Reactivity of a Novel Antibody ZCH-2B8a on Normal and Malignant Hematopoietic Cells.
Authors: Tang Yongmin , Guo Li , Zhang Haizhong , Zhang Yi , Shen Hongqiang , Qian Baiqin , Ning Botao , Luo Cunfang 
Published: October 2019

Zhejiang Children’s Hospital (ZCH)-2B8a antibody was recently generated in our laboratory using leukemia cell line KG1a as immunogen. This antibody was submitted to the 8th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the results showed that the antibody recognized an unknown molecule on the surface of some blood cells. In this study, the reactivity of the antibody on normal and malignant hematopoietic cells was investigated and its diagnostic and therapeutic significances were explored. Materials and Methods 3 specimens each of normal peripheral blood, bone marrow/G-CSF mobilized peripheral blood CD34+ stem/progenitor cells (PBSCs), 13 hematopoietic cell lines and the cells from 100 leukemia patients (male 58: female 42, 33 cases of children and 67 adults, aged 11 month ~ 77 yrs) were enrolled into this study. A multi-parameter flow cytometry with CD45 gating strategy was used to analyze the cells. Results 2B8a weakly reacted with a few B lymphocytes (34.3±15.91%), monocytes(44.4±37.77%), granulocytes (26.4±22.40%), but not reacted with other blood cells such as T cells (0.6±0.37%), NK cells (2.6±1.56%), DC cells (8.4±8.18%), erythrocytes (0.4±0.21%) and platelets (0.3±0.28%). It was reactive with 38.0±9.09% of bone marrow CD34+ cells with 21.0±22.00% of CD38+ and 25.3±22.6% of CD38- subsets (P = 0.5). In G-CSF mobilized PBSCs, 2B8a recognized 11.7±18.73% of CD34+ PBSCs with 9.7±15.20% of CD38+ and 33.3±57.74% of CD38- subsets. The positive rates of 2B8a on a panel of hematopoietic cell lines were shown in Table 1. The fresh leukemia cell analysis showed that the antibody primarily reacted with lymphoid leukemias, of which acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL) accounted for 29%(9/31) and 40%(2/5), respectively. It did not react with most cases of myeloid leukemias, with only 5%(3/57) of acute myeloid leukemia(AML) and 0% (0/6) of chronic myelogeneous leukemia (CML) cases, in which significant difference between myeloid and lymphoid leukemias (χ2= 10.29, P < 0.01) was found; In ALL, 2B8a only reacted with B lineage (32%, 9/28), while not with T and NK lineage leukemias. In B lineage leukemia, early Pre-B ALL was the most reactive type of disease, positive in 60% of cases, significantly higher than cALL cases (5/21, 24%, P = 0.0173) with declining expression pattern with cell maturation. There was no reactivity in mature B-ALL but it was strongly reactive with lymphoma cells suggesting the possibility for this antibody as a potential targeting therapy for patients with B lineage lymphoma. Conclusions 2B8a may be used in distinguishing lymphoid from myeloid leukemia. It might hold a potential application for targeting therapy for patients with leukemia and malignant lymphoma. (This work was supported by the grant (No. 398427) from Zhejiang Provincial Scientific Fundation).

Table 1. Reactivity of 2B8a on hematopoietic cell lines Cell lines Specificity Positivity ±% CEM T cell leukemia 5.0±0.67 JM T cell leukemia 5.1±0.84 Molt-3 T cell leukemia 8.3±3.59 Molt-4 T cell leukemia 4.3±3.29 Nalm-6 B cell leukemia 94.9±0.78 Raji B cell lymphoma 98.8±0.22 SMS-SB B cell lymphoma 95.6±2.17 KG1a Myeloblastic leukemia 16.7±1.93 U937 Monoblastic leukemia 77.4±17.36 HL60 Promyelogenous leukemia 30.7±3.17 K562 Erythroleukemia 27.9±0.58 HEL92.1.7 Erythroleukemia 11.4±5.89 Meg01 Megakaryocytic leukemia 33.0±3.33