IE873098L - Murine hybridoma lym-2 - Google Patents

Description

FIELD OF INVENTION The field of the invention is hybridomas and monoclonal antibodies. More specificallythis invention relates to hybridoma-produced monoclonal antibodies which identify B» lymphocyte surface antigens, and which are useful in the diagnosis and therapy of B-cell derived human lymphomas and leukemias.

BACKGROUND OF INVENTION The fusion of mouse myeloma cells and spleen cells from immunized mice by Kohler and Milstein in 1975 (Mature 256: 495-497, 1975) demonstrated for the first time that it was possible to obtain a continuous cell line making homogeneous (so-called "'monoclonal") antibody. Since this seminal work, much effort has been directed to the production of various hybrid cells (called "hybridomas") and to the use of the antibody made by these hybridomas for various scientific investigations .

The analysis of lymphocyte populations in human lymphoid tissues has been greatly facilitated by the availability of monoclonal antibodies directed against lymphoid differentiation antigens. These reagents have been used to localize lymphocyte subsets topographically in the lymph node, spleen, and thymus and to phenotype lymphoid malignancies for the diagnosis and classification of the non-Kodgkin's lymphomas and leukemias.

An increasing number of monoclonal antibodies directed at B-cell surface antigens have been reported. Among the commercially available products are monoclonal antibodies to each of the heavy and light chain inmunoglobulin classes. Other available reagents include BA-1 (Abramson, C.S., Kersey, J.H. , and LeBien, T.W. , J. Irasaunology 125:83-88, 1981), B1 (Nadler, L.M.t Ritz, J., Hardy, K., Pesando, J.M., J.Clin. Invest. 67:134-140,, 1981), B2 (Nadler, L.M. , Stashenko, P., Hardy, R. , Van Agthoven, A. , Terhorst, C. , and Schlossmann, S.F., J. Immunol. 126:1941-1947, 1981), BL1, BL2, and BL3 (Wang, C.Y., Azzo, w., Al-Katib, A., Chiorazzi, N., and Knowles, D.M., J. Immunol. 133:684-691, 1984), OKB1, CKB2, 0KB4, and 0KB7 (Mittler, R.S., Talle, M.A., Carpenter, K., Rao, P.E., and Goldstein, G., J. Immunol., 131:1754-1761, 1983) and others. Although these monoclonal antibodies have been found to identify B-cell differentiation antigens, many cross-react with non~1ymphold tissues, have relatively low avidity of binding, or are directed against antigens which are shed into the blood. Hence, B-cell specific monoclonal antibodies with in vivo diagnostic or therapeutic potential have not been described to data.

SUMMARY OF INVENTION A hybridoma clone, designated Lym-2, was produced from the fus ion of primed mouse splenocytes and mouse myeloma NS-1 cells. Hybridoma Lym-2 produced a murine IgGl monoclonal antibody which recognizes a cell surface protein expressed in normal and malignant B lymphocytes. Immunoperoxidase staining of a panel of normal human tissues shows that Lym-2 reacts with germinal center and mantle zone B lymphocytes and interdigieating histiocytes of the lymph node. A subset of peripheral blood B cells are positive and no reactivity has g been observed in human bone marrow by flow cytometric analysis. Because of the remarkable specificity of Lya-2 for hu- * man B-cells and derived malignancies, these data suggest chat, * Lym-2 will be an appropriate reagent for in vivo diagnosis 10 and therapy of the human B-cell lymphomas and leukemias.

DETAILED DESCRIPTION The antigenic preparation used in obtaining the hybrid-IS oma, Lym-2 consisted of the nuclei of human chronic lymphocytic leukemia (CLL) biopsy cells. See Epstein, et al.» in J. Immunol.p 133:1028-1036„ 1984, for the nuclei preparation procedure. The purified CLL nuclei were used to prepare che murine hybridoma according to well known procedures. Briefly, hybridoma clone Lym-2 was produced by the fusion of mouse myeloma NS-1 cells and EALB/c splenocytes obtained from a mouse hyperimmunized with che nuclei of CLL cells. These 23 cell lines are generally available in che United States and ether countries.

