A novel monoclonal antibody specific for human pre-B cell leukemia/lymphoma

Nakamura N, Morimura Y, Abe M, Wakasa H. A novel monoclonal antibody specific for human pre-B cell leukemia/lymphoma. Eur J Haematol 1991: 47: 168-173.

Abstract: A novel monoclonal antibody, designated WH 14-antibody (WH14-Ab), was produced by using a non-T ALL cell line (HBL-3) as an immunogen. 35S-labelled immunoprecipitate revealed that the antigen reacting with WHlCAb was estimated to be 30 Kd. Immunoglobulin isotype of WH14-Ab was IgG1. In the normal hematopoietic tissue, WH14-Ab reacted with a small number of monocytes (< 30%) in the peripheral blood, but neither with the lymphocytes nor granulocytes. WH 14-Ab reacted with HBL-3 and REH, but not with other B-cell leukemia/lymphoma and EBV-transformed cell lines. In addition, WH 14-Ab reacted with most non-T ALL and pre-B lymphoblastic lymphoma. WH14-Ab did not react with all T-cell lymphomas. These findings indicate that the WH14-Ab may recognize the cell surface determinant shared by immature B cells, especially pre-B cells, in the B-cell lineage. WH14-Ab may be useful not only for the detection of pre-B cell leukemia/lymphomas but also for the investigation

I of maturation and differentiation of B-cell lineage.

Introduction

The understanding of human normal and leukemic hematopoiesis has been markedly advanced by the development of monoclonal antibodies reactive with hematopoietic cells during their differentiation and maturation (1-4). A great variety of antibodies re- acting with determinants on B cells have been re- ported by many investigators. However, specific monoclonal antibodies which recognize only deter- minants on immature B cells, particulary on pre-B cells, have rarely been developed (1-4). We report a novel monoclonal antibody, designated WH 14-Ab, which is mainly specific for pre-B derived leukemia/ lymphoma cells.

Material and methods Production of murine monoclonal antibody

A novel monoclonal antibody (mAb) was produced by somatic-cell hybridization according to the pre- viously reported methods ( 5 ) . In brief, BALB/C mice were immunized with neoplastic cells of HBL-3 cell line which was established in our department from non-T ALL cells (6). The immunized murine spleen cells were fused with murine myeloma cells (NS-1) as usual. Hybridomas, which reacted specifically with HBL-3 cells but neither with Raji (human Bur- kitt’s lymphoma cell line) nor HBL-1 (human B-cell

Naoya Nakamura, Yutaka Morimura, Masafumi Abe and Haruki Wakasa Department of Pathology, Fukushima Medical College, Fukushima, Japan

Key words: monoclonal antibody - pre-B cell leukemia/lymphoma - 30 Kd

Correspondence: Naoya Nakamura, M.D., Department of Pathology, Fukushima Medical College, 960-1 2, 1-Hikarigaoka, Fukushima, Japan

Accepted for publication 1 March 1991

large cell lymphoma cell line (7)), were selected. These hybridomas were cloned twice by the limiting dilution method. mAb was prepared from ascitic fluid of mice injected with hybridoma cells. Immu- noglobulin subclass was determined with methods by Ochtalony and co-workers (8).

Normal and neoplastic hematopoietic tissue

Normal mononuclear cells and leukemic cells ob- tained from peripheral blood or bone marrow were separated by density gradient sedimentation (9). Fresh materials of the lymph nodes, tonsils, spleen and thymus obtained surgically had been kept as frozen tissues.

lmrnunohistological study

All fresh tissues were fixed in PLP (periodate-lysin- paraformaldehyde) fixative for 4 h and snap-frozen at - 70 O C. 5 p frozen sections were cut and placed on albumin-coated slides before immunostaining. Immunostaining was performed by Streptavidin Biotin complex procedure (10).

lmmunofluorescence assay

Cell surface antigens were detected by indirect im- munofluorescence technique. Fluorescin-isothio- cyanate (F1TC)-conjugated F(ab’ ), goat anti-mouse

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IgG and anti-mouse IgM (Tago, Burlin, CA) were used as the secondary antibody. Fluorescence cells were examined on Axiophooa (Zeiss, Germany) or on a FACScan (Becton Dickinson, USA), and pos- itive cells were determined by epi-illumination with Axiophooa.

