IL84466A - Murine hybridoma lym-1 and diagnostic antibody produced thereby - Google Patents

Description

b Lym-1 monoclonal Murine Hyjiridoma Lym-iand- diagnostic/antibody produced thereby * UM'On >00 U ΑΝ>Ί/ ΠΑ 1 J 1 1 - D ■ ί? T33)> ΟΒΠ ί'Π Northwestern University -WUfttNE -HY&RtDGMAr -IrYW-t -AND -DIAGNOSTIC- -ANTIBODY- -PRODUCED -THEREBY FIELD OF INVENTION The field of the invention is hybridomas and monoclonal antibodies. More specifically, this invention relates to hy-bridotna-produced monoclonal antibodies which identify B-lymphocyte surface antigens, and which are useful in the diag nosis and therapy of B-cell derived human lymphomas and leu-kemias .

BACKGROUND OF INVENTION The fusion of mouse myeloma cells and spleen cells from immunized mice by Kohler and Milstein in 1975 (Nature 256 : 495-497, 1975) demonstrated for the firs 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 hy brid cells (called "hybridomas"). and to the use of the antibody made by these hybridomas for various scientific investig tions .

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 dia nosis and classification of the non-Hodgki ' s lymphomas and leukemias .

An increasing number of monoclonal antibodies directed a B-cell surface antigens have been reported. Among the commer cially 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. , JL_ Immunol. , 133:684-491, 1984), 0KB1, 0KB2 , 0KB4, and 0KB7 (Kit- tier, 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.

S UMMARY OF THE INVENTION A hybridoma clone, designated Lym-1, was produced from the fusion of primed mouse splenocytes and mouse myeloma NS-1 C^uj. fc& i v^ "*-- cells. Hybridoma Lym-1 produced a murine IgG2a monoclonal 3-r- c* v~»*av*H» antibody which recognizes a 31,32,33, and 35 kilodalton cell surface protein expressed in normal and malignant B lymphocytes. Immunoperoxidase staining of a panel of normal human tissues shows that Lym-1 reacts with germinal center and mantle zone B lymphocytes and interdigitating histiocytes of the lymph node, medullary dendritic cells of the thymus, and weak- ly with surface epithelium of the colon. A subset of peripheral blood B cells are positive and no reactivity has been observed in human bone marrow by flow cytometric analysis. The antigen recognized by Lym-1 is not shed from the surface of lymphoma cells either in cell culture or in patients and is not modulated after Lym-1 binding. Lym-1 itself has been shown to have high avidity to human lymphoma cells in vivo as demonstrated by radionuclide binding studies in lymphoma patients using 1-123 conjugates. Binding to normal tissues such as the bone marrow, spleen, lymph node, liver, kidney, lung or central nervous system has not been demonstrated in over 30 patients studied. Lym-1 has further been found to be highly stable to radionuclide conjugation methods and may be prepared as F(ab')2 or F(ab) fragments without significant loss of anti body activity. Collectively, these data suggest that Lym-1 will be an appropriate reagent for in vivo diagnosis and thera py of the human B-cell lymphomas' and leukemias.

DETAILED DESCRIPTION The antigenic preparation used in obtaining the hybridoma Lym-1 consisted of the nuclei of the human lymphoma cell line Ra i, which cell line is generally available in the United States and other countries. See Epstein et al. in J . Immuno1. 133:1028-1036, 1984 for the nuclei preparation procedure. The purified Raji nuclei were used to prepare the murine hybridoma according to well known procedures. Briefly, hybridoma clone Lym-1 was produced by the fusion of mouse myeloma NS-1 cells and BALB/c splenocytes obtained from a mouse hyperimmunized with the nuclei of Raji cells.

The nionoclona,! antibodies produced by the hybridoma Lym-1 were tested to determine the properties and specificity of Lym-1, These tests a,nd the results are described below.

