UA61065C2 - Method for treating epithelioma - Google Patents

Abstract

The invention concerns a method of diagnosing and treating epithelioma, the method being based on the expression of the variant exon v6 of the CD44 gene as the molecular target. In a preferred embodiment, v6-specific antibody molecules, in particular the monoclonal antibody BIWA-1 (VFF-18), are used for this purpose.

Description

Description of the invention

The invention relates to a method of diagnosis and treatment of squamous cell cancer, based on the expression of the variable exon of the mb gene CO44, means for implementing such a method, as well as its application.

Recently, it was shown that the expression of CO44 surface glycoprotein variants is necessary and sufficient to induce so-called spontaneous metastatic behavior not only in a non-metastatic rat pancreatic adenocarcinoma cell line, but also in a non-metastatic rat fibrosarcoma cell line (Sypiiey et al. 1991). While the smallest isoform of CO44, the standard form of CO44 5, is ubiquitously expressed in a number of different tissues, including epithelial cells, certain splice variants of CO44 (CO44m) are expressed only on a subset of epithelial cells. CO44 isoforms are produced through alternative splicing in such a way that the sequences of 10 exons (m1-y10) in CO445 are completely cut out, however, with large variants, they can exist in different combinations (Zsgeayp! et al. 1992; Neideg et al., 1993; Noitapp et al. , 1991).

The variants differ in that different amino acid) sequences are inserted into a certain position of the extracellular part of the protein.

Such variants could be detected in various human tumor cells and in human tumor tissue. Thus, the expression of CO44 variants in the course of colorectal carcinogenesis was recently investigated (Neiadeg et al. 1993).

Expression of CO44 variants is insufficient in normal human colon epithelium, and only weak expression is detected in proliferating crypt cells. At late stages of tumor progression, for example, in the case of adenocarcinoma, all malignant degenerate variants of CO44 are expressed. Further, the expression of splicing variants of CO44 in activated lymphocytes, as well as in the case of non-Hodgkin's lymphomas, has recently been shown (Koortagp et al., 1993).

Different approaches are known that are suitable for differential expression of the variant exon of the CO44 gene in tumors and normal tissues for methods of diagnosis and treatment (international patent applications 94/02633, p. 29 94/12631, 95/00658, 95/00851, European patent 0531300 ). Gee)

The expression of variant CO44 molecules in squamous cell carcinoma was also investigated. Zayti and others. (1993) found, with the help of the specific anti-ub antibody Mag3.1, a weakening of the expression of mb in tumor cells in comparison with normal cells. VgooK5 and others. (1995) obtained heterogeneous staining in nasopharyngeal cancer with the help of 11.9 specific anti-UB antibody. Strong staining was achieved only in 2 out of 12 cases, while in most cases only weak focal expression of mb could be detected immunohistologically. Gee)

The task of the presented invention was the development of new methods of diagnosis and treatment of squamous cell cancer, as well as the development of means for the implementation of such methods. at

This task could be solved with the help of the presented invention. It concerns the methods of diagnosis and treatment of squamous cell cancer based on the expression of the variant exon mb of the CO44 gene as a molecular marker or target. In particular, the present invention relates to methods based on a strong and homogeneous EE expression of mb in squamous cell carcinoma, which was unexpectedly able to be firmly established, contrary to the theory known in the existing state of the art. Antibody molecules with the appropriate specificity are suitable, in particular, as a drug base to selectively target squamous cell carcinoma. "

At the same time, preference is given to methods that differ in that an antibody molecule C is used, which recognizes the amino acid sequence -OMUEOMAKUUNESUKOT, especially preferably the amino acid sequence MMEOMAKUUNESUK. In this case, the monoclonal antibody VIMUA-Y is especially preferred

Iz" (MERE-18 clone), which is formed from a hybridoma cell line, deposited on June 7, 1994 under Mo OBM ACC2174 in Germany

Collections of Microorganisms and Cell Cultures (OZM) GmbH, Mascheroder Weg 16, 0-38124 Braunschweig, Germany (international patent application 95/33771), or a derivative of this antibody.

Other aspects of the present invention relate to the use of such antibody molecules, with the help of which

The method according to the invention is carried out, as well as the means for implementing this method. - The nucleotide and amino acid sequence of the variant exon mb of the CO44 gene is known (Zsgeayp et al., 1992;

Tbid et al., 1993). The existence of degenerate or allelic variants is irrelevant to the implementation of the invention; so similar options are definitely included.