The monoclonal antibodies produced fay che hybridoma Ly: 2 were tested to determine the properties and specificity of Lym-2. - These tests -and the results are described below.

For the purpose of this patent application, cultures of che hybridoma Lym-2 have been placed on deposit with, the 35 Americas Type Culture Collection, 12301 Farklawa Driire, Rock-ville, Maryland, 20852. Hybridoma Lym-2 has been assigned i the ATCC accession Ho. KB 8613. This deposit has been conformed to the requirements of Che Budapest Treaty. The primary characteristics of hybridoma Lysi-2 are as follows: 4 1. Origin; It was produced by fusion of NS-T mouse myeloma cells with EALB/c souse splenocytes prised with human CLL nuclei„ 5 2. Cultivation: The Lym-2 hybridoma can be cultivated in RPMI-1640 medium containing 15% fetal calf serum, 100 units/ml penicillin-C, and 100 ug/ml streptomycin sulfate. 3. Properties: The Lym-2 hybridoma is not phytopatho- 10 genie and is not known to have any dangerous properties. It is tumorigenic in BALB/c mice. 4. Antibody: Lym-2 produces a murine IgGl monoclonal ^2 antibody which specifically stains the germinal center, mantle zone, and interfollicular histiocytes of human lymph nodes and derived malignancies. It is negative on T-ceils, myeloid cells, and other human tissues studied to date. 20 Routine ismmnaprecipitation and immunoblot methods have failed to identify the antigen recognized by Lym-2. 5. Testing: The production of Lym-2 antibody by the hybridoma cells can be tested by indirect Immunofluorescence 25 on viable cells or 2% paraformaldehyde fixed B~cell lines„ such as SU-DHL-6 or toy inammoperoacidase staining on frozen sections of human lymph nodes.

Hie Lyra-2 hybridoma may be procagated in vitro at an 30 * """" " initial cell concentration of 2 at 10^ cells/ml in RPMI-1640 medium containing 15X fetal calf serum, 100 units/ml penicil-lin-Gf and 100 ug/ml streptomycin sulfate. The cells are <■ 2$ grown in stationary suspension culture at 37°C in a well- humidified 5% C09 incubator and are transferred ©very 3-4 days.

Using the culcuring procedure described above, the Lym-2 antibody may also be produced. The antibody is obtained by 5 cantrifuging the cell culcure medium ac 1.000 rpm for 10 minutes at 46,C Co pellet che cells. The supernatant:, which contains approximately 10 ug/nl of IgGl monoclonal antibody, is then frozen at -lO^C in small aliquots for use in the immunofluorescence and immunoperoxiaase procedures.

To obtain larger yields of higher concentration Lym-2 antibody for the radioimmunolocalization studies, che hybridoma nay be injected into BALB/c mice. The injected hybridoma will cause che formation of antibody-producing tumors after a suitable incubation time, which will result in a high concentration of the desired antibody in the bloodstream and the peritoneal exudate (ascites) of the host mouse. The Lym-2 antibody is recovered from the mice by removing the ascites fluid with a needle and syringe. The ascites is then spun ac 1,COO rpm for 15 minutes ac 4°C to pellet the cells and the supernatant is filtered sequentially through a 0.8 micron and 0.22 micron filter units to remove residual debris. Using sterile technique, the filtered ascites is then stored at -S0a'C for long-term stability. From this preparation, approximately 2»3 mg/mi of IgGl can be recovered and purified by standard methods. Literature references describing the foregoing procedures are: Hoogearaad, W,, HeXman. T.. and Hoogeraraad, J..: J. laaamol-. Methods. 61:317-320, 1983. Coding. J.W., J. Immunol. Methods, 39:285-303, 1980.