Determination of antigen molecular weight recognized by WH 14-Ab

THP-1 cells internally radiolabelled with 0.5 mCi 35S-methionine were resuspended in the lysis buffer (0.5% Nonidet P-40 (Sigma, USA), 10 niM Tris- HCl PH 7.6, 1 mM MgCl,, 0.1 mM phenylmethyl- sulfonyl fluoride (Sigma) and 0.02% sodium azide) at 4' C for 30 minutes and centrifuged at 1 1 000 rpm for 25 min. The supernatant was immunoprecipi- tated with protein A Sepharose CL-4B (Pharmacia, Sweden) according to the previously described tech- nique (11). The sample was electrophoresed by means of sodium dodecylsulphate polyacrylamide gel previously described by Laminlie et al. (12) and was subsequently visualized by radioautography.

Response of HBL-3 cells to WH14-Ab

The response of the HBL-3 cells to WH14-Ab was determined by mixing 100 p1 of the cell suspension (1 x 105/ml) with either 100 pl of medium alone or 100 pl of medium containing 1.3 - 13 pg of WH14- Ab. An induction of the cell proliferation by WH14- Ab was measured by incorporation of 3H-thymidine. Radioactivity was measured in a liquid scintillation counter and the proliferation was evaluated as cpm (mean standard deviation, n = 3).

Results 1) Immunological characteristics of HBL-3 cell line (6) (Table 1)

HBL-3 cell line was established from the bone mar- row of 9-year-old female with non-T ALL (L2). HBL-3 cells showed the earliest B-cell characteris- tics committed to B-cell lineage (CIg - , SIg - , TdT+ , Ia+ , B4(CD19)+, Bl(CD20)-, JS(CDlO)-, IgHR, TcRG). Karyotype of the HBL-3 cell line was 46, XX, - 3, - 9, - 9, +der(3)t(3;?), +der(9)t(9;?), + der(9)t(1;9).

2) Hybridization and screening

After somatic cell hybridization, supernatants ob- tained from 2 (WH2-Ab and WH14-Ab) among 100 different hybridomas reacted only with HBL-3 cells. Normal mononuclear cells of peripheral blood re- acted with WH2-Ab but not with WH14-Ab. There- fore WH14 hybridoma was cloned and passaged into BALB/C mice to produce malignant ascites.

Table 1. Characteristics of HBL-3 cells as an immunogen

1) lmmunophenotype Surface lg - Leu6 (CD 1) - MY7 (CD13) Cytoplasmic lg - Leu5b (CD 2) - MY4 (CD14) C3 receptor - Leu4 (CD 3) - LeuMl (CD15) Fcy receptor - Leu3a (CD 4) - MY9 (CD33) HLA-DR t Leu1 (CD 51 - B4 (CD19) t Leu9 (CD7) - CALLA (CDIO) E l (CD20) - Leu1 1 (CD16) - IL-2R (CD25) B2 (CD21) - Leu7 (CD57) - BAl (CD24) t Leu8 - TdT

2) Karyotype 46XX. - 3, - 9, -9, tder(3)t(3;?), tder(9)t(9;?), tder(9)t( 1 ;9]

3) Genotype

Immunoglobulin heavy chain (JH) Immunoglobulin light chain (JK) T-cell receptor (Ca 1) T-cell receptor (Jy 1)

EcoRl BamHl R R G G G G G G

t

R: Rearrangement G: Germline.

3) Distribution of WH 14 antigen on normal hematopoietic cells (Table 2)

The reactivity of WH14-Ab on normal hemato- poietic cells is shown in Table 2; WH14 antigen was observed in approximately 20% of monocytes and platelets. WH 14-Ab reacted with only alimited num- ber of bone marrow mononuclear cells but not with any cells of lymph node, tonsil, spleen, or thymus. WH14-Ab showed virtually no reactivity with the lymphocytes and granulocytes.

