Production of recombinant humanized monoclonal anti-human neutrophil antigen (HNA) antibodies with potential applicability as standard antibodies.

BACKGROUND: Antibodies against human neutrophil antigen (HNA) are involved in the pathogenesis of neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. The present methods for anti-HNA antibody identification strongly depend on the presence of standard antisera with known allo/isospecificities. Here, we aimed to produce recombinant humanized antibodies to HNA from available mouse monoclonal antibodies (MoAbs). STUDY DESIGN AND METHODS: RNAs were extracted from available hybridoma cells producing mouse anti-HNA antibodies recognizing HNA-1a (TAG-1), -1b (TAG-2), -2 (TAG-4), and FcγRIIIb, and the cDNA was synthesized. Recombinant fragments consisting of the variable regions of the H and L chains of the mouse MoAb ligated to the constant region of human IgG were incorporated into an expression vector and transfected into CHO cells. Antibody specificity of the selected humanized monoclonal antibodies was confirmed, and tested by the participants of the ISBT Granulocyte Immunobiology Working Party (GIWP) workshop 2020. RESULTS: GIFT results confirmed the specific reactivity of TAGH-1 to -4, except for a cross-reactivity of TAGH-2 with HNA-1a/a neutrophils, only in flow-cytometry. MAIGA results showed clear specificity of all humanized antibodies, but the selection of the appropriate capture monoclonal antibody was essential for the test. The results of the ISBT GIWP showed high concordance among the labs. CONCLUSIONS: These are the first humanized monoclonal antibodies to HNA-1 and HNA-2 antigens produced and they will be important standard reagents for laboratories testing for neutrophil antibodies. We plan to have these humanized MoAbs available through WHO.

Clinical significance of human neutrophil antigen-1 antibodies in children with neutropenia.

Primary autoimmune neutropenia in young children is characterized by chronic neutropenia and positivity for antibodies against human neutrophil antigens (HNAs). This study analyzed the clinical characteristics of 402 children with neutropenia to identify differences between those with and without HNA-1 antibodies (HNA1abs). HNAabs in sera were detected by granulocyte immunofluorescence testing using flow cytometry. Relative fluorescence intensity (RFI) values were used to divide patients into positive (PG, n = 302), borderline (BG, n = 34), and negative (NG, n = 66) groups. The antibodies reacted to HNA-1a alone (59%), HNA-1b alone (1%), and HNA-1a/1b (40%). The PG had a significantly lower absolute neutrophil count before definitive diagnosis and a 1.6- to 2-times greater risk of hospitalization during neutropenia than the other groups. The median duration of neutropenia was longest in the PG at 25 months, followed by 20 months in the BG and 14 months in the NG. This large-scale cohort characterizes clinically distinct groups using the RFI value for HNA1abs in young children with neutropenia. Detection of HNA1abs may aid in understanding the clinical characteristics of children with neutropenia.

Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

BACKGROUND: Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. STUDY DESIGN AND METHODS: We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. RESULTS: The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. CONCLUSION: EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN.

[Sustained complete remission for one year following low-dose rituximab therapy for chronic autoimmune neutropenia].

A 78-year-old male with a 5-year history of neutropenia was admitted to our hospital with high fever. Peripheral blood and bone marrow findings showed severe granulocytic hypoplasia. Granulocyte-colony stimulating factor and steroid pulse therapy did not improve the clinical conditions at all, and severe sepsis persisted. After he was diagnosed with autoimmune neutropenia by detection of anti-neutrophil antibody in sera, low-dose infusion of rituximab was performed. Neutrophil count promptly increased and normalized one month after the start of treatment. He has remained in remission for 1 year. Compared with the standard dose, low-dose rituximab therapy is promising for the treatment of chronic autoimmune neutropenia because of the lower cost as well as the lower incidence of adverse reactions.

[Influence of monoclonal antibodies to human neutrophil antigens, HNA-1a/b and HNA-2a on phagocytosis].