EXPERIMENTAL EXAMPLES The scientific basis of the present invention wilt be <•> ware fully understood from the following description of the research investigations which led to the. invention. 6 Materials and Methods Antigen preparation. Nuclei from the CLL cells were prepared. Two ml of packed cells were thawed and washed once with Ca/PIPES buffer (0.01 M CaCl2, 2 jc 10"3 H piperazine-N. N'-bis (2-ethanesulfonic acid) in a 50-ml centrifuge tube. The sedinent was then resuspended in 40 ml of Ca/PIPES buffer and thoroughly homogenized by using a nocor-driven Teflon pes tie co disrupt the swollen cells. The nuclei were then sedimented and resuspended in Ca/PIPES buffer containing 1% Nonidet P-40. The nuclei were then rehomogenized and checked by phase contrast microscopy to be free of contaminating cytoplasmic and membranous debris. Nuclei were then washed twice in Ca/PIPES buffer co remove the detergents„ resuspended in 10 ©1 of PBS, and sonicated three times for 15™sec intervals to produce a more homogeneous suspension. The nuclei preparations were then frozen in l~ml aliquots at ~85°C until use.

Immunization protocol. A l~ml aliquot of the nuclear preparation was chawed, resonicated to reduce viscosity, and emulsified in 1.5 ml of complete Freuad's adjuvant (Difco Laboratories, Detroit, Michigan) by using two glass syringes and a 20-gauge microemuls ifying needle (Bolab). Three 10-wk-" old BALE/c female mice were injected subcutaneously at multiple sites by using a 22-gauge needle and glass syringe. Two weeks later, Che mice were rainoculated as above except the nuclear extracts were prepared in incomplete adjuvant. Ten days later, the sic® received a third inoculation of antigen, this time without adjuvant and by i.p. injection.

Four days later,, the mice were sacrificed by cervical dislocation and the spleens were removed by aseptic techniques. 7 Cell fusion and cloning procedures.. Spleen cells were fused with 8-azaguanine-resiscant mouse myeloma NS-l cells at a ratio of 5:1, respectively, by using 401 polyethylene glycol 1540 m.w. as described by de Sc. Groth and Scheidegger, J. Immunol. Kechods „ 35:1, 1980. Culture supematants from wells with active call growth were tested by indirect immunofluorescence with fixed cell preparations as described below. Positive cultures were cloned twice on 0.5% Noble agar containing RPMI 1640 medium, 201 fetal calf serum, and antibiotics, as described by Epstein and Kaplan, Cancer Res.. 39:1748, 1979.

Serologic characterisation of monoclonal antibody iso-types. Hybridoma supernatant® from 4-day .cultures were concentrated 10 to ZOx in B15 minicon concentrations (Amicon, Lexington,, HA) and tested in double diffusion Ouchterlony plates against rabbit anri-mouse immunoglobulin heavy chain specific antisera. The precipitin bands were read after 2 .to 3 days of incubation in a well-humidified 37°C incubator.

Live cell indirect immunofluorescence. Cells were washed twice wich PBS (0.2 g SH^?04„ 0.1 g CaCl7.2H20, 1.15 g NA2S?04, 0.1 g MgCl2.6H20. 0.2 g KC1, 8.0 g NaCI/liter) containing 1 sag/ml beirine serun albumin (ESA:RIA grade, Sigma Chemical, St. Louis, MO) and 0.02* sodium aside.• Single cell 6 suspensions containing I x 10 cells were incubated for 30 rain with 100 ul of monoclonal antibody supernatant at 4°C. Cells were then washed to remove excess antihody and incubated with a 1/20 dilution of fluorescein~conjugated goat anti-mouse IgG F (ab*)2 fragment specific (Cappel, Cochran-ville, PA) for 30 min at 4*C. After two additional washes, two drops of mounting solution composed of 1:1 glycerol and PBS, pH 8.0 and 21 paraformaldehyde (#40X8, Polysciences, Harrington, PA) were added co each cube. The cells were mounted onto a glass slide and examined within 24 hours by epifluorescence microscopy with a Leitz Orthoplan microscope with a ploetaopak 2.1 fluorescence illuminator, HBO 100 mercury lamp, and 50x water immersion objective. A minimum of 200 cells were examined for immunofluorescence staining by two independent observers. Supernatant from NS-l myeloma cultures was used as a control to determine the background staining of each cell line.