For the. purpose of filing United States patent application issued as U.S. Patent 4,724,213 Serial No. 738, 084, /cultures of the hybridoma Lym-1 were placed on deposit with the American Type Culture Collection, 123Q1 Parklawn Drive, Rockville, Maryland, 2Q852, U.S.A. The deposit was on a restricted basis during the pendenc of the United States application but was otherwise in accordance with the Budapest Treaty, and the viability of the deposited cultures was confirmed by the depository. Hybridoma Lym-1 was assigned the ATCC accession No. HB 8612. The primary characteristics of hybridoma Lym-1 are as follows: 1. Origin ; It was produced b fusion of NS-1 mouse myeloma cells with BALB/c mouse splenocytes primed with Raj i human lymphoma nuclei. 2. Cultivation ; The Lym-1 hybridoma can be cultivated in RPMI-1640 medium containing 15% fetal calf serum, 100 units/ml penicillin-G, and 100 yg/ml streptomycin sulfate. 3. Properties : The Lym-1 hybridoma is not phytpathogenic and is not known to have any dangerous properties. It is tu-morigenic in BALB/c mice. 4. Antibody Lym-1 produces a murine IgG2a monoclonal antibody which specifically sta,ina th.e germinal center, ma,ntle zone, and interfollicular histiocytes of h an lYmJ?h nodes and derived malignancies. It is negative on T-cells, myeloid cells, and other human tissues studied to date. Lym~l recognizes a 31,32,33 and 35 fciJLQdalton cell membrane protein in R,a.ji cells. 5» Testing; The production of Lym-1 antibody by the hybridoma cells can be tested by indirect immunofluorescence on viable cells or 2% para.formaldeh.yde fixed Βτ-cell . lines , such as Raji, or by immunoperoxidase stai.ning on frozen sections of human lymph nodes.

The Lym-1 hybridoma may be propagated in vitro at an in-itial cell concentration of 2 x 10 cells/ml in RPMI-1640 medium containing 15% fetal calf serum, 100 units/ml penicillin-G, and 100 pg/ml streptomycin sulfate. The cells are grown in stationary suspension culture at 37°C in a well-humidified 5% CC>2 incubator and are transferred every 3-4 days.

Using the culturing procedure described above, the Lym-1 antibody may also be produced. The antibody is obtained by centrifuging the cell culture medium at 1,000 rpm for 10 minutes at 4°C to pellet the cells. The supernatant, which contains approximately 10 ng/ml of IgG2a monoclonal antibody, is then frozen at -20 °C in small aliquots for use in/the immuno- r fluorescence and immunoperoxidase procedures.

To obtain larger yields of higher concentrations Lym-1 antibody for the radioimmunolocalization studies, the hybridoma may be injected into BALB/c mice. The injected hybridoma will cause the formation of antibody-producing tumors after a suitable incuba,ti,on 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 Lymr-1 antibod is recovered from the mice by removing the ascites fluid with a needle and syringe. The ascites ia then spun a 1 , OQQ rpm for 15 minutes at 4QC to pellet the cells and the supernatant is filtered sequentially through, a, 0,8 -micron And a Q,22 micron filture units to remove residua,! debria, Using sterile technique, the filtered ascites is then stored at -8Q°C for long-term stability. From this preparation, approximately 2-3 mg/ ml of lgG2a can be recovered and purified by standard methods. Literature references describing the foregoing procedures are: Hoogenraad, N., Helman, T., and Hoogenraad, J.: J. Immunol . Methods, 61:317-320, 1983; Goding, J.W., J. Immunol. Methods, 39:285-308, 1980, EXPERIMENTAL 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.

Materials and Methods Antigen preparation. Nuclei from the Raji cell line were prepared. 2 ml of packed cells were thawed and washed once with C/PIPES buffer (0.01 M CaCl2, 2 x 10~3 M piperazine-Ν, ' -bis ( 2-ethanesulfonic acid) in a 5Q ml centrifuge tube, The sediment was then resuspended in 40 ml of Ca/PIPES buffer and thoroughly homogenized by using a motor-driven Teflon pestle to disrupt the swollen cells, The n celi were then sedimented and resuspended in Ca/PIPES buffer containing 1% Npnidet P-4Q, The nuclei were then rehompgenized and checked by phase contrast microscopy to be free of contaminating cytoplasmic and membranous debris. Nuclei were then washed twice in Ca/PIPES buffer to remove the, detergents, resuspenced in 3,0 ml of PBS , and sonicated three times for 15-sec intervals to produce a, more homogeneous suspension. The nuclei preparations were then frozen in 1-ml aliquots at -85°C u til use.

Immunization protocol,, A aliquqt of the nuclear preparation was th w , resonicated to reduce viscosity, and emulsified in 1,5 ml of complete. Freund's adjuvant (Difco Laboratories, Detroit, Michigan) by using two glass syringes and a 20-gauge microemulsifying needle (Bolab) . 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.

