IE59771B1 - Murine hybridoma lym-2 and diagnostic antibody produced thereby - Google Patents

Description

Tne field of the invention is hybridomas and raonoclonal antibodies. More specifically,, this invention relates to hybrIdoma-produced monoclonal antibodies which Identify B5 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 10 immunized mice by Kohler and Milstain in 1975 (Nature 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 15 hybrid cells (called hybridomes”) 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 7q 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 j diagnosis and classification of the non-Hodgkin’s lymphomas O{- and leukemias. -2An 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 immunoglobulin classes. Other available reagents include BA-1 (Abramson, C.S., Kersey, J.H., and LeBien, T.W., J. Immunology 125:83-88, 1981), Bl (Nadler, L.M. , 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 Schlossman, 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, OKB4, and OKB7 (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-lymphoid 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 date.

SUMMARY OF INVENTION A hybridoma clone, designated Lym-2, was produced from the fusion 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 3 lymphocytes and interdigitating hisciocyces of “he lymph node. A subset of peripheral blood 3 cells are positive and no reactivity has ί- been observed in human bone marrow by flow cytometric analysis. Because of the remarkable specificity of Lym-2 for hu- * man B-eells and derived malignancies, these data suggest chat, Lym-2 will be an appropriate reagent for in vivo diagnosis -G and therapy of the human B-cell lymphomas and leukemias. > DETAILED DESCRIPTION The antigenic preparation used in obtaining the hybrid15 ©ma Lym-2 consisted of the nuclei of human chronic lymphocytic leukemia (CLL) biopsy cells. See Epstein, et al., in J. Immunol., 133:1028-1036, 1984, for the nuclei preparation procedure. The purified CLL nuclei were used to prepare the - aurin* hybridoma according co «ell knovn procedures. Briefly, hybridoma clone Lym-2 was produced by the fusion of mouse myeloma NS-l cells and B&LB/c splenoeytes obtained from a t souse hynerimmunized with the nuclei of CLL cells. These cell lines are generally available in the United States and other countries.

The noncclonal antibodies produced by the 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 the hybridoma Lym-2 have been placed on deposit with the American Type Culture Collection, 12301 Farklawn Drive, Rockvilla, Maryland, 20852. Hybridoma Lym-2 has been assigned i the ATCC accession Ho. KB 8613. This deposit has been conformed to the requirements of the Budapest Treaty. The primary characteristics of hybridoma Lym-2 are as follows: 1. Origin: Ic was produced by fusion of KS-T mouse myeloma cells with EALB/c souse splenocytea primed with human CLL nuclei» 2. Cultivations The Lym-2 hybridoma can be cultivated in RPMI-1640 medium containing 15% fecal calf serum, 100 unics/ml penicillin-C, and 100 ug/ml streptomycin sulfate. 3. Properties : The Lym-2 hybridoma is not phytopatho10 genic 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 antibody which specifically stains the germinal center, mantle zone, and interfollicular histiocytes of human lymph nodes and derived malignancies. It is negative on T-cells, myeloid cells, and other human tissues studied to date.

Routine immunoprecipitation and immunoblot methods have failed to identify the antigen recognized by Lym-2.

. 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 3-cell lines p such as SU-DHL-6 or by immunoperoxidase staining on frozen sections of human lymph nodes.

Tne Lym-2 hybridoma may be propagated In vitro at an 30 ' ' initial ceil concentration of 2 x 10^ cells/ml in RPMI-1640 medium containing 15% fetal calf serum, 100 units/ml penicilIln-G, and 100 ug/ml streptomycin sulfate. The cells are * 3S grown in stationary suspension culture at 37°C in a wellhumidified 5% C09 incubator and are transferred every 3-4 days.

Using the culturing procedure described, above, the Lym-2 antibody may also be produced. The antibody is obtained by centrifuging the ceil culture medium at 1,000 rpm for 10 minutes at 46,C to pellet the cells. The supernatant, which contains approximately 10 ug/ml of IgGl monoclonal antibody, is then frosen at -2CPC in, small aliquots for use in ch® immunofluorescence and immun,operoxidase procedures.