Fixed cell Indirect Immunofluorescence. To examine cells for the presence of intracellular antigens, fixed cell preparations were used. Cells were washed twice with PBS containing 1 mg/ial BSA and 0.02X sodium aside and were pipetted aropwise ac a concentration of 5.0 * 10° cells/ml onto Teflon-coated printed microscope slides containing 10 5-nna wells/slide. After che cells settled to the surface of the glass, the overlying fluid was quickly removed by aspiration and the cells -were dried to the slide by a gentle stream of warm air. The slides were then immediately fixed in 2% paraformaldehyde in PBS for 15 mir. at room teocpetature.

After fixation, the slides were rinsed is PBS and placed in acetosae at -20°C for 3 mis Co make che cells permeable.

After a final rinse to remove the acetone, the slides were stored at 4*C in PBS containing 0.02Z sodium aside.

For the immunofluorescence assay, 35 ul of hybridoma supernatant were pipetted onto each well 0f che printed microscope slide preparations. After 60 min of incubation at 37°C in a humidified chamber, che slides were rinsed three times in PBS and again incubated for 30 min at S/'C with 20ul 01 3. 1/20 d j*lu s- 3»OSs, i i tiO 4. © S C © A" COS1J «,!^S,Ei5id! gOSC •sUfi.C A, —wBOUS «® IgG F(ab')2 fragment specific. The slides %rere Chan rinsed three times in PBS, counterstained with Evans blue for S min at room temperature by using a freshly prepared solution containing 50 ul of a IX stock solution of Evans blue in 80 ©1 of PBS, rinsed a final tine in PB5„ and nouncad with cover-slips by using a 1:1 solution of glycerol and PBS, pH-8.0.

Iranunoperoxidase staining. Frozen sections were prepared from human tissue biopsies obtained froa che Section of Surgial Pathology, Northwestern Memorial Hospital, from specinans submitted from pathologic diagnosis. The sections were stained with the monoclonal antibody Lym-2 by using the avidin-biotin complex imsmnoperoxidase staining procedure as described by Hsu, ec al. „ J. Kistochem.' Cytochem.. 29:577-580, 1981. For chase experiments a 1/4 dilucion of Lym-2 supernatant was used. As a negacive control. MS-1 supernatant which is unreactive in frozen sections, was used with each run.

Lytn-2 Purification: A summary of Lysa-2 purification is described as follows? 1. raise ascites in pristane prised BALB/c mice. 2. harvest: ascites aseptieally fro® peritoneal cavity. 3. remove cells by centrifugation (1,500 rpss for 20 lain) . 4. filter co sterilise and resove debris (0.2 micron). 5. SO" ammonium sulfate precisication. 6. dialysis against PBS overnight as 4 Eluate at pH 5.6-5.7. 8. dialyze against PBS overnight at 4°C. . 1 0 9. ultracentrifuge at 30,000 rpm for I hr at 4°C. 10. membrane filter (0.2 micron). 11. snore in aliquots at -80°C until use.

RESULTS Hybridoma clone Lym-2 was produced by che fusion of souse myeloma NS-1 calls and BALB/c splenocyces obtained from a mouse hyperimmunized with nuclei from CLL cells. Isotypic analysis revealed that mono clonal antibody Lytn-2 was of the IgGl heavy chain subclass. The Lym-2 antibodies were identified by indirect immunofluorescence techniques with the use of paraformaldehyde-acetone-fixed cell preparations.