4) Reactivity of WH14-Ab with human hematopoietic cell lines (Table 3)

WH14-Ab reacted with HBL-3, Molt-3 and THP-1 cell lines, but not with other leukemia or lymphoma cell lines or EBV-transformed cell lines. The fluo-

Table 2. Reactivity of WH14 AB with various hematopoietic systems

Examined cases

Positive rate (%)

Peripheral blood Mononuclear cells Monocytes Granulocytes Platelets

Bone marrow cells Thymus cells

Tonsil cells

4

3 1

3

1,121.1 20.8f5.7

1.120.4 tl-•

2.1 f 1.1 - -

Reactivity of WH14-Ab was assayed by indirect immnofluorescence. Percentage of positive cells by immnofluorescence in individual cases (mean f standard deviation). * Some flurorescence cells visible on microscopy.

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Table 3. Reactivity of WH14 Ab against human hematopoietic cell lines

1) 8-cell leukemia/lymphoma HBLJ (pre-B cell leukemia) REH (common ALL)

Raji (Burkitt's lymphoma) TL-1 (Burkitt's lymphoma) HBL-1 (large cell lymphoma) HBL-2 (large cell lymphoma) HBL-4 (Burkitt's lymphoma) HBL-5 (Burkitt's lymphoma) EBV-transformed cell line 1

BALL-1 (B-ALL)

L

HBL-6 (myeloma)

2) T-cell leukemia/lymphoma Molt-3 (T-ALL) HSB-2 (T-ALL)

3) Monocytic leukemia u937 THP- 1

4) Others HL-60 KG-1

Reactivity of WH 14-Ab with cultured cell lines by indirect immunofluoresence. f: most cells were positive. t/-: a partial reactivity (see Fig. 1). -: negative. +: small number cells (< 20%) were weak positive.

rescence intensity of HBL-3 cells is shown in Fig. 1.

5) Reactivity of WH14-Ah with various leukemias and lymphomas (Table 4)

WH14-Ab reacted with 10 of 11 cases of non-T ALLs, 1 case of B-lymphoblastic lymphoma and 2 of 7 cases with small cleaved cell lymphoma. WH 14- Ab did not react with other types of B-cell lympho- mas (24 cases) or T-cell lymphomas (15 cases). WH 14-Ab reacted with Reed-Sternberg (RS) cells from 2 of 7 cases of Hodgkin's disease. It is partic- ularly interesting that all WH 14-Ab-positive RS cells also expressed B-cell phenotype by pan-B mono-

' = O 7

FL 1

Table 4. Reactivity of WH14 Ab against Ieukemiaflymphomas (positive caseslexamined cases)

1) Non-T ALL

2) B-cell lymphomas Follicular small cleaved cell

mixed Diffuse small

small cleaved cell mantle zone lymphoma mixed large immunoblastic Burkitt's

3) T-cell lymphomas Diffuse lymphoblastic

medium mixed large pleomorphic

CD20- CD3- RS-cell CD20' CD3- RS-cell

5) Acute myeloid leukemia M 1 & M2 M4 M5 M6

6) Elastic crisis of CML Lymphoid crisis Myeloid crisis Monocytoid crisis

4) Hodgkin's disease

l o l l 1

0/2 012 014 215 01 1 013 0113 013 013

015 o/ 1 015 012 012

013 212

016 214 213 01 1

111 013 111

Binding of WH 14-Ab with various leukemia/lymphomas were assayed by indirected immunoflurosence or immunostaining. Positive reactivity was defined as 9 30% specific fluorescence cells by indirect immunofluroscence or staining of surface of cells by im- munostaining.

clonal antibody. In addition, WH 14-Ab reacted with 5 cases of acute myeloid leukemia (AML): none of 7 cases of AML (M1 and M2), 2 of 4 cases of AMMoL (M4), 2 of 3 cases of AMoL (M5) and 1 case of lymphoid crisis of chronic myelogenous leu- kemia.

