EP1090107A1

EP1090107A1 - Human monoclonal antibodies against the tumor antigen uk114 and lymphocyte cells and hybridomas for their production

Info

Publication number: EP1090107A1
Application number: EP99931141A
Authority: EP
Inventors: Alberto Bartorelli; Gianni Bussolati; Ada Funaro
Original assignee: Zetesis SpA
Current assignee: Zetesis SpA
Priority date: 1998-06-26
Filing date: 1999-06-23
Publication date: 2001-04-11
Also published as: IT1301815B1; JP2002519020A; WO2000000591A1; ITMI981467A1; AU4775899A; CA2331762A1

Abstract

Human monoclonal antibodies against the tumor antigen UK114 and use thereof in diagnostic and therapeutic applications.

Description

HUMAN MONOCLONAL ANTIBODIES AGAINST THE TUMOR ANTIGEN UK114 AND LYMPHOCYTE CELLS AND HYBRIDOMAS FOR THEIR PRODUCTION

The present invention refers to human monoclonal antibodies against the tumor antigen UKl 14, to immortalized lymphocytes and to hybridomas secreting said antibodies.

A new antigen of 14 kDa has been recently purified from the perchloric acid-soluble f action of goat liver (Bartorelli, A. et al., J. Tumor

Marker Oncology, 9:37, 1994). The primary structure of the UKl 14 protein

(Ceciliani, F., et al., FEBS, 1996) shows high sequence similarities with the protein family defined as YER057c/YJGF (Schmiedeknecht, G., et al., Eur.

J. Biochem. 242:339, 1996). The UKl 14 protein is localized in the cytoplasm of normal cells while it is apparently expressed on cell membrane after neoplastic transformation acquiring the characteristics of tumor- associated antigen (Bartorelli, A. et al., Int. J. Oncol. 8:543, 1996).

Antibodies specific for the UKl 14 protein have been isolated from the sera of cancer patients and in many cases the specific antibody titer turned out to be significantly increased in patients treated with UKl 01 , the drug containing as the active component the heterologous UKl 14 protein (Bartorelli, A., et al., J. Tumor

Marker Oncology 9:57, 1994). Said antibodies react with a membrane tumor antigen and elicit complement-dependent cytolysis in vitro and in urine models.

For the preparation of specific immunocompetent cells secreting the monoclonal antibodies of the invention, samples of peripheral blood from cancer patients treated with UK101 , responsive to said treatment, were used.

In this way, it has been possible to isolate cells secreting the desired monoclonal antibodies with high efficiency, as detailed hereinbelow. The process used for the preparation of said antibodies is the following: blood samples were obtained from patients affected by carcinomas unresponsive to chemotherapy and successfully subjected to repeated treatments with UKl 01 in the context of a trial authorized by the Italian Ministry of Health. Peripheral patients' lymphocytes enriched in the B population were immortalized with EBV. Eight cell lines have been selected out from the numerous polyclonal lines according to the reactivity with purified UKl 14 protein and were cloned. The positive clones were expanded and assayed repeatedly to avoid the frequent loss of secreting activity described for all the EBV-transformed lines. The antibodies of interest have been analyzed on a panel of tumor cell-lines of different origin and of normal cells so as to evaluate the selective recognition of the tumor-associated antigen. The human antibodies, in addition to the purified native protein, all recognized a membrane antigen expressed on gastric tumors (HT-29, Kato III), colon tumors (Colo 684, LoVo), breast tumors (MCF-7, MDA), and on some neuroblastomas (LAN-1, SY5Y) as well as on breast and lung tumors from patients subjected to surgery. In contrast, - they consistently displayed no reactivity with cells of hematopoietic origin (Molt-4, HPB-AII, HI-60, K562), with normal cells (peripheral lymphocytes, fibroblasts, Chang cells) and with normal cells surrounding the neoplasia as shown by cytofluorimetric analysis. A first embodiment of the invention refers therefore to the human monoclonal antibodies directed against the UKl 14 protein.

A second embodiment of the invention refers to immortalized lymphocyte cells secreting the monoclonal antibodies against UKl 14.