Cell fusion and cloning procedures. Spleen cells were fused with 8-azaguanine-resistant mouse myeloma NS-1 cells at a ratio of 5:1, respectively, by using 40% polyethylene glycol 1540 m.w. as described by de St, Groth and Scheidegger, J , Immunol , Methods, 35 -,l, 1980, Culture supernatants from wells with active ceil grpwth were tested by indirect immunofluores-cence with fixed cell preparations as described below. Positive cultures were cloned twice on 0.5% Noble agar containing RPMI 1640 medium, 20% fetal calf serum, and antibiotics, as described by Epstein and Kaplan, Cancer Res. 39:1748, 1979.

Serologjc chaffacterjzation of monoclonal antibody isotypes. Hybridoma supematanta from 4-day cultures v/ere concentrated 1Q to 20x in B15 mi.nicon concentrators (Amicqn, Lexington, MA) and tested in double diffusion Ouchterlony plates against rabbit anti-mouse immuno lobulin heavy ch i specific ant sera. The precipitin bands we e read after 2 to 3 days of incubation in a well-humidified 37 °C incubator.

Live cell indirect immuno luorescence. Cells were washed twice with PBS (0.2 g KH2P04, 0.1 g CaCl2,2H2), 1.15 g a^HPA^ 0.1 g MgCl2.5H20, 0.2 g Cl, 8.Q g NaCl/liter) containing 1 mg/ ml bovine serum albumin (BSA: RIA 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 4°C. 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 PBS, pH 8,0 and 2% paraformaldehyde (#4018, Polysciences , Warrington, PA), were added to each tube. The cells were mounted onto a glass slide and examined within 24 hours by epi.flourescence microscopy with a Leitz orthoplan microscope wit a ploemopak 2,1 fluorescence illuminator, HBO 100 mercury lamp, and 5Ox 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 stain- ing of each cell lin.e, Fixed cell ndirect iinmuno luorescence . To examine cells for the presence of intracellular antigens:, ffcxed cell preparations were used. Cells were washed twice wit PB containing 1 mg/ml BSA and Q.Q.2% sodium azide and were pipetted dropwise at a concentration of 5,0 x 1Q6 cells/ml onto Teflon-coated printed microscope slides containing 10 5-mm wells/slide. After the 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 min at room temperature. After fixation, the slides were rinsed in PBS and palced 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 0.02% sodium azide.

For the immunofluorescence assay, 35 μΐ of hybridoma supernatant were pipetted onto each well of the printed microscope slide preparations. After 60 min of incubation at 37°C in a humidified chamber, the slides were rinsed three times in PBS and again incubated for 3Q min at 37PC with. 20. jjl of a 1/2Q dilution of fluorescein conjugated goat a.ntimouse IgG F(.ab')2 fragment specific. The slides were then rinsed three times in PBS , counterstained with Evans blue, fo-t- 5 jnin at room temperature by using a freshly prepared solution containing 50 yl of 1% stock solution of Evans blue in 80 ml of PBS, rinsed a final time in PBS, and mounted with coverslips by using a 1:1 solu- -IQ-tion of glycerpl a d PBS, pH 8.0. ljTununoperoxi;daae staining , Frozen, sections we e r a ed from human tissue biopsies obtained from th.e. Section of Surgical Pathology, Northwestern Memorial Hospital, from specimens submitted from pathologic diagnosis. The section were stained with the monoclonal antibody Lym-l by using the avidin-biotin complex immunoperoxidase staining procedure as. described by Hsu, et al., J. Histochem. Cytochem. 29; 5.77-580, 1981. For these experiments a 1/2 dilution of Lym-l supernatant was used. As a negative control, NS-1 supernatant which is unreactive in frozen sections was used with each run.

