AU594305B2 - Monoclonal antibodies, a process for their preparation and their use - Google Patents

Abstract

New antigens (AG 1-4) with at least one common epitope, and monoclonal antibodies recognising these antigens, are claimed. AG1 is a protein antigen with a molecular wt. of 72,000 +- 3000 under reducing and non-reducing conditions. AG2 is a glycoprotein antigen with a molecular wt. of more than 200,000 under non-reducing conditions, and is serologically not identical to F VIIIR:AG. AG3 is a complex of three glycoprotein antigens with molecular wts. of 180,000 +- 10,000, 95,000 +- 10,000 and 55,000 +- 10,000 under non-reducing conditions. AG 4 is a glycoprotein antigen with a molecular wt. of more than 200,000 under non-reducing conditions, and is not identical to EMA.

Description

~444 5943 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Application Number: Lodged: Int. Class Complete Specification Lodged: Accepted: Published: Priority: SFejqlated Art: i 0 This document contais tlhe amendmes i. mde or i Section 49 and is correct L i nig dle d I,:tc t «J+ Q 201J Na'ae of Applicant: BEHRINGWERKE AKTIENGESELIS CHAFT Addressof Applicant: D-3550 Marburg 1, Federal Republic of Germany

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Actual Inventor: 4i f Si I o S A~llress for Service KLAUS BOSSLET and HANS-HARALD SEDLACEK EDWD. WATERS SONS, QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

r Complete Specification for the invention entitled: MONOCLONAL ANTIBODIES,A PROCESS FOR THEIR PREPARATION AND THEIR USE The following statement is a full description of this invention, including the best method of performing it known to US 1 i-I 4 4 2 HOE 84/8 009 The invention relates to monoclonal antibodies (MAB) which bind to defined cytoplasmic, membrane-associated and secreted antigens. These antibodies can be used as a diagnostic aid or as an active compound or carrier of 'active compounds.

The technique of immunization using defined, isolated antigens, and the production of antibodies against such antigens, is known. It is also known to immunize using unpurified antigenic material and to select those antibodies which recognise a particular component in such a mixture of antigens.

In the attempt to induce such antibodies, we have succeeded in selecting monoclonal antibodies which react, 6 under non-reducing or reducing conditions, with the follow- 15 ing protein antigens or particular epitopes on these proe tein antigens.

Antigen 1: A protein with a molecular weight (MW) of 72 3 Kilo-Daltons (KD) under non-reducing conditions or under reducing conditions.

Antigen 2: A glycoprotein complex of MW 200 KD under non-reducing conditions.

SAntigen 3: Three glycoproteins of MW 180 10 KD, MW 10 KD and 55 10 KD under non-reducing conditions or under reducing conditions.

S 25 Antigen 4: A glycoprotein complex of MW 200 KD under non-reducing conditions.

Antigen 1 carries an epitope which, on the cell membrane of the Oat-75 small-cell lung carcinoma cell line (Cancer Research (1977), 37, 3088-3095) and on the tumors derived from Oat-75 and transplanted onto the nude mouse, is accessible to the MAB 278/97 of the IgG 1 isotype, that is to say the MAB binds to the native cell line, or the corresponding transplanted tumor tissue, carrying the epitope. It has not been possible to detect the epitope either on another small-cell Lung carcinoma Id -:l r r 1

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04* 40* I £e C 3 cell line (SHP-77) or on 6 other non-small-celL lung carcinoma cell lines (CaLu-1, E-14, B109, A549, ChaGo, Bro-CaHof; ATCC lines). Moreover, it has not been possible to detect the epitope on 5 pancreatic tumor cell lines (PaTu-I, II, IIL, MIA Pa-Ca-2, PANC-1), 2 gynecological tumor cell lines (HeLa, PA-1), 3 other, nonrelated carcinoma cell lines (MCF-7, MDA-1, Co-Wi), 6 human fibroblast lines, 3 non-human cell lines (Vero, rat fibroblasts, NMRI mouse fibroblasts) or leukocytes from peripheral human blood.