In addition to immortalized cells, particularly EBV-immortalized cells, having the drawbacks of instability and low yield in produced antibody, human hybridomas were obtained. To this purpose, three EBV lines, having the best growth and secreting capacity, were selected.

However, even though the somatic fusion techniques in the murine model is widely known, it is presently available only a very low number of stabilized human plasmacytomas, none of which being a good partner of somatic fusion for the generation of human-human hybridomas. Better results were obtained by the use of heteromyelomas derived from the fusion of a human cell with a murine myeloma.

Said lines yield generally more stable hybridomas, able to better grow in nude mice in form of ascitic tumor. Three EBV lines selected on the basis of their growing characteristics were fused with the hetero-myeloma K6H6 (Carrol, WL, et al., J. Immunol. Meth. 89:61-69, 1986) obtainable from ATCC and the obtained clones were analyzed for reactivity against the UKl 14 antigen by an ELISA method. The fusion system was very efficient and more than 70% of the obtained clones were reactive. The selected hybridomas were expanded in vitro and cloned by limit dilution. The final result was the selection of three hybridomas: UK#l/2G3hy, UK#l/lAl lhy and UK#l/3D8hy. The first two hybridomas are of IgM-k isotype and the third of IgM- λ. The hybridomas maintained the tumor specificity characteristic of the parental lines, as shown in the following Table:

TABLE

Cells and cell lines UK#l/2G3hy UK#l/l/3D8hy UK#l/lAllhy

KatoIII 50%+ 75%+ 23% +

(gastric carcinoma)

HT-29 63%+ 40%+ 84% +

(colon carcinoma)

Colo-684 47%+ 44%+ 42% +

(colon carcinoma)

LoVo 89%+ 78%+ 48% +

(colon carcinoma)

MCF-7 61%+ 53%+ 56% +

(breast carcinoma)

MCF-10 (non-malignant breast)

MOG-G-UVW 87%+ 86%+ 71% +

(astrocytoma)

LAN-1 90%+ 82%+ 86% +

(neuroblqstoma)

SY5Y 10%+ 20%+ 22% +

(neuroblastoma)

Chang -

(liver cells)

Supt-1

(T cell leukemia)

Molt-4

(T cell leukemia)

K562 (erythroleukemia)

HL-60

(promyelocytic leukemia) The reactivity of human monoclonal antibodies on the different tumor lines also showed high differences in terms of number of positive cells and of fluorescence intensity thereby suggesting that different epitopes, recognized by the tree reagents, exist on the target antigen expressed on the membrane, as showed in figure. The three hybridomas have been adapted to grow in vivo in nulnu mice and so are able to grow as ascites tumor, and this allows to increase the specific immunoglobulin concentration of some order of magnitude. Then, the hybridomas have been adapted to grow in synthetic serum-free medium: a main stage to purify antibody eventually destined to clinical use.

The lytic capacity that the UK#l/2G3hy and UK#l/lAl lhy are able to exert on tumor targets in the presence of human complement (enclosed figure) is a very promising characteristic for possible clinical uses.

Therefore, a further object of the invention refers to human hybridomas that are able to secrete anti UKl 14 human monoclonal antibodies. One of such hybridomas (UK#1/3D8) was deposited at ECACC (European Collection of Cell Cultures) under n° 9706 2409 on June 24^th, 1997.

The human antibodies herein described are an optimal clinic/diagnostic tool: first of aspect at present all, they permit to test the human antibody answers to the UKl 01 treatment, at present unknown and different from what is reproducible in the animal models; secondly, the produced reagents could be used in diagnostics as vectors of radioactive isotopes useful in radio-pilot surgery, or in therapy as toxins or drugs vectors.

