1a-specific human mAb with naturally paired H and L chains. Specific binding of mAb 26.4, both native and recombinant forms, to platelets and to purified integrins αIIbβ3 (from platelets) and αVβ3 (from trophoblasts) from HPA-1a(+) donors was demonstrated by flow cytometry and surface plasmon resonance technology, respectively. No binding to HPA-1a(-) platelets or integrins was detected. Moreover, the Ab binds with higher affinity to integrin αVβ3 compared with a second HPA-1a-specific human mAb, B2G1. Further in vitro experimentation demonstrated that mAb 26.4 can opsonize HPA-1a(+) platelets for enhanced phagocytosis by monocytes, inhibit binding of maternal polyclonal anti-HPA-1a Abs, and weakly inhibit aggregation of HPA-1a-heterozygous platelets, the latter with no predicted clinical relevance. Thus, mAb 26.4 is highly specific for HPA-1a and could potentially be explored for use as a prophylactic or therapeutic reagent for FNAIT intervention and as a phenotyping reagent to identify women at risk for immunization.
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Affiliation(s)
- Mariana Eksteen
- Immunology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø - The Arctic University of Norway, N-9037 Tromsø, Norway
| | - Heidi Tiller
- Immunology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø - The Arctic University of Norway, N-9037 Tromsø, Norway; Department of Obstetrics and Gynecology, University Hospital of North Norway, N-9038 Tromsø, Norway
| | - Maria Averina
- Department of Laboratory Medicine, Diagnostic Clinic, University Hospital of North Norway, N-9038 Tromsø, Norway
| | - Gøril Heide
- Immunology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø - The Arctic University of Norway, N-9037 Tromsø, Norway
| | - Mette Kjaer
- Immunology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø - The Arctic University of Norway, N-9037 Tromsø, Norway; Department of Laboratory Medicine, Diagnostic Clinic, University Hospital of North Norway, N-9038 Tromsø, Norway; Prophylix Pharma AS, Forskningsparken, N-9294 Tromsø, Norway
| | - Cedric Ghevaert
- Department of Haematology, University of Cambridge, Cambridge CB2 0XY, United Kingdom; National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, United Kingdom
| | - Terje E Michaelsen
- The Norwegian Institute of Public Health, N-0403 Oslo, Norway; and School of Pharmacy, University of Oslo, N-0316 Oslo, Norway
| | - Øistein Ihle
- The Norwegian Institute of Public Health, N-0403 Oslo, Norway; and
| | - Anne Husebekk
- Immunology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø - The Arctic University of Norway, N-9037 Tromsø, Norway; Department of Laboratory Medicine, Diagnostic Clinic, University Hospital of North Norway, N-9038 Tromsø, Norway
| | - Bjørn Skogen
- Immunology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø - The Arctic University of Norway, N-9037 Tromsø, Norway; Department of Laboratory Medicine, Diagnostic Clinic, University Hospital of North Norway, N-9038 Tromsø, Norway; Prophylix Pharma AS, Forskningsparken, N-9294 Tromsø, Norway
| | - Tor B Stuge
- Immunology Research Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø - The Arctic University of Norway, N-9037 Tromsø, Norway;
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Kickler T. From an early National Blood Foundation award to our ongoing studies in fetal and neonatal alloimmune thrombocytopenia. Transfusion 2005; 45:64S-66S. [PMID: 16086790 DOI: 10.1111/j.1537-2995.2005.00533.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Affiliation(s)
- Thomas Kickler
- Hematology Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
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Abstract
Phage display, which exploits fundamental tools and principles of immune repertoire diversity, antigen-antibody interactions, and clonal and immunologic selection, is used increasingly to advance experimental and clinical hematology. Phage display is based on the ability of bacteriophage to present engineered proteins on their surface coat. Diverse libraries of proteins such as peptides, antibody fragments, and protein domains corresponding to gene fragments or cDNAs may be displayed. Interactions between phage-displayed proteins and target antigens can be identified rapidly and characterized using high throughput methodologies. Peptide and gene fragment libraries are particularly useful to characterize binding interactions between proteins, such as ligand-receptor interactions. This approach allows rapid generation of human antibodies, often against nonimmunogenic, conserved proteins. Phage antibodies against surface and intracellular antigens are used as reagents for flow cytometry, in vivo imaging, and therapeutic targeting. Phage-derived antibodies also facilitate analyses of the humoral antibody response. Finally, cellular delivery of phage-displayed peptides and gene fragments can be used to modulate functional pathways and molecules in vitro and in vivo. The combinatorial power of phage display enables identification of candidate epitopes without knowledge of the protein interaction, a priori. Overall, these capabilities provide a versatile, high-throughput approach to develop tools and reagents useful for a plethora of experimental hematology applications. This paper focuses on current and future applications of antibody and epitope phage display technology in hematology.
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Affiliation(s)
- B P Mullaney
- Department of Laboratory Medicine, University of California at San Francisco, San Francisco, Calif., USA.
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Yu ZC, Ding J, Nie YZ, Fan DM, Zhang XY. Preparation of single chain variable fragment of MG 7 mAb by phage display technology. World J Gastroenterol 2001; 7:510-4. [PMID: 11819819 PMCID: PMC4688663 DOI: 10.3748/wjg.v7.i4.510] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To develop the single chain variable fragment of MG7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator.