Furthermore, the epitope is undetectabLe on 12 lung carcinomas transplanted onto the nude mouse and 4 transplanted pancreatic tumors as well as 7 lung carcinomas taken directly from the patient, one carcinoma of stomach and one of the colon, 5 normal lung tissues, or one sample in each case of normal tissue from the pancreas, stomach, colon and thyroid.

Following fixation with formaldehyde and embedding in paraffin of the Oat-75 tumor transplant, the epitope, 20 which is detectable on cryopreserved material, is modified such that MAB 278/97 no longer binds.

Antigen 2 carries an epitope which, on the cell membrane of the Oat-75 small-cell lung carcinoma cell line and on the tumors derived from the Oat-75 and transplanted onto the nude mouse, is accessible to MAB 278/105 of the IgG 3 isotype. The epitope can be detected in vessels, for example in cryostat sections as well as on materials from normal and tumor tissue which have been treated with formaldehyde and embedded in paraffin. In 30 this case, it is found preferentially in endothelial cells, but it can also be found in extracellular regions such as, for example, in parts of the basal membrane or the internal elastic lamina. It is undetectable in megakaryocytes, but it is detected in the capillary loop endothelium of the glomeruli (in contrast to F VIIIR:AG). The epithelium in Bowmann's capsule is unreactive. By its nature, it is particularly concentrated in tumors of the vascular system. Moreover, the epitope is detectable in, for example, endothelial cells of human umbilical cords and 4 on the cell membrane (after fixing with glutaraldehyde) as well as in the cytoplasm (punctate components around Sthe nucleus and intracytoplasmic granular distribution).

Furthermore, endothelial cells of human iliac crest veins express the epitope while, in contrast, the epitope is undetectable in endothelial cells of the pulmonary aorta of cattle. The epitope is undetectable on the cell membrane of leukocytes from human peripheral blood. In addi- !tion to the resistance of the epitope in tissue to fixai| 10 tion with formaldehyde and embedding in paraffin, which i was indicated above, it is also resistant to treatment i with neuraminidase but is sensitive to periodic acid oxil dation. On the basis of its properties, MAB 278/105 i appears to be suitable for the diagnosis of tumors derived i 15 from the endothelium and for chronic inflammations.

SAntigen 3 is, because of its strong cross-reaction I with heterologous anticarcinoembryonal antigen antisera, similar to the carcinoembryonal antigen (CEA) which was isolated in 1965 by Gold and Freedman (J.exp.Med. (1965), 122, 467). A relatively large number of epitopes are loca- S ted on this complex glycoprotein, and these also occur on i other molecules which are not identical to CEA. These are the antigens NCA 2 (non-specific crossreacting antigen; J.Immun. III (1973), 1926), NCA 1 (non-specific crossreacting antigen; Ann.N.Y.Acad.Sci. (1975), 259, 389), BGP-I (Int.J.Cancer (1976), 17, 588) and CEAIow (Scand.

J.Immunol. (1978), Vol. 8, Suppl. 8, 423-428).

i Seven new epitopes have been discovered on antigen 3, and some of these also occur on the molecules mentioned immediately above (Table The antigen has been detected by immunoprecipitation in, for example, a colon car- J cinoma cell line (DE-TA) and its molecular weight there is significantly lower than the molecular weights reported in the literature for CEA.

1- Table I MAB CEA CEALow NCA 2

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I II III IV V VI VII The epitopes on the CEA molecules which are recognized by MABs I-VII differ from the epitopes defined, using monoclonal antibodies, in MoL.Immunol. (1982), 19,12, 1641-1648 and European Patent Appl. 0,098,162 A2 (1983) by differences in the distribution on the abovementioned moLecules.

The characteristics of MAB VII (BW 431/31) are described in detail below: MAB BW 431/31 recognizes an allotypical epitope on the CEA (180 KD) purified from human colon, pancreatic, 20 stomach and lung carcinomas. This epitope is resistant Sto fixation with formaldehyde and embedding in paraffin of the tissue used for the investigation. Furthermore, the epitope is resistant to periodate oxidation and treatment with neuraminidase.