Radioimmunoassay for the Detection of Shed Antigen. In order to determine if the antigen detected by Lym-l is shed from the surface of human lymphoma cells in vitro and in patients, a sandwich solid phase radioimmunoassay was developed, In this assay, purified Lym-l is attached to plastic immulon walls by incubation for 1 hr at room temp. After removing excess antibody by washing with PBS, the wells are further incubated for 30 min at room temperature with PBS containing 10 mg/ml bovine serum albumin to block the surface of the immulon wells from further protein binding, After removing the excess BSA, concentrated supernatant from cultured Raji cells,. Raji membrane preparations (positive control) , or human ser m from lymphoma patients were incubated for 30 minutes at room temp. After washing, 1Q0,Q00 cpm of I-125-Lym-l was added to determine the amount of bound antigen. After a 30 minute incubation period and thorough washing with PBS, the wells were counted in gam- -lima counter Antige Modulation Studies. Antigenic modulation after Lym-1 binding was, determined by indirect immunofluorescence procedures as described by Ritz et al. (Bloo , 58:141-152, 1981).

Lym-1 Purification. A summary of Lym-1 purifi.ca.tion is described as follows: 1. raise ascites in pristane primed BALB/c mice. 2. harvest ascites aseptically from peritoneal cavity, 3. remove cells by centrifugation (1,500 rpm for 20 min). 4. filter to sterilize and remove debris (Ό.2 microns). 5. 50% ammonium sulfate precipitation. 6. dialysis against PBS overnight at 4°C. 7. affinity purification on Protein-A sepharose. Eluate at pH 5.6-5.7. 8. dialyze against PBS overnight at 4°C. 9. ultracentrifuge at 30,000 rpm for 1 hr at 4°C. 10. membrane filter (0.2 microns). 11. store in aliquots at -80°C until use, F(ab) and F(ab')2 fragments of the above preparation were prepared by papain and pepsin digestions, respectively, using standard procedures.

Radioiodination Procedures, For the animal studies, purified Lym-1 was radiolabeled by a solid phase system us.ing lo-dogen (Pierce Chemical, Rockford, IL)", In a typical reaction, Iodogen (0.5 - 2.0 g) was plated into test tubes using methylene chloride. A mixture of Lym-1 (0.1 - 1.0 mg/ml) and 1-131 sodium iodid C.5Qr50O yCi). was added to the tubes containing lodogen and incubated at 4°c for 15 in. The reaction was terminated by decanting the solution, Th radiolabeled Lym-was purified using gel exclusion chromatography with Biogel P-10, 50-500 mesh (Bio-Rad Labs, Richmond, CA) , For the patient studies, Lym-1 and F b} and F('ab')2 fragments were radiolabeled with 1-123 using a modified chloramine T method (Mills, S.L., DeNardo, S.J., DeNardo, G.L , , Schlom, J., Epstein, A.L., and Lagunas-Solar , M. , J. Nuclear Medicine, submitted) . I-123-Lym-l was evaluated by cellulose acetate (pH 8.6) electrophoresis and by HPLC-TSK 3000 chromatography.

Lym-1 Binding Assay. The radioimmunoreactivity of the radiolabeled monoclonal antibody was determined using a live cell assay consisting of 1 x 106 Raji cells, CEM cells from a T-cell acute lymphoblastic leukemia cell line were used to assess the extent of non-specific binding. Purified radiolabeled Lym-1 was added to 1 ml of the respective cell suspension at antigen excess and incubated at room temeprature for 60 minutes. The cell suspension was then washed 3x with PBS and counted using appropriate gamma scintillation spectrometry to assess the degree of binding, Radjoimaging of Tumor-Bearing Mice, Athymic nude mice, each bearing a right thigh human lymphoma induced by- an intramuscular 7 injection of 1 x 10 Raji cells were given Lugols solution orally 24 hr prior to initiation of the study. Between 150-400 Ci of I-131-Lym-l was then injected intravenousl into the hetero- transplanted nude m ce. Posterior gamma scintillation images (.100,00.0 counts! were obtained up to 7 days after injection using a gamma scintillation camera, with; a pinhole collimeter interfaced to a computer system. Immediately after animal imaging, an appropriate 1-131 standard s counted with the same geometry in order to guantitate the animal data. At 7 days after injection, the animals were sacrificed and organ biodistribution studies were performed using appropriate gamma scintillation counting, Radioimaging of Cancer Patients. Volunteer breast cancer and lymphoma patients were injected with 1-5 Ci of 1-123 labeled Lym-1 or fragments. Serum sample obtained at 5 min, 30 min, 1 hr, 2 hr, 6 hr, 18 hr, 24 hr, and 48 hr post-injection were evaluated by HPLC-TSK 3000 in order to assess the size of the circulating radiolabeled molecules. Urine samples were also taken to determine the total 1-123 excreted over 24, 48 and 74 hr, post-injection. On selected urine samples, HPLC-TSK 3000 analysis was performed to examine the size of the 1-123 molecules being excreted. Planar images of the head, chest, and anterior and posterior abdomen were obtained for 750,000 counts with a medium energy coliimeter at 0.2 hrs, 4-6 hr, and 18-24 hr post-injection. Single photon emission tomographic, images (SPECT). were obtained at 2-4 hr and 24 hr post-injection over the chest and abdomen (64 views on a 128 x 129 matrix) . Computer reconstruction and attenuation corrections were performed on these images and uptake was compared to 1-123 calibration pin phantom (Macey et al., J.' Nuclear Med. 25:105, 1984).