For all the applications mentioned above, the antibodies of the invention do not cause the undesirable side effects typical of the murine reagents, as they are of human nature. The following example shows in more detail the steps for the preparation of the immortalized cells, of the hybridomas and of the ascitis, as well as the functional characteristics belonging to the lytic power, i) B cell immortalization Human B cells purified from peripheral blood from a carcinoma lung cancer patient successfully treated with UK101 , were enriched by conventional density gradient. The T lymphocytes were removed from the resulting lymphocytic cells by rosetting with sheep erythrocytes. The purified B cells (10⁷ ml) were infected by incubation with Epstain Barr virus (EBV) obtained from the B95--8 cell line (10% in the colture medium). After one night of incubation at 37°C, the cells were centrifuged, washed twice with culture medium and plated (along allogenic lymphocytes irradiated-2000 rads) at a concentration of 5000/well in Iscove medium containing 10% FCS. ii) Selection of specific clones for UKl 01 After 20 days of colture the obtained clones were tested for their capacity, to produce specific immunoglobulins for the protein UKl 14 by enzymatic test on plates pre-coated with the purified protein (Bartorelli, A., et al., M. J. Tumor Marker Oncology 1 1 :67, 1996). The reactive clones were expanded in vitro and the supernatants were used to determine the isotype and to study the distribution in immunofluorescence. iii) Human hybridoma production

Among the obtained lymphoblastoid cell lines, three were selected, on the basis of the best growth and secretion characteristics, to be used as partner in the somatic fusion with the myeloma. Since the availability of suitable human myeloma as tumor partners in the cell fusion is very scarce and the products resulting are also very unstable, an hetero-myeloma cell line (human- mouse) provided from ATCC able to fuse with high efficiency and with a better products stability was selected. The used cell line (K6H6/B5) was obtained from the fusion of a murine myeloma with normal human B lymphocytes and selected for being non-secreting and aminopterin-sensitive. The EBV cell lines were fused, in a 2: 1 ratio, with the K6H6/B5 line using polyethylene glycol (pH 7) as promoter agent following a conventional technique. After the fusion the cells were plated on irradiated allogenic lymphocytes constituting the support cells. After 24 hours the selectors were added in the following ratio: hypoxanthine l OOμM, aminopterin 800nM, thymidine 15 μM, ouabain 0.1 μM. The specificity of the obtained clones was evaluated with enzymatic assay, as described (Bartorelli, A., Biancardi, C, Cavalca, V., Clemente, C, Ferrara, R., Arzani, C, Bailo, M., M.J. Tumor Marker Oncology 1 1 :67, 1996). iv) Cytofluorimetric analysis The cells of the different cell lines analyzed (2x10⁵) were resuspended in phosphate-buffered saline and incubated for one hour at 4°C with the examined antibody. After two washes, the cells were incubated at 4°C for 30 minutes with fluorescein labeled anti-human Ig. Fluorescence was analyzed by means of a FACSort ( using as software Lysis II). Selected experiments were performed with an analogous process on cells from fresh tumor, obtained from surgical samples, v) Hybridoma growth in nude mouse

2x10 cells of each hybridoma were resuspended in 200 μl of physiological solution and inoculated in the peritoneal cavities of nude mice, previously inoculated with 0,5 ml of pristane. After 15-18 days, 2-4 ml of ascites were taken from each mouse. vi) Complement-dependent cytotoxicity

KATO III (gastric carcinoma), MOG-G-UVW (astrocytoma) and HT-29 (colon carcinoma) selected as tumor target, were labeled with 3,7 MBq of Na₂ [⁵¹ C_R]0₄ for 1 hour at 37°C. After repeated washes the cells were incubated at 37°C for 1 ,5 hour in the presence of the human antibodies anti-UK114 (ascites diluted 1 : 10) and of 10 μl of human serum. As controls of the dependence of the lytic activity from the complement, human serum deprived of C8 C9 factors, or heat-inactivated, were used.

Claims

1. Human monoclonal antibodies against UKl 14 tumor antigen.

2. Immortalized lymphocytic cells able to produce the antibodies, according to claim 1.

3. Cells according to claim 2, immortalized with EBV virus.

4. Hybridomas according to claim 1 , secreting monoclonal antibodies.

5. Hybridoma according to claim 4, deposited under No. 97062409 on June 24th, 1997.

6. A method for the preparation of the antibodies according to claim 1 , comprising: the preparation of lymphocytic cells samples from patients responsive to the treatment with UK101 ; the immortalization of such cells and the selection of the lines producing antibodies; the fusion of such cells with heteromyelomas from the fusion of a human cell with a murine myeloma; the selection of active clones.

7. The use of the antibodies according to claim 1, for the preparation of reagents, diagnostics, therapeutic agents, said antibodies being optionally bound/combined with radioactive isotope, toxins or drugs.