Objective To screen the anti-CD22-specific nanobodies to provide a basis for immunotherapy agents. Methods The naive phage nanobody library was constructed and its diversity was analyzed. Three rounds of biotinylated streptavidin liquid phase screening were performed by using biotinylated CD22 antigen as the target, and the sequence of nanobodies against CD22 were identified by ELISA and gene sequencing. Results The capacity of the constructed naive phage nanobody library was 3.89×10⁹ CFU/mL, and the insertion of effective fragments was higher than 85%. Based on this library, seven anti-human CD22 nanobodies were screened, and the amino acid sequence comparison results showed that the overall similarity was 70.34%, and all of them were hydrophilic proteins. The results of protein-protein complex docking prediction showed that the mimetic proteins of the five nanobody sequences could be paired and linked to CD22, and the main forces were hydrophobic interaction and hydrogen bonding. Conclusion This study provided a basis for the study of chimeric antigen receptor T cells targeting CD22, successfully constructed the natural phage nanobody library and obtaining five anti-CD22-specific nanobodies.
Camelus/immunology* ; Single-Domain Antibodies/chemistry* ; Peptide Library ; Humans ; Animals ; Sialic Acid Binding Ig-like Lectin 2/genetics* ; Amino Acid Sequence ; Molecular Docking Simulation

This study was aimed to investigate clinical and prognostic significances of 4 target antigens (CD19, CD20, CD22 and CD33) for antibody-based immunotherapy and to evaluate the applications of these antibody-based target therapy to adult acute lymphoblastic leukemia (ALL). The immunophenotype of 220 adult patients with ALL were analyzed by four-color flow Cytometry, and cytogenetic and molecular parameters were detected by conventional cytogenetics, fluorescence in situ hybridization, real-time quantitative PCR, nested PCR and DNA sequencing. The results showed that CD19 positive (CD19(+)) cases were more in female (46.4% vs. 23.4%, P = 0.006), elderly patients aged > 60 years (14.4% vs. 2.1%, P = 0.022), CD33(+) co-expression cases (47.8% vs. 12.0%, P = 0.001) and genetic high-risk group (55.8% vs. 20.8%, P = 0.002) compared with CD19 negative (CD19(-)) cases; CD20(+) cases had lower co-expression of CD13 than CD20(-) cases (31.6% vs.67.1%, P = 0.000) and no significant prognostic indications for CD20(+) was observed; CD22(+) cases had higher relapse rate at 12-month than CD22(-) cases (93.9% vs.57.1%, P = 0.041) in B-ALL patients; CD33(+) cases had higher incidence of Ph(+) than CD33(-) cases (43.5% vs.19.4%, P = 0.007) and significantly correlated with Ph(+) (r = 0.261, P = 0.006). It is concluded that elucidation of the characteristics of the target antigens (CD19, CD20, CD22, CD33) used for antibody-based immunotherapy will help hematologists making the correct decision whether and when to use these antibody-based target therapies.
Adolescent ; Adult ; Aged ; Antigens, CD19 ; immunology ; Antigens, CD20 ; immunology ; Child ; Female ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; Sialic Acid Binding Ig-like Lectin 2 ; immunology ; Sialic Acid Binding Ig-like Lectin 3 ; immunology ; Young Adult