Neutrophil antibodies frequently cause severe conditions such as transfusion-related acute lung injury, alloimmune/autoimmune neutropenia. As it was thought that surviving neutrophils would also be damaged from antibodies binding with the neutrophil membrane, we studied the functional influence of neutrophil antibodies on natural phagocytosis and immune phagocytosis by using neutrophil-specific monoclonal antibodies (MoAbs), TAG1: HNA-la on FgammaRIIIb, TAG2: HNA-1b on FgammaRIIIa/b, TAG3: FgammaRIIIa/b and TAG4: HNA-2a. In an inhibition assay of carbon particle phagocytosis as a representation of natural phagocytosis, neutrophils binding with TAG3 or TAG4 were inhibited from carbon particle-phagocytosis by 42.5% and 53.2% (% inhibition), respectively, but in an inhibition assay using TAG3 MoAb, HNA-2a strongly positive neutrophils were more weakly inhibited than HNA-2a weak-positive neutrophils, 39.2% and 54.0% (% inhibition), respectively. These results suggested that HNA-2a salvaged the inhibition process of natural phagocytosis by anti-FcgammaRIII antibodies. These results also suggested that natural phagocytosis would be inhibited when antibodies combined with every antigen on the cell membrane. In an inhibition assay of EA-rosette formation as a representation of immune phagocytosis, FcgammaRIII-specific MoAbs, TAG1, TAG2 and TAG3 inhibited EA-rosette formation, but HNA-2a-specific MoAb, TAG4, did not markedly inhibit EA-rosette formation. It was thought that neutrophil immune-phagocytosis would be inhibited by antibodies binding with FcgammaRIII, and that HNA-2a was not related to immune phagocytosis. Further investigation of the relationship between clinical symptoms and antibody specificity or antibody quantity is needed.

Monitoring neutrophil engraftment in allogeneic stem cell transplantation by flow cytometric analysis of neutrophil-specific antigens NA1 and NA2.

Neutrophil-specific antigen (NA) expression on neutrophils was analysed in 18 Japanese children before and after allogeneic stem cell transplantation (allo-SCT) with myeloablative regimen. Donor-recipient NA-incompatibility was present in one of eight NA1/NA2 heterozygous patients and eight of 10 NA1/NA1 or NA2/NA2 homozygous patients. After allo-SCTs from NA-incompatible donors, a neutrophil recipient-to-donor conversion was confirmed in all cases. Conversion to donor NA type was complete before the absolute neutrophil count reached 0.1 x 10(9)/l. These observations indicate that flow cytometric analysis of NA antigens is a simple and useful method for monitoring neutrophil engraftment in NA-incompatible allo-SCT.

Albumin attenuates neutrophil activation induced by stimulators including antibodies against neutrophil-specific antigens.

As neutrophil activation is related to several adverse transfusion reactions, we studied about the activation induced by anti-neutrophil antibodies and the stabilizing effects of albumin pretreatment by means of flow cytometry. Anti-neutrophil monoclonal antibody (anti-HNA-1a, 1b, 2a) alone induced CD11b expression and shedding of L-selectin, and anti-HNA-1a reinforced reactive oxygen species (ROS) production. Albumin pretreatment significantly reduced CD11b expression and L-selectin shedding induced by fMLP and ROS production induced by PMA, G-CSF combined with PMA or LPS-fMLP, or anti-HNA-1a combined with PMA. These findings suggest that anti-HNA-1a is related to adverse reactions and albumin has a regulating effect on neutrophil activation.

Significance of human neutrophil antigen-2a (NB1) expression and neutrophil number in pregnancy.