The epitope can be detected by immunohistological methods in the following human tissues fixed with formaldehyde and embedded in paraffin: 80-90% of the primary tumors of colon carcinoma and the liver metastases result- S, ing from them show a strong membranaceous reaction which 30 increases toward the lumen. In contrast to this, there is no reaction with normal liver tissue surrounding the metastases. An additional significant finding is that c* there is no significantly detectable reaction by the entire normal colon tissue, including the mucosa. This observation distinguishes MAB BW 431/31 from all the monoclonal antibodies described in the literature, especially from Mab 35 described in Int.J.Cancer (1984) 33, 643, which shows a significantly stronger reaction with the outer mucosa and, additionally, with the intestinal crypts.

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p~ P Ililjr~ ~u rr i~4iL~ua ra~-~ 6 No significant reaction with MAB BW 431/31 is shown by any of the other normal tissues tested, such as normal lung, normal pancreas, inflamed pancreas, normal stomach, normal spleen, tonsils, lymph nodes, and leukocytes from peripheral blood. In addition to the majority of colon carcinomas, about 30-50% of the tested pancreatic carcinomas and about 40-60% of the tested stomach carcinomas showed a marked reaction with BW 431/31.

MAB BW 431/31 is a mouse immunoglobulin of the Y 1 isotype associated with a light chain of the )type. The Fc part of the molecule is able to bind to protein A.

CEA can be isolated in a highly pure form from tissue solubilizates and body fluids using MAB BW 431/31 immobilized on solid carriers.

The CEA thus purified undergoes a change following adsorption onto nitrocellulose, so that the binding of MAB 431/31 is now inefficient. In contrast to this, under identical conditions, on nitrocellulose the epitope on CEA which is recognized by Mab 35 (Int.J.Cancer (1984) 33, 643) remains accessible to Mab 35, so that Mab 35 binds very efficiently. Furthermore, the molecular weight of the MAB BW 431/31 which has been fractionated under non-reducing conditions by SDS-PAGE is significantly below that of Mab (about 7000 KD). This difference in molecular weight of the 25 X-light chain of Mab BW 431/31 as revealed by SDS-PAGE under reducing conditions (MW of C-light chain of Mab BW 431/31 25 KD, of Mab 35 29 KD).

MAB VIII recognizes an epitope which is to be found only on a subpopulation of CEA molecules Antigen 4 carries an epitope which, on the cell membrane of the Pa-Tu-I pancreatic carcinoma cell line and on the tumors of the Pa-Tu-I cell line transplanted onto the nude mouse, is accessible to MAB 406/14 of the IgG 1 isotype (light Kchain). The MAB reacts with pancreatic tumor tissue which has been cryopreserved or fixed with formaldehyde and embedded in paraffin, and with the duct epithelium of the healthy and inflamed pancreas and with colon carcinomas.

The MAB reacts weakly with the alveolar epithelium of normal 0 44 o o 4 0 o 0* r0 o *0 o 4 04 0PPI 040 S.00 0.0 a S* 0 64 o 4 00 4 :r r-.

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0 d ca 24 Uaar~l-l-------~gPIIL ~~DI~1 -r I I r |,r i 7 or with Leukocytes from human peripheral blood. The MAB reacts weakly with the intestinal crypts.

The MAB reacts with not only the duct epithelium of the pancreas, but also the duct epithelium of the liver and of the mammary gland, and in the latter case specifically in the apical part of the epithelial cells.

Furthermore, in the region of the normal mammary gland, the epithelium of the acini and the coating connective tissue cells in the region of the acini are positive.