To obtain larger yields of higher concentration Lym-2 antibody for the radioismunolocalisation studies, the hybrid10 oraa may be injected into BALB/c mice. The injected hybridoma will cause the formation of antibody-producing tumors after a suitable incubation time, which will result in a high concen15 cracion of the desired antibody in the bloodstream and che peritoneal exudate (ascites) of che 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 1,000 rpm for 15 minutes at 4eC 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 *7 e “ -80a,C for long-term stability. From this preparation, approximately 2»3 mg/ml of IgGl can be recovered and purified by standard methods. Literature references describing the foregoing procedures are; Soogecraad, $., Helman, T. , and .. Eoogemraad, J..; J. laammol·. Methods, 61:317-320, 1983. Coding, J.W., u. Immunol., Methods., 39:285-303, 1980.

EXPESIHENXAL EXAMPLES The scientific basis of the present Invention will be more fully understood from the following description of the research investigations which led to the Invention. β Materials and, Methods Antigen preparation. Nuclei from the CLL cells were prepared. Two ml of packed ceils were thawed and washed once with Ca/PIPES buffer (0.01 M CaCl9, 2 x 10 M piperazine-N, N’-bis (2-echanesulfonic acid) in a 50-ml centrifuge cube.

Tne sediment was chen resuspended in 40 ml of Ca/PIPES buffer and thoroughly homogenized by using a motor-driven Teflon pestle co disrupt che swollen cells. The nuclei were chen sedimented and resuspended in Ca/PIFES 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 C&/BIPES buffer co remove che detergents , resuspended in 10 ml of PBS, and sonicated three times for 15-sec inter20 vals co produce a more homogeneous suspension. The nuclei . preparations were chen frozen in 1-ml aliquots at »85°C until use.

Immunization protocol. A 1-ml aliquot of the nuclear ·»« preparation was thawed, resonicated to reduce viscosity, and emulsified in 1.5 ml of complete Freund4s adjuvant (Difco Laboratories, Detroit, Michigan) by using nra glass syringes aud a 20-gauge microemulsifying needle (BolabK Three 10-wk-“ old BALB/c female mice were injected subcutaneously at multiple sites by using a 22-gauge needle and glass syringe. Two weeks later, the mice were reinoculated as above except the nuclear extracts were prepared in incomplete adjuvant.

Ten days later, the mice 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. Ί Cell fusion and cloning procedures. Spleen cells were fused with 8-azaguanine-resiscant mouse myeloma NS-1 cells at a ratio of 5:1, respectively, by using 40% polyethylene gly5 col 1540 m.w. as described by de St. Groth and Scheidegger, J. Immunol. Methods, 35:1, 1980. Culture supernatants from wells with active cell growth were tested by indirect immunofluorescence with fixed cell preparations as described below. IQ Positive cultures were cloned twice on 0.57. Noble agar containing RPMI 1640 medium, 20% fetal calf serum, and antibiotics, as described by Epstein and Kaplan, Cancer Res . , n 39:1748, 1979.

Serologic characterication of monoclonal antibody isotypes. Kybridoma supernatants from 4-day .cultures were concentrated 10 to 20x in B15 minieon concentrations (Amicon, Lexington, MA) and tested in double diffusion Ouchterlony plates against rabbit anti-mouse immunoglobulin heavy chain specific antisera. The precipitin bands were read after 2 to days ef incubation in a well-humidified 37aC incubator.

Live cell indirect iaaasnofluorescenee. Cells were washed twice with PBS ¢0.2 g 0.1 g CaCl7.2H,0, 1.15 g NA2HP0a, 0.1 g MgCl2.6H20. 0.2 g KCX, 8.0 g NaCl/liter) containing 1 mg/ml bovine serum albumin (BSA:5XA grade, Sigma ' Chemical, St. Louis, MO) and 0.02® sodium azide.* Single cell suspensions containing 1 x 10 cells were incubated for 30 min with 100 ul of monoclonal antibody supernatant at 4eC. Cells were then washed to remove excess antibody and incubated with a 1/20 dilution of fluorescein-conjugated goat anti-mouse IgG F (ab’)2 fragment specific (Cappel, Cochranville, PA) for 30 min at 4®C. After two additional washes, two drops of mounting solution composed of 1:1 glycerol and BBS, pH 8.0 and 27, paraformaldehyde (#4018„ Polysciences, Warrington, PA) «era added to each tube. The cells were mounted onto a glass slide and examined within 24 hours by epifluorescence microscopy with a Leicr Orthoplan micro* scope with a ploemopak 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-1 myeloma cultures was used as a control to determine the background staining of each cell line.