METHODS: mRNA was isolated from MG7 producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOIII of highly expressing MG7-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG7 ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG7 ScFv. Finally, the relative molecular mass of soluble MG7 ScFv was measured by SDS-PAGE.
RESULTS: The V-H, V-L and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 × 106 and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG7 antibody for binding to the antigen expressed on KATOIII cells. Within 2 strong positive phage clones, the soluble MG7 ScFv from one clone was found to have the binding activity with KATOIII cells. SDS-PAGE showed that the relative molecular weight of soluble MG7 ScFv was 32.
CONCLUSION: The MG7 ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.
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Affiliation(s)
- Z C Yu
- Department of Gastroenterology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
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Lubenow N, Eichler P, Albrecht D, Carlsson LE, Kothmann J, Rossocha WR, Hahn M, Quitmann H, Greinacher A. Very low platelet counts in post-transfusion purpura falsely diagnosed as heparin-induced thrombocytopenia. Report of four cases and review of literature. Thromb Res 2000; 100:115-25. [PMID: 11108897 DOI: 10.1016/s0049-3848(00)00311-x] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Differential diagnosis between post-transfusion purpura (PTP) and heparin-induced thrombocytopenia (HIT) can be difficult in the initial stages of thrombocytopenia, as the early clinical presentations are often similar. Four patients are described who were suspected clinically of suffering from HIT. All four patients had recent blood transfusions and platelet alloantibodies, thus the diagnosis of PTP was made. One lethal gastrointestinal and one retroperitoneal hemorrhage developed in two of the four patients. Unusually, one patient was male and two different platelet alloantibodies were present in his serum; in another patient platelet alloantibodies and HIT-antibodies were detectable. To arrive at the right diagnosis as quickly as possible is vitally important since treatment, which has to be initiated promptly, is very different for the two syndromes. Thus, we suggest that in patients where HIT is suspected, additional information should be sought. If features consistent with PTP (such as a recent blood transfusion or a marked drop in platelet count to below 15 Gpt/L) are present, we recommend parallel testing for platelet alloantibodies to rule out PTP.
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Affiliation(s)
- N Lubenow
- Institute for Immunology and Transfusion Medicine, Ernst-Moritz-Arndt University, Greifswald, Germany.
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Abstract
The development of phage display technology has facilitated the development of many new and sometimes novel antibody based reagents for scientific research. However, present methods for selection from phage-sFv display libraries are limited to selection against purified antigens or ex vivo cells of known origin and phenotype. Existing methods therefore preclude the isolation of sFv against unknown molecules in their natural environment, where expression is complex and subject to diverse control mechanisms. Since such a complex environment is difficult to mimic in vitro, the development of an in vivo selection procedure would greatly enhance the selection from phage display antibody libraries and lead to the development of reagents against cell surface molecules in their natural environment. This would be particularly advantageous for isolation of sFv against vascular endothelium which can readily change phenotype when cultured and is believed to express molecules in a tissue specific manner and in response to different stimuli. We describe here the development of an in vivo selection procedure in the mouse and demonstrate its potential for the selection of sFv from a phage-sFv library. The target antigen for one sFv is expressed solely on the thymic endothelium, while the second, a 165-170 kDa molecule in present on both thymic endothelium and the perivascular epithelium.
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Affiliation(s)
- M Johns
- Department of Immunology, Division of Medicine, Commonwealth Building, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, W12 0NN, London, UK
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Watkins NA, Armour KL, Smethurst PA, Metcalfe P, Scott ML, Hughes DL, Smith GA, Williamson LM, Clark MR, Ouwehand WH. Rapid phenotyping of HPA-1a using either diabody-based hemagglutination or recombinant IgG1-based assays. Transfusion 1999; 39:781-9. [PMID: 10413288 DOI: 10.1046/j.1537-2995.1999.39070781.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
BACKGROUND The HPA-1 system is carried on the beta3 integrin. HPA-1a (Zw(a), Pl(A1)) is immunogenic in an HPA-1b homozygote (HPA-1b1b). In pregnancy, 1 of 365 women forms anti-HPA-1a, which causes severe thrombocytopenia in 1 in 1100 neonates. Identification of women at risk of forming anti-HPA-1a and the screening of donors to obtain HPA-1a-negative platelets for therapy need reliable, low-cost, automated assays. STUDY DESIGN AND METHODS A diabody with dual specificity for HPA-1a x D and an IgG1 anti-HPA-1a have been constructed by the use of the genes encoding the first anti-HPA-1a fragment. With these reagents, two complementary HPA-1a phenotyping assays have been developed. RESULTS This diabody was used in a simple hemagglutination technique to perform HPA-1a phenotyping on soluble glycoprotein IIb/IIIa from EDTA plasma samples. Over 1000 unselected donors have been correctly HPA-1a-phenotyped by use of the diabody. The human recombinant IgG1 anti-HPA-1a was produced in a rat myeloma cell line and was fluorescein labeled for use in a whole-blood flow cytometric HPA-1a phenotyping assay. This IgG1 anti-HPA-1a shows a clear differential between HPA-1a-positive and HPA-1a-negative platelets at nM antibody concentrations. CONCLUSIONS The two recombinant reagents described are highly suitable for screening and confirmatory HPA-1a phenotyping. They permit rapid determination of the HPA-1a phenotype and are amenable to automation.
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Affiliation(s)
- N A Watkins
- Department of Haematology, University of Cambridge, UK
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