The mammary sarcomas derived from these mesenchymal cells also have a positive reaction. The coating connective tissue cells represent a mesenchymal component. This reactivity with both mesenchymal and epithelial components distinguishes MAB 406/14 from antibodies which react with the "epithelial membrane antigen" (EMA) (J.Clin.Pathol. (1979) 32, All the mammary carcinomas derived from the duct epithelium (ductal mammary carcinomas) which have been investigated to date (12/12) show a pronounced cytoplasmic reaction, and some also show a perinuclear reaction with MAB 406/14, as do the mammary carcinomas derived from glandular tissue (lobular mammary carcinomas; In addition, the lymph nodes (the tumor cells in them) affected by ductal mammary carcinomas show a strong reaction with 25 MAB 406/14 All the squamous cell carcinomas of the lung which have been tested (10/10) show a pronounced cytoplasmic as well as a membranaceous reaction. 6 of tested adenocarcinomas of the lung, 4/8 large-cell carcinomas of the lung and 2/5 small-cell carcinomas of the lung react with MAB 406/14.

Furthermore, MAB 406/14 shows no detectable binding to tissue from normal spleen, lymph nodes or stomach while, in contrast, there is weak binding to the mucosa, some cells of the glands and some parts of the muscle in the region of the normal colon. In addition, the MAB binds to a subpopulation of the tested colon carcinomas (4/17) and exocrine pancreatic carcinomas (11/17). The epitope which occurs in tissues which are positive to MAB 406/14 is resistant not only to fixation t~~ri 4- i z i .1 iI 8 with formaldehyde and embedding in paraffin but also to periodic acid oxidation and treatment with neuraminidase.

One application is the non-invasive detection of lymph node metastases of mammary carcinoma by injection of the radioactively labelled MAB into the interdigital webs, into the draining lymph tracts or intravenously.

Additional areas of use are immunoscintigraphy ano immunotherapy of, in particular, squamous cell carcinoma of the Lung, as well as other histological types of carcinoma of 10 the lung, and ductal mammary carcinoma and certain exocrine pancreatic carcinomas. The detection in body fluids of the epitope defined by MAB 406/14 is a method for the early diagnosis and for monitoring the progress of those tumors which secrete the epitope in amounts which are readily detectable.

The method used for the production of the abovementioned monoclonal antibodies is described on pages 3, 4 and 5 of German Offenlegungsschrift 3,329,184.

The invention relates to monoclonal antibodies which recognize a protein antigen which has a molecular weight of approximately 72 3KD under non-reducing or reducing conditions, or a glycoprotein antigen which has a molecular weight than 200 KD under non-reducing conditions, and which is not identical to F VIIIR:AG, or 8 defined epitopes on three glycoprotein antigens which have a molecular weight of 180 10 KD, 95 10 KD or 10 KD under non-reducing conditions, or a glycoprotein antigen which has a molecular weight than 200 KD under non-reducing conditions, and which is not identical to EMA.

These antigens are unambiguously characterized by the occurrence, indicated above, on particular cells and tissues and by the characteristics of the epitopes which occur on these antigens and are defined by the particular MABs.

The invention also relates to a process for the preparation of one of the monoclonal antibodies described above, which comprises the immunization of mammals with cells of the Oat-75 cell Line, of the Pa-Tu-I cell line or of a CEA-positive adenocarcinoma, or carcinofmbryonic antigen

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i I- 9 (CEA), the removal of spleen cells from an animal immunized in this way and fusion with the cell line X 63-Ag8653 or SP-2, selection of the hybridomas and obtaining of the monoclonal antibodies.

The immunogen used for the induction of MAB 278/97 and 278/105 is the Oat-75 cell line, and that for the induction of MAB 406/14 is th. Pa-Tu-I cell line. The immunogen used for the induction of the MABs defined by the epitopes on antigen 3 is a CEA-positive adenocarcinoma 10 of the lung; however, it is also possible to use commercially available CEA.

Mammals, preferably mice, are immunized with cells or antigens of one or more of these cell lines, and the spleen cells from these animals are fused with the X 63 15 Ag8653 cell line.

It is also possible to remove, for example, human lymphoid cells from tumor patients or healthy subjects, and to carry out in vitro immunization and fusion with a human myeloma cell. It is also possible to use the technique of EBV transformation or transfection with DNA (oncogene transfer) in order to stimulate the human lymphoid cells to permanent division.

The resulting hybridomas are tested to find whether they contain antibodies of the desired specificity.