NMR Enhancement, Studies, In collaboration with Dr, Michael McNamara at the University of California at San Francisco WR Research Institute, Lym-1 has been conjugated to gadolinium using anhydrous DTPA. Raji lymphoma implants in nude mice were used to study the effects of gadolinium conjugated Lym-1 on NMR relaxation times and image enhancement, Immunotoxin Conjugation. In collaboration with Clin Midy/ Sanofi in Montpellier, France, Dr, Guy Laurent has conjugated purified Lym-1 with ricin toxin A chains to determine its cytotoxic effects on three target cell lines (Raji, Daudi, and SB) . Using a protein synthesis inhibition assay, the IC50 (amount of A chain to obtain 50% inhibition of leucine incorporation) was measured in order to determine the potential effectiveness of this immunotoxin.

RESULTS Hybridoma clone Lym-1 was produced by the fusion of mouse myeloma NS-1 cells and BALB/c splenocytes obtained from a mouse hyperimmunized with nuclei from Raji cells. Isotypic analysis revealed that monoclonal antibody Lym-1 was of the IgG2a heavy chain subclass. The Lym-1 antibodies were identified by indirect immunofluorescence techniques with the use of paraformal-dehyde-aceton-iixed cell preparations, The reactivities of monoclonal antibody Lym-1 on established human malignant lymphoma, and leukemia cell lines are shown in Tables I and II, respectively.

TABLE I Reactivity of Lytn-1 with human malignant lymphoma cell lines by indirect immunofluorescence Cell Line Lym Burkitt's lymph Raji EB3 RAMOS SU-AmB SU-AmB Diffuse histiocytic lymph SU-DHL-1 SU-DHL-2 SU-DHL-4 SU-DHL-6 SU-DHL-7 SU-DHL-8 SU-DHL-9 NU-DHL-1 U-937 Undifferentiated lymphoma MU-DUL-1 a Fixed cell indirect immunofluorescence assay, b Data expressed as (-) negative, (+) positive.

TABLE II Reactivity of Lym-1 with human leukemia and lympho- blastoid cell lines by indirect immunofluroescence Cell Line Lym-la Acute lymphoblastic leukemia T Cell b Molt-4 CE HSB-2 HPB-ALL JM Null cell REH + ALL-1 KM-3 B-cell BALM-2 + NALM-1 (pre-B from CML) Myeloid leukemia K562 (erythroid-CML) HL-60 (promyelocytic) ML-2 (myeloid) TPH-1-0 (monocytic) G1 (myeloid) Myeloma U-266 ARH-77 Lymphoblastoid BL-1 MU-LB + NU-LB + a Fixed cell indirect immunofluorescence assay. b Data expressed as (-) negative; (+) positive.

In Table III, the staining reactivity of Lym-1 on human malignant lymphoma and chronic lymphocytic leukemia biopsies is shown. Indirect immunofluorescence studies showed that Lym-1 was positive on the majority of B-cell derived tumors.

TABLE III .

Indirect immunofluorescence staining of human lymphoma and chronic lymphocytic leukemia biopsy cells Lym-1 Reactivity Diagnosis (positive cases/total cases) Lymphoma3 (frozen sections of lymph node biopsies) well-differentiated lymphocytic 1/3 poorly-differen iated lymphocytic 1/5 mixed lymphocytic and histiocytic 8/9 histiocytic (B-cell type) 12/17 T-cell 0/2 Leukemia (cytospins of peripheral blood) Chronic lymphocytic B-cell type 4/10 T-cell type 0/5 a Rappaport classification.