Fixed cell indirect immunofluorescence. To examine IS -cells for th® presence of intracellular antigens, fixed cell preparations were used. Cells were washed twice with PBS containing I mg/ml BSA, and 0.02X sodium aside and were s pipetted dropwise at a concentration of 5.0 » 10 cells/ral onto Teflon-coated printed microscope slides containing 10 -nsa wells/slide. After the cells settled to the surface of the glass, the overlying fluid was quickly removed by aspiration and tne ceils were dried to the slide by a gentle scream of warm air. The slides were then immediately fixed in 27 paraformaldehyde in PBS for 15 min ac room temperature.

After fixation, the slides were rinsed in PBS and placed in acetone at -20**C for 3 min to make the cells permeable.

After a final rinse to remove the acetone, the slides were stored at 4*C in PBS containing 0.027 sodium aside.

For the immunofluorescence assay, 35 ul of hybridoma supernatant were pipetted onto each well cf the printed microscope slide preparations. After 80 min ©f incubation at 37°C in a humidified chamber, the slides were rinsed three times in PBS and again incubated for 30 min at 37eC with 20uI of a 1/20 dilution of fluorescein conjugated goat anti-mouse IgG F(ab’)2 fragment specific. The slides were than rinsed three times in PBS, counterstained with Evans blue for 5 min at room temperature by using a freshly prepared solution containing 50 ul of a IX stock solution of Evans blue in 80 ml of PBS, rinsed a final tine in PBS, and mounted with coverslips by using a 1:1 solution of glycerol and PBS, pH-8.0.

Immunoperoxxdage staining. Frozen sections were prepared from human tissue biopsies obtained from che Section of Surgial Pathology, Northwestern Memorial Hospital, from specimens submitted from pathologic diagnosis. The sections were stained with the monoclonal antibody Lym-2 by using the avidin-hiocin complex itsmtmoperoxidase staining procedure as described by Hsu, et al., J. Histochem.' Cytochem., 29:57720 580, 1981. For these experiments a 1/4 dilution of Lym-2 supernatant was used. As a negative control, NS-1 supernatant which is unreactive in frozen sections, was used with each run. «3 is “ Lym-2 Purification: A summary of Lym-2 purification is described as follows: 1. raise ascites In pristane primed BALB/c mice. 2. harvest ascites aseotically from neritoneal cavity. 3. remove cells by centrifugation ¢1,500 rpm for 20 min) . 4. filter to sterilise and remove debris (0.2 micron). . 50* ammonium sulfate precipitation. 6. dialysis against PBS overnight at 4SG. 7. affinity purification on Frotein-A sepharose.

Eluate at pK 5.6-5.7. 8. dialyze against FBS overnight at 4a'Cs . ο 9. ultracentrifuge at 30,000 rpm for 1 hr at 4°C. . membrane filter (0.2 micron). 11. store in aliquots at -80°C until use.

RESULTS Hybridoma clone Lym-2 was produced by the fusion of ιθ mouse myeloma HS-1 cells and BALB/c splenoeytes obtained from a mouse hyperimmunized with nuclei from CLL cells. Isotypic analysis revealed that monoclonal antibody Lym-2 was of the IgGl heavy chain subclass. The Lym-2 antibodies were idenci13 tied by indirect immunofluorescence techniques with the us® 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 IT, respectively. 1 table: x Reactivity of Lym-2 with human malignant lymphoma cell lines by indirect immunofluorescence Cell Line Lym-2a Burkitc's lymphoma Raji EB3 10 ramos SU-AmB-1 SU-AmB-2 4* NU-AmB-1 4* NK-9 — 13 Diffuse histiocytic lymphoma SU-DHL-1 RB SU-DHL-2 « SU-DHL-4 SU-DHL-5 » Su-DHL-6 + SU-DHL-7 - 20 SU-PHL-8 ·* SU-DHL-9 NU-DHL-l - U-937 25 Undifferentiated Lymphoma NU-BUL-l . a b Fixed cell indirect inmsnoflixorsscenca assay. Data expressed as (-) negative, (+) positive.

Reactivity of Lym-2 with human leukemia and lymphoblastoid cell lines by indirect immunofluorescence TABLE II Cell Line Lym-2a Acute lymphoblastic leukemia T Cell Holt-4.