Among the hybridoma supernatants tested, there were some which contained antibodies of the specificities described above. The hybridomas secreting these antibodies were cloned, and the monoclonal antibodies obtained from these hybridoma clones were used to carry out immunochemical characterization of the antigen recognized by them. The molecular weight is determined by comparing with commercially available markers. The antigens can be purified by affinity chromatography,, In addition, the binding of the monoclonal antibodies to histological specimens was measured using the indirect immunoperoxidase technique (J.Clin.Pathol. (1979), 32, 971).

Furthermore, the molecular characterization of

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4_ i 1: i: -I r II 10 the antigens, or subpopulations of antigenic molecules, recognized by the monoclonal antibodies was carried out, in addition to the Western blot analysis, by radioimmunoprecipitation (Behring Institute Communications (1984), 74, 27-34), and the epitope analysis for the 180 10 KD CEA, or 10 KD and 55 10 KD CEA related molecules was carried out by means of an ELISA under blocking conditions (Clin.

Chim.Acta (1983), 135, 13-22, specifically page The molecular characterization of the epitopes on human tissue which are recognized by the MABs was carried out as described below: 4-6 um thick sections of cryopreserved human tissue carrying the appropriate epitopes were dried on the slide (for 30-60 min at room temperature) and then fixed in acetone for 1-2 sees. They were then a) for the periodate oxidation, washed in PBS, pH 7.2, containing 1 g/l BSA for 10 min, briefly immersed (rinsed) in PBS, pH 7.2, and incubated with 1 g/100 ml periodic acid in PBS, pH 7.2, for 1 hour at room temperature in a humidity cabinet. The sections are then washed 3 x 5 min in 1 g/1 BSA in PBS, pH 7.2, and incubated with normal goat serum, further treated, as in J.Clon.Pathol. (1979) 32, 971, with the MABs which are to be tested, and their binding to the tissue is detected using the indirect immunoperoxidase 25 technique.

b) For the treatment with neuraminidase, washed 2 x 10 min in 0.05 M Na Ca++ acetate buffer, pH 5.5, and incubated for 1 hour with various amounts of Vibrio cholerae neuraminidase (100mU/ml, 10mU/ml,lmU/ml, 0.lmU/ml) in the abovementioned acetate buffer, at room temperature in a humidity cabinet. After washing the sections twice in acetate buffer and twice in PBS, pH 7.2, containing 1 g/l BSA, they are incubated with normal goat serum, further treated, as in J.Clin.Pathol. (1979) 32, 971, with the MABs which are to be tested, and their binding to the tissue is detected using the indirect immunoperoxidase technique.

To test the resistance to formaldehyde and paraffin of the epitope recognized by the particular MAB, the 4-6 um .4 44 4 4 r* 4t 4 4 4

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thick cryopreserved tissue sections are replaced by tissue which is fixed in formaldehyde and embedded in paraffin and then sectioned in a microtome, the paraffin is removed Romeis, Mikroskopische-Techniken (Techniques of Microscopy) 16th edition, page 145, section 546, 1968) and further treated as described in J.Clin.Pathol. (1979) 32, 971 for the indirect immunoperoxidase technique.

Because of their reactivity with antigen 1, the characterized monoclonal antibodies can be used for the diagnostic recognition of small-cell lung tumors carrying the 72 KD antigen and for differentiating them from other tumors without antigen 1.

The monoclonal antibodies characterized on the basis of their reactivity with antigen 2 can be used for the diagnostic recognition of tissue carrying the 200 KD antigen (endothelial tissues and vascular tumors) and for differentiating it from other tissue without antigen 2.

The monoclonal antibodies defined by their reactivity with 8 different epitopes on the CEA related molecules 180 10 KD, 95 10 KD and 55 10 KD in size can be used for the diagnostic recognition of CEA-positive tissue (for example colon carcinomas, pancreatic carcinomas, stomach carcinomas, mammary carcinomas and lung carcinomas).