6) Determination of antigen recognized with WH14-Ab (Fig. 2)

The immunoprecipitation by autoradiography re-

1 5 0

-TL 1 I

FL 1

Fig. 1 . WH14-Ab reacts with most of HBL-3 cells and a partial number of REH cells, but not with Raji cells.

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7) Response of HBL-3 cells to WH14-Ab

The incorporation of 3H-thymidine by HBL-3 cells was 67 695 2 278 - 73453 2 25261 cpm in compari- son to 46461 2 6378 cpm in control.

Discussion

We produced a novel monoclonal antibody, desig- nated WH14-Ab, which shows a relatively specific reactivity with pre-B cell leukemia/lymphoma cells. WH14-Ab did not react with any lymphoid cells in peripheral blood, tonsil, spleen and thymus. In lym- phoid malignancies, WH 14-Ab reacted only with non-T ALLs and B-lymphoblastic lymphoma lack- ing surface immunoglobulin (SIg). WH 14-Ab did not react with SIg-positive B-cell lymphomas and T-cell lymphomas, except 2 cases of small cleaved cell lymphoma with SIg. In addition, WH 14-Ab re- acted with pre-B cell line and non-T-ALL cell line (HBL-3 and REH) but not with mature B-cell lines or early B-cell lines (BALL-1, Raji, TL-1, HBL-4

However, WH14 antigen is not strictly specific for the B-cell lineage because of a slight positive reac- tion with approximately 20% Of the monocytes in the peripheral blood and myelomonocytic (M4) or monocytic (M5) leukemia cells. The reactivity of WH14-Ab might be similar to that of a few mAb such as OKTlO (CD38) (1, 4), which reacts with immature lymphoid cells and plasma cells as well as > 95 % of thymic cells. However, WH 14-Ab failed to react with any normal lymphoid cells including thymic cells. In addition, an immunoprecipitate re- vealed that molecular weight of antigen recognized

(1, 3), HBL-5 (1, 3), HBL-1 (7) and HBL-2 (7).

Fig. 2. SDS-PAGE analysis of "S-methionine labelled immuno- precipitates. (a) a blank of non-specific binding to protein A sepharose CL4B. (b) antigen molecule recognized by WH14-Ab (arrow).

vealed that the molecular weight of the antigen re- cognized by WH 14-Ab was approximately 30 000 dalton single band (reduced).

Fig. 3. Non-T ALL. (left) Leukemic cells diffusely infiltrate in the bone marrow (H.E. x 260). (right) Leukemic cells (Ia+, CD19-, CD20-, CD10+, SIg-) are positive for WH14-Ab (immunohistological staining, x 260).

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Fig. 4 . Hodgkin’s disease. (left) Some popcorn-like Reed-Sternberg cells scattering in the reactive lymphocytes (H.E. x 260). (right) WH14-Ab reacts only with RS-cells, but not with small lymphocytes around RS-cells (immunohistological staining x 260).

by WH14-Ab was 30 kd, which is clearly different from that of CD38.

These findings indicate that WH14-Ab is some- what different from CD38(OKT10) and the anti- genic determinant recognized by WH 14-Ab may be considered to be expressed at an early stage of B-cell maturation. This mAb may be quite useful for the detection of leukemia/lymphoma of pre-B cell ori- gins.

The origin of Reed-Sternberg giant cell (RS cell) in Hodgkin’s disease is controversial (1, 5). Many studies have suggested a T-lymphocyte origin (1, 6), B-lymphocyte origin (1, 7) and monocyte- macrophage origin (1, 8). In the present study, RS cells expressing B-cell associated antigen (CD19 or CD20) were positive for WH14-Ab, but RS cells lacking B-cell associated antigen were negative for WH14-Ab. Although this positive reaction could not be well evaluated, it aroused some interest in the cellular origin of RS cells, suggesting a heterogenous possibility in Hodgkin’s disease.

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