The immunoperoxidase staining reactivity of Lym-1 on frozen sections of normal human biopsy tissues is shown in Table IV. Lym-1 was found to be specific to B-cell lymphocytes and histiocytes in lymphoid tissues. No reactivity was demonstrated in human bone marrow or in non-lymphoid human organs.

TABLE IV Reactivity of Lytn-1 with normal human tissues Tissue Lym-l Reactivity lymph node + B cell zones tonsil + B cell zones thymus + medullary dendritic cells bone marrow* blood* + subset of, E lymphocytes adrenal brain breast colon + surface epithelium, macrophages hear liver lung pancreas salivary gland skin (macrophages positive) skeletal muscle smooth muscle thyroid ^Determinations made by flow cytometric analysis on viable cells in suspension.

The immunoreactivity of Lym-1 on human solid tumor cell lines v/as determined by indirect immunofluorescence techniques on fixed cell preparations. As shown in Table V, Lym-1 was not found reactive on the cell surface of any of the 26 cell lines tested, but did have weak reactivity on a small number of cell lines in the cytoplasm or nucleus.

TABLE V Immunoreactivity of Lyra-l with human solid tumor cell lines Solid Tumors Lym-1 Reactivity CaCL-74-36 (melanoma) BM-166 (neuroblastoma) Y79 (retinoblastoma) HeLa (ovarian Carcinoma) SU-CGS-1 (clear Cell sarcoma) Colo 38 (melanoma) C-399 (colon carcinoma) A-172 (glioblastoma) NCI-H69 (small cell carcinoma of lung) IMR-5 (neuroblastoma) Hutu-80 (colon carcinoma) HT-29 (colon carcinoma) 734B (breast carcinoma) S -80 (rhabdomyosarcoma) SW-1503 (metothelioma) SW-733 (papillary carcinoma of bladder) + weak cytoplasmic speckling U118-MG (glioblastoma) + sparse cytoplasmic speckling SW-872 (liposarcoma) + discrete nuclear speckling SW-780 (transitional cell carcinoma of bladder) SW-1045 (synovial cell carcinoma) SK-608 (astrocytoma) - cytoplasmic speckling SVJ-1353 (chondrosarcoma) SW-451 (squamous cell carcinoma of + nuclear specklesophagus) ing SW-156 (hypernephroma) HU-04 (glioblastoma) + nuclear speckling SW-579 (squamous cell carcinoma of thyroid) -megative; +:positive by indirect immunofluorescence microscopy on fixed cell preparations.

Table VI below summarizes the major characteristics of Lym-1 antibody.

TABLE VI Characterization of Monoclonal Antibody Lym-1 Lym-1 Immunogen Raji nuclei Isotype IgG2a Antigen 31,32,33,35 kilodalton protein Antigen site cell surface Lymphoid Reactivity lymph node and tonsil B-cell zones and histioc bone marrow no e blood subset of E lymphocytes thymus medullary dendritic cell on-Lymphoid Reactivity none Tumor specificity B-cell lymphomas and leu kemias Antigenic modulation experiments using Raji cells showed that the antigen recognized by Lym-1 does not modulate on the cell surface but is stably expressed for several days. A sandwich radioimmunoassay technique used to determine if the antigen detected by Lym-1 was shed from the surface of human lymphoma cells either in vitro or in patients clearly gave negative results as shown in Table VII.

TABLE VII Detection of Shed Antigen by P.adioimmunoassay 1-125-Lym-l Antigen Source Radioimmunoassay Binding (cpm)* Raji cells (1x10°) live cells 51,582 Raji membranes solid phase, direct 6,060 Raji membranes solid phase, sandwich 19,274 Raji supernatant solid phase, sandwich 619 (20x cone.) Lymphoma patient sera #1 solid phase, sandwich 115 #2 " " " 111 #3 " " " 168 #4 " " " 385 #5 fi " " " 269 CEM cells (1x10 ) live cell 214 * 1x10 cpm/test Animal radioimaging studies with Lym-1 shoed that Lym-1 bound specifically and rapidly to the Raji tumor implant with essentially no binding seen to normal mouse organs. Biodistri-bution studies at day 7 with 1-131-Lym-l antibody is shown below in Table VIII.