The study was to investigate the biological characteristics of hyperleucocyte acute leukemia (HAL) and its clinical significance. Immunophenotyping was performed in 48 HAL patients and 73 NHAL patients by three-color flow cytometry analysis using CD45/SSC gating, meanwhile the cytogenetic analysis was performed in 74 patients. The results showed that as compared with NHAL group, HAL group had lower proportion of eryth-lineage in bone marrow (P < 0.05); in AML, the CD14 expression of HAL group was apparently higher than that of NHAL group (P < 0.05); in ALL, HAL group had higher expression of CD8 and lower expression of CD22, cCD79a compared with NHAL group (P < 0.05); the two groups had no significant difference in expression of special lineage antigens and overlapping lineage antigens (P > 0.05). The CR rate of HAL group was lower than that of NHAL group. It is concluded that bone marrow inhibition of HAL group is more severe than that of NHAL group. In AML, monocytic leukemia is easier to become into HAL than other leukemias. In ALL, T-lineage antigens of HAL group are more easily expressed than those of NHAL group; the leukemia cells of HAL group are naiver than those of NHAL group, meanwhile the prognosis of HAL is poor.
Adolescent ; Adult ; Aged ; CD79 Antigens ; biosynthesis ; genetics ; CD8 Antigens ; biosynthesis ; genetics ; Child ; Child, Preschool ; Female ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; drug therapy ; mortality ; pathology ; Leukocyte Count ; Lipopolysaccharide Receptors ; biosynthesis ; genetics ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; mortality ; pathology ; Prognosis ; Sialic Acid Binding Ig-like Lectin 2 ; biosynthesis ; genetics

OBJECTIVETo report abnormal expression of cCD79a/cCD22 in four cases of acute myeloid leukemia (AML) with t (8;21).

METHODSThe characteristics of morphology, immunophenotype, chromosome karyotype (MIC) and clinical manifestations of 4 AML patients with t (8;21) expressing cCD79a/cCD22 were analyzed.

RESULTSThe features of the 4 patients were: (1) no difference in gender; (2) young age; (3) exmedullary infiltration may be present; (4) normal number of white blood cells in peripheral blood; (5) morphology showed acute myeloid leukemia with high percentage of blast cells; (6) B-lymphoid and myeloid immunophenotype, and high expression of CD34; (7) frequent depletion of Y chromosome and complex changes of chromosomes; (8) positive for AML1/ETO fusion gene; (9) response well to chemotherapy regimen which simultaneously treated myeloid and lymphocytic leukemia.

CONCLUSIONAbnormal expression of cCD79a/cCD22 in AML with t (8;21) (q22;q22) suggested that this kind of leukemia might be related with abnormal expression gene of B cell.

Adolescent ; Adult ; CD79 Antigens ; metabolism ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Female ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; genetics ; Male ; Middle Aged ; Sialic Acid Binding Ig-like Lectin 2 ; metabolism ; Translocation, Genetic ; Young Adult

To evaluate the expression of cytoplasmic CD79a (CyCD79a) and other commonly used B-lymphoid immunomarkers including cytoplasmic CD22 (CyCD22), CD19, CD20 and CD10 in various acute leukemia cells and to define the most sensitive and specific markers in the diagnosis of precursor B-cell acute lymphoblastic leukemia (pB-ALL), the immunophenotypic data from 221 de novo adult and pediatric acute leukemia patients as studied using multi-parameter flow cytometry in addition to routine morphologic and enzyme cytochemical assay, were retrospectively analyzed. Cytogenetic and/or molecular biological data in all 45 cases of acute promyelocytic leukemia (APL) and 13 cases of acute leukemia suspected as AML with the fusion genes such as AML1/ETO and CBFbeta/MYH11 were investigated. The results showed that CyCD79a and CyCD22 were the most sensitive and specific markers respectively for pB-ALL. Expression of CyCD79a was seen in 100% of 58 cases of pB-ALL. At the same time, none (0%) of all 147 cases of acute myeloid leukemia (AML) and 15 cases of precursor T-cell acute leukemia (pT-ALL) was positive for CyCD22. The conclusion is made that united detection of CyCD79a and CyCD22 is the optimal immune combination for the diagnosis pB-ALL and the distinguishing pB-ALL with AML and pT-ALL.
Acute Disease ; B-Lymphocytes ; immunology ; Biomarkers, Tumor ; immunology ; CD79 Antigens ; immunology ; Cytoplasm ; immunology ; Flow Cytometry ; Humans ; Immunophenotyping ; Karyotyping ; Leukemia, Myeloid ; genetics ; immunology ; pathology ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; metabolism ; pathology ; Sialic Acid Binding Ig-like Lectin 2 ; immunology