CEM HSB-2 HPB-ALL JM 7- Null cell REH NALL-1 KM-3 B-cell BALM-2 NAIM-6 (pre-B) NALM-1 (pre-B from CHL) Myeloid leukemia K562 (erythroid-CML) HL-60 Cpromyelocytic) ML-2 (myeloid) TPH-1-0 (monocytic) KG1 (myeloid) Myeloma U-266 AEH-77 Ψ Lymphoblastoid BL-1 NU-LB-1 Nu-LB-2 + .j.

Fixed call indirect immunofluorescence assay. Data expressed as (-) negative, (4-) positive. 3 In Table III, che sGaining 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 the majority of B-cell derived tumors.

TABLE III Indirect immunofluorescence staining of human 10 lymphoma and chronic lymphocytic leukemia biopsy cells Lym-2 Reactivity Diagnosis (positive cases/total cases) Lymphoma (frozen sections of lymph node biopsies) well-differentiated lymphocytic 3/3 poorly-differentiated lymphocytic 5/5 mixed 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 Rappaport classification The immunoperoxidase staining reactivity of Lyra-2 on 30 frozen sections of normal human biopsy tissues is shown in Table X¥e Lym-2 was found to be specific to B-cell lymphocytes and histiocytes in lymphoid tissues. No reactivity was demonscrated in human bone marrow or in non-lymphoid human organs» 4 table iv Reactivity of Lym-2 with normal human tissues Tissue Lyra-2 Reactivity lymph node + B cell zones tonsil + 3 cell sones thymus » bone marrow* blood* + subset of B lymphocytes adrenal brain - breast colon - heart - 1 iver lung pancreas salivary gland - skin - skeletal muscle smooth muscle thyroid ^Determinations made by flow cytometric analysis on viable cells in suspension.

Tne inEBianoreactivity of Lym-2 on human solid rumor cell lines was determined by indirect ijnnunofluorescence tech2S niques en fixed cell preparations. As shewn in Table V, Lym-2 was net found reactive on the cell with any of the 26 cell lines tested.

TABLE V Immunoreactivity of Lym-2 with human solid tumor cell lines Solid Tumors Lym-2 Reactivity CaCL-74-36 (melanoma) BM-166 (neuroblastoma) • Y79 (retinoblastoma) - HeLa (ovarian carcinoma) SU-CCS-1 (clear cell sarcoma) Colo 38 (melanoma) C-399 (colon carcinoma) A-172 (glioblastoma) 1¾ J?CI-H69 (small cell carcinoma of lung) - IMR-5 (neuroblastoma) Hutu-80 (colon carcinoma) - HT-29 (colon carcinoma) - 734B (breast carcinoma) - SV-80 (rhabdomyosarcoma) - SV-1503 (mesothelioma) - SV-733 (papillary carcinoma of bladder) - U11S-MG (glioblastoma) - SV-872 (lioosarcoma) - SV-780 (transitional cell carcinoma of bladder SV-1045 (synovial cell carcinoma) - SV-608 (astrocytoma·) SV-1353 (chondrosarcoma) - SV-451 (squamous cell carcinoma of esophagus) & SV-15 6 (hyp ern ep hr oma) - I‘nJ-04 (glioblastoma) SV-5 79 (squamous cell earcimts. of thyroid) a - : negative; + : positive by indirect immunofluorescence microscopy on fixed cell preparations. i 6 Table Vi below summarizes tne major characteristics of Lym-2 antibody.

TABLE VI Characterization of Monoclonal Antibody Lym-2 Lym-2 Immunogen CLL nuclei Is©type IgGl Antigen unknown Antigen sice cell surface Lymphoid Reactivity lymph node and tonsil B-cell zone and histiocytes bone marrow none blood subset of B lymphocytes thymus none Non-Lymphoid Reactivity none Tumor Specificity B-cell lymphomas and leukemias

Claims (8)

1. ei&iss

1. The hybridoma cell line Lym-2 (ATCC No. 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 No. 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 10 line (ATCC No. HB 8613) or clones thereof, harvesting the supernatant, and recovering the Lym-2 antibodies therefrom.

4. A method for producing Lym-2 antibodies, comprising injecting hybridoma cells of the Lym-2 cell line (ATCC 15 No. H3 8613) or clones thereof Into SALS/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. H3 8613), or clones 20 thereof.

6. The hytaridoma 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 antibody, substantially as hereinbefore described.

8. A Lym-2 antibody, whenever produced by a method claimed in a preceding claim.