TABLE VIII Biodistribution of I-131-Lym-l in heterotransplanted nude mouse organ " injected dose % inj ected dose gram blood 1.441 heart 0.048 0.355 lung 0.088 0.335 liver 0.403 0.296 spleen 0.044 0.720 kidney 0.159 0.382 tumor 8.216 3.191 Patient Radloimaging Studies In studies performed over the last 20 months in collaboration with Dr. Sally DeNardo at the University of California at Davis, 5 breast cancer patients (negative controls) and 25 lymphoma patients have been imaged using planar films and SPECT in strumentation after injection of I-123-Lym-l radioconjugate (1-5 mCi) . Images of the lymphoma lesions were successfully ob tained in all 25 lymphoma patients in 4 hr and 18 hr postinjec-tion. In the 5 breast cancer patients and in all lymphoma patients, binding of I-123-Lym-l to normal tissues was not seen. Metastatic tumors identified by biopsy, by NMR or CAT scan, or radiographically were successfully imaged with I-123-Lym-l. Unsuspected tumor, such as those in the bone marrow or occult locations were seen in some patients. HPLC analysis showed that I-123-Lym-l remained unbound and free in the patient circulation conclusively showing the absence of circulating antigen. The antibody was principally secreted as F(ab) fragments by the kidney. Non-specific uptake of I-123-Lym-l by the liver could be avoided by preloading the patients with 5 mg of cold Lym-1 prior to the injection of radiolabeled antibody. Whole antibody as well as F(ab) and F(.ab')2 fragments conjugated to 1-123 were used successfully to image lymphoma lesions in these patients. No anti-mouse immune response and no adverse side effects were noted in any of the patients, even those receiving multiple injections over a several months period.

NMR Studies Gadolinium conjugated Lym-1 was found to shorten the NMR relaxation times and hence produce enhanced NMR images in Raji implants in nude mice 3 days after injection. This is the first successful demonstration of NMR enhancement of tumors using spin label conjugated monoclonal antibodies. After gadolinium conjugation with DPTA, over 90% of the antibody reactivity was retained as shown by the live cell binding assay.

Immunotoxin Studies A 1C60 of 10 ^ was obtained with ricin toxin A chain conjugated to Lym-1. By comparison, an anti-HLA-Dr antibody-ricin A chain conjugate had a 2 log higher 1C50. The lower 1C50 of Lym-1 may be due to the fact that the immunotoxis is not effectively entering the tumor cell as is shown for antibodies with modulating antigens.

In summary, the advantages of Lym-1 as an iri vivo diagnostic and therapeutic reagent are shown below.

TABLE IX Advantages of Lym-1 as a Radio imaging and Immuno- therapeutic Reagent Antigen 1. small antigen reservoir 2. not shed or modulated 3. expressed on the majority of B-cell lymphomas and leukemias Antibody 1. highly stable after radiolabeling or spin labeling procedures 2. radioimages lymphomas but not normal lymphoid organs 3. radiolabeled antibody is excreted by kidney 4. does not complex in serum of patients by HPLC analysis 5. is retained by lymphomas for long periods of time (7 days in mouse model) 6. can be used to shorten NMR relaxation times and give tumor enhancement by NMR 7. can be used theoretically to deliver a therapeutic dose of radiation to tumor tissue without significant toxicity to normal organs or tissues 8. does not appear to produce an anti-mouse response in lymphoma patients 9. does not produce toxic effects in patients - 25 - 84466/2

Claims (5)

1. The hybridoma cell line Lym-1 (ATCC No. HB 8612) and clones thereof.

2. A method for producing murine IgG2a Lym-1 antibodies, comprising culturing hybridoma cells of the Lym-1 cell line (ATCC No. HB 8612) or clones thereof, and recovering the Lym-1 antibodies produced.

3. A method for producing murine IgG2a Lym-1 antibodies, according to claim 2, comprising culturing in a suitable medium hybridoma cells of the Lym-1 cell line (ATCC No. HB 8612) or clones thereof, harvesting the supernatant, and recovering the Lym-1 antibodies therefrom.

4. A method for producing murine IgG2a Lym-1 antibodies, according to claim 2, comprising injecting hybridoma cells of the Lym-1 cell line (ATCC No. HB 8612) or clones thereof into BALB/c mice , removing the ascites fluid, and recovering the Lym-1 antibodies therefrom. •

5. The murine IgG2a Lym-1 monoclonal antibody produced by the hybridoma cell line Lym-1 (ATCC No. HB 8612) and clones thereof. FOR THE APPLICANT: Dr. Yitzhak Hess