The monoclonal antibodies characterized by their reactivity with antigen 4 can be used for the diagnostic recognition of tissue carrying the 200 KD antigen (mammary carcinomas) and for differentiating it from other tissue without antigen 4.

Furthermore, all the antibodies described above 30 which recognize antigens can be used to detect in body fluids the antigens which they recognize, or, in the radio-actively labelled form, to bind in vivo to the tissue carrying the particular antigen and to detect the antigens (immunoscintit t I graphy). In addition, these monoclonal antibodies can be used as carriers of active compounds and be employed for the therapy of malignant diseases. Another possible use is represented by the inhibition of tumor cell metastasis following in vivo administration and binding I certify that this and the preceding 3 pages are a tr and ex act copy of pages 1 of ti spcification originally lodged.

he 7 (C~05 fr ~d ~4 12 of the monoclonal antibodies to antigen-carrying metastasizing tumor cells. In addition, these monoclonal antiodies can, without the presence of other toxins, be toxic for the antigen-carrying tumor cells or, after binding to the tumor cell membrane, lead to differentiation processes which allow malignant tumor cells to become benign cells.

For diagnostic purposes, the monoclonal antibodies can be used in a concentration of from 0.001 jg/ml to 100 pg/ml, depending on the assay.

Therapeutic effects are attained in amounts of 200 pg-100 mg/kg body weight.

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Claims (8)

1. A process for the preparation of a monoclonal antibody selected from Monoclonal antibodies which recognize a protein antigen (AG 1) having a molecular weight of 72 3 KD under non-reducing conditions or reducing conditions, Monoclonal antibodies which recognize a glucoprotein antigen (AG 2) having a molecular weight 200 KD under non-reducing conditions, which is not identical to F VIIIR:AG by serology, Monoclonal antibodies which recognize glycoprotein antigens (AG 3) having a molecular weight of 180 10 KD, 95 10 KD or 55 10 KD under non-reducing conditions, or Monoclonal antibodies which recognize a glycoprotein antigen (AG 4) having a molecular weight 200 KD under non-reducing conditions, which is not identical to EMA, which comprises the immunization of mammals with cells of the Oat-75 cell line, of the Pa-Tu-I cell line or of a CEA-positive adenocarcinoma, or carcino-embryonic antigen (CEA), the removal of spleen cells from an animal immunized in this way and fusion of them with the cell line X 63-Ag8653, selection of the hydridomas and obtaining of the monoclonal antibodies.

2. The process as claimed in claim 1, wherein the mammals are mice.

3. Monoclonal antibodies which recognize a protein antigen (AG 1) having a molecular weight of 72 3 KD under non-reducing conditions or reducing conditions produced by the method of claim 1 or 2. .d -14-

4. Monoclonal antibodies which recognize a glycoprotein antigen (AG 2) having a molecular weight 200 KD under non-reducing conditions, which is not identical to F VIIIR:AG by serology produced by the method of claim 1 or 1 2. i 1 Ds 9 r. a (t I a i 'U"LO1 i) o i i i ~pan j 4a ii ra a arr rBbe tit i i I 'bi ,1 t rr r r r cit 1 i titE'-. Monoclonal antibodies which recognize glycoprotein antigens (AG 3) having a molecular weight of 180 10 KD, 10 KD or 55 ±10 KD under non-reducing conditions produced by the method claim 1 or 2.

6. Monoclonal antibodies which recognize a glycoprotein antigen (AG 4) having a molecular weight 200 KD under non-reducing conditions, which is not identical to EMA produced by the method of claim 1 or 2.

7. The use of a monoclonal antibody as claimed in any one of claims 3 to 6 in an in vitro diagnostic aid.

8. The use of a monoclonal antibody as claimed in any one of claims 3 to 6 as a carrier for a pharmaceutical active compound.

9. The use of a monoclonal antibody as claimed in any one of claims 3 to 6 as a therapeutic agent. DATED this 8th day of December, 1989. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT ATTORNEYS 2ND FLOOR 290 BURWOOD ROAD HAWTHORN VIC. 3122 1.68/135:C.K. c~ 1 4~