The reactivities of monoclonal antibodies Lym-2 on established human malignant lymphoma and leukemia cell lines are shown in Tables I and II, respectively. 11 TABLE I Reactivity of Lym-2 with human malignant lymphoma cell lines by indirect immunofluorescence Cell Line Lym-23, Barkletc s lymphoma Raj i EB3 RAMOS SU-AmB-1 SU-AmB-2 NU-AmB-l NK-9 Diffuse histiocytic lymphoma SU-DHL-1 - SU-DHL-2 « SU-DHL-4 + SU-DHL-5 5U-DHL-6 + 5U-DHL-7 SU-PHL-8 4- SU-DHL-9 + NU-DHL-1 0-937 - Undifferentiated lymphoma NU-DUL-1 a Fisted cell indirect immunofluorescence assay, b Data expressed as (-} negative, (+•) positive. 12 TABLE II Reactivity of Lym-2 with human leukemia and lymphobl&stoid cell lines by indirect imnruno fluorescence Cell tine Lym-2a Acute lymphoblastic leukemia T Cell HoIt-4 cm H5B-2 HPB-ALL JM 13 Null cell REH NALL-1 KM-3 B-cell 20 BALM-2 NALM-6 (pre-B) NALM-1 (pre-B from CHL) Myeloid leukemia K562 (erythroid-CML) KL-60 (promyelocytic) ML-2 (myeloid) TPH-1-0 (monocytic) KGl (myeloid) ■ Myeloma 30 U-266 ARH-77 Lyraphofelastoid 35 BL-1 + NU-LB-1 + NU-LB-2 a Flsced call indirect immunofluorescence assay, b Basa expressed as (-) negative, (+) positive. 1 3 In Table III, che staining reactivity of Lym-2 on human malignant lymphoma and chronic lymphocytic leukemia biopsies is shown. Indirect immunofluorescence studies showed that Lym-2 was positive on che majority of B-cell derived tumors.

TABLE III Indirect immunofluorescence staining of human lymphoma and chronic lymphocytic leukemia biopsy cells Lym-2 Reactivity Diagnosis (positive cases/total cases) Lymphoma0 (frozen sections of lymph node biopsies) well-differentiated lymphocytic 3/3 poorly-differentiated lymphocytic 5/5 miised lymphocytic and histiocytic 7/9 histiocytic (B-cell type) 12/17 T-cell 0/2 Leukemia (cytospins of peripheral blood) Chronic lymphocytic B-cell type 8/10 T-cell type 0/5 a R&ppsport classification The imanmoperoxidas& staining reactivity of Lym-2 on frozen sections of normal human biopsy tissues is shown in Table IV. Lym-2 was found to be specific to B-cell lymphocytes and histiocytes iss lymphoid tissues. Mo reactivity was demonstrated in human bone marro« or in non-lymphoid human organs. 1 4 |RW .Ti ftBO «•> Wft ym-2 on human solid tumor cell 1e « o •§ ® TABLE VI Characterization of Monoclonal Antibody Lym-2 Lym-2 Immmogarx Isotype Antigen Antigen sice Lymphoid Reactivity unknown cell surface CLL nuclei IgGX lymph sioda and tonsil bone marrow blood 3-cell zone and histiocytes none subset of B lymphocytes thymus Non-Lymphoid Reactivity Tumor Specificity none none B-cell lymphomas and leukemias

Claims (8)

17 ei&iss

1. The hybridoma cell lira® Lym-2 (ATCC M©„ HB 8613) and clones thereof.

2. A method for producing Lym-2 antibodies, comprising 5 culturing hybridoma cells of the Lym-2 cell line (ATCC So- HB 8613)- or clones thereof, and recovering the Lym-2 antibodies produced.

3. A method according to claim 2, comprising culturing in a suitable medium hybridoma cells of the Lym-2 cell 1Q line (ATCC No. HB 8613) or clones thereof, harvesting the supernatant, and recovering the Lym-2 antibodies therefro®,,.

4. A method for producing Lym-2 antibodies, comprising injecting hybridoma cells of the Lym-2 cell line (ATCC 15 No. EB 8613) or clones thereof into BALB/c mice, removing the ascites fluid, and recovering the Lym~2 antibodies therefrom.

5. The Lym-2 monoclonal antibody produced by the hybridoma cell line Lym-2 (ATCC No. HB 8613), or clones 20 thereof. r 18

6. The hybridoma cell line Lym-2 according to claim 1 or a clone thereof,, substantially as hereinbefore described.

7. A method according to claim 2 for producing a Lym-2 antibodyp substantially as hereinbefore described.

8. A Lym-2 antibody„ whenever produced by a method claimed in a preceding claim. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.