BACKGROUND: Expression of human neutrophil antigen-2a (HNA-2a) is greater in women than in men. The size of the HNA-2a-positive neutrophil population increases with pregnancy. STUDY DESIGN AND METHODS: T he relationship between HNA-2a expression on neutrophils and monocytes, and their relative numbers, was investigated. HNA-2a expression shown as the size of the HNA-2a-positive cell population and the fluorescence intensity on the cells was analyzed using flow cytometry. This investigation was done among 165 pregnant women during pregnancy and postpartum. RESULTS: In normal pregnancy, numbers of neutrophils and monocytes changed in relation to HNA-2a expression. HNA-2a was also expressed intensively on monocytes from some pregnant women in the first and second trimesters. HNA-2a expression and the number of cells markedly decreased postpartum. In threatened premature labor, the number of neutrophils decreased earlier than in normal pregnancy. CONCLUSION: HNA-2a expression may increase soon after fertilization. In this study, the results indicate that the change in the number of neutrophils and monocytes is related to HNA-2a expression.

Efficacy of prophylactic use of trimethoprim-sulfamethoxazole in autoimmune neutropenia in infancy.

PURPOSE: Most children with autoimmune neutropenia (AIN) have a benign clinical course because of the spontaneous resolution of neutropenia. The authors observed the clinical course of AIN in infancy accompanied by the prophylactic use of trimethoprim-sulfamethoxazole (TMP-SMX) during neutropenia. PATIENTS AND METHODS: Eight infants with AIN were followed by serial tests for antineutrophil antibodies and management of infectious complications. RESULTS: The spontaneous disappearance of antineutrophil antibodies that preceded the normalization of the neutrophil count was found in all patients. Until the resolution of neutropenia, TMP-SMX was administered in five patients, resulting in a reduction in the incidence of infection with no adverse effects. CONCLUSIONS: These observations demonstrate the possibility of the safety and usefulness of TMP-SMX treatment in patients with AIN.

Human neutrophil antigen-2a expression on neutrophils from healthy adults in western Japan.

BACKGROUND: Human neutrophil antigen-2a (HNA-2a)-specific MoAb, which is necessary to prepare panel neutrophils and to determine the phenotype of patients' neutrophils, has been produced in many laboratories in Japan. The frequency of the HNA-2a-positive population in western Japan is unknown. STUDY DESIGN AND METHODS: A cell line secreting an HNA-2a-specific MoAb, TAG4, was established. TAG4 was characterized by flow cytometry, and the reactivities of TAG4 and another HNA-2a-specific MoAb, 7D8, were compared in an inhibition assay and two-color flow cytometry. TAG4 was tested against granulocytes from 408 healthy adult volunteers in western Japan. RESULTS: TAG4 reacted with neutrophils, but not with the other blood cells. The reactivity of TAG4 with granulocytes treated with glycosyl-phosphatidylinositol-specific phospholipase C was markedly reduced. The reactivity of TAG4 and 7D8 was similar, but 7D8 did not prevent TAG4 from binding. A total of 406 of 408 donors were TAG4 positive. The mean percentage of HNA-2a expression of females was 70.2 percent, and that of males was 66.8 percent. CONCLUSION: TAG4 and 7D8 seem to bind to different epitopes of HNA-2a. The frequency of the HNA-2a-positive population is 99.5 percent of healthy adults in western Japan. The mean percentage of HNA-2a expression of females was greater than in males.

Significance of the detection of antineutrophil antibodies in children with chronic neutropenia.

We evaluated the clinical characteristics of 41 children with chronic neutropenia based on the quantitative analysis of antineutrophil antibodies in serum by flow cytometry. According to the strength of antineutrophil antibodies, the patients were divided into 3 groups: 12 patients presented negative antibodies, 13 patients showed weak positive antibodies, and 16 patients showed strong positive antibodies. No significant differences were seen in age of diagnosis, severity of neutropenia, and infectious complications associated with neutropenia among the 3 groups. The spontaneous resolution of neutropenia was observed in all patients with negative antibodies and in 22 of 29 patients with positive antibodies. The age of the recovery of neutropenia and the duration until spontaneous resolution of neutropenia were significantly dependent on the antibody strength at the time of diagnosis. These results demonstrate that the quantification of antineutrophil antibodies at the time of diagnosis may be useful in considering the clinical course of chronic neutropenia in childhood.