Gee! 20 The sequence of exon mb of the human CO44 gene is:

I A TOR 88 tt z ET AT sl TI SVO OSA VST SST AT AST ASA ASO SAA BALA SA OST SS SAS

Kk E i y t 2 KE I M v g o ih g io)

ARO OAA SK TO TT OZOS AAS AbA tob SAT OA sb TAS SOS SAHA that t v A EE 5 5 EE 5 tot ХХ А

ГФ) АСА ССС АБА САК ОСС ТОС SAT ОТО АСА АСС pos zom ost b. d The invention can be implemented with polyclonal or monoclonal antibodies specific to the epitope encoded by the mb exon, in particular, to the epitope within the amino acid sequence in OMUROMAMUMUNESUKOT, especially preferably within the amino acid sequence UUEOMAMU/NESUK.

Obtaining antibodies against known amino acid sequences can be carried out by known methods (Satsu, 1989). For example, a peptide with such a sequence can be obtained synthetically and introduced as an antigen to the immunization protocol. Another way involves obtaining a component protein containing the desired amino acid sequence in such a way that the nucleic acid (synthetic or, for example, that can be obtained as a result of polymerase chain reaction /LLR/ from a suitable sample) encoding this sequence, integrated into an expression vector, and the fusion protein component is expressed in the host organism. If necessary, the purified component protein can then be inserted as an antigen into the immunization protocol, and insert-specific antibodies or, in the case of monoclonal antibodies, hybrids expressing insert-specific antibodies, selected appropriately. Such methods are determined by the state of the art. Neiaeg and others. (1993, 1996a) and Koortap et al. (1993) describe the production of antibodies against variant epitopes of CO44.

For the method according to the invention, however, antibody molecules that are derivatives of poly- or monoclonal antibodies can also be used, for example, Par- or P(ar)"-fragments of immunoglobulins, recombinantly obtained single-chain antibody derivative (csEm), chimeric or humanized antibody , as well as other molecules specifically bound to the epitope encoded by the mb exon. Rar- or E(ab')" fragments or other fragments can, for example, be obtained from the complete immunoglobulin of the antibody VIMUA-II (MEB-18) 70 or other antibodies (Kheytap et al., 1993). The expert, in addition, is able to obtain recombinant mb-specific antibody molecules. In particular, he can, according to the analysis of the amino acid sequence of the VIMUA-I (MEE-18) antibody and/or when using a hybridoma cell line that produces this antibody, especially according to the genetic information contained in it, receive recombinant antibody molecules of the same idiot type, as , for example, VIMUA-I (MEBE-18), i.e., /5 antibody molecules, which in the region of the antigen binding position (areas that determine complementarity, KVD) have the same amino acid sequence as the VIM/A-I antibody (MEE-18). Appropriate methods are determined by the state of the art. Such recombinant antibody molecules can, for example, be humanized antibodies (5pip et al., 1989; Bazvom and Zeetapp, 1991), bispecific or bifunctional antibodies (Uueipeg et al., 1993; boodmip, 1989, Reaegwiope, 1996), a single-chain antibody derivative (zsEm, dopypsop and Viga, 1991), complete or fragmented immunoglobulins (Soiota et al., 1992; Mevrii et al., 1992; Vagbraz et al., 1992) or antibodies obtained by shuffling the chains (Umipieg et al., 1994) .

Humanized antibodies can, for example, be obtained by transplantation of KVD (European patent 0239409). Frame sections can also be modified (European patent 0519596; international patent application 9007861). To humanize antibodies, it is now possible to use such methods as PCR (see, for example, European patent 0368684; European patent 0438310; international patent application 9207075), or computer modeling (see, for example, international patent application 9222653 ). There can also be i) obtained and used component proteins, for example, a component protein from a single-chain derivative of an antibody and a toxin (Spatsanagu et al., 1990; ERgiedtap et al., 1993). The broad term "antibody" or "antibody molecule" should include, in addition to polyclonal and monoclonal antibodies, all the compounds discussed in this section, as well as other compounds structurally derived from immunoglobulins and obtained by known methods.

It is within the capabilities of an average specialist to obtain information about the epitope (see fig. 1, fig. 4) and s) of an equivalent VIMUA-I (MEE-18) antibody with the same binding specificity. Such antibodies are therefore also included in the invention.

For a method of diagnosing antibody molecules, preferably VIMUA-Y antibody molecules, their fragments or - recombinant antibody molecules with a similar idiotype, can be combined, for example, with radioactive "o isotopes, such as, for example, 71291, 1911, 111/d, Z9pTs, or with radioactive compounds (I aggop et al., 1991; Trotazg et al., 1989; Zgimaziama, 1989), with enzymes, for example, peroxidase or alkaline phosphatase (Satsu et al.

Kaukipaaiia, 1989), with fluorescent dyes (Doppzop, 1989) or with biotin molecules (Spcezaop et al., 1979). For therapeutic use, Mb-specific antibody molecules, preferably molecules of antibody 40. VIMA-Y (MEB-18) or molecules of an antibody derived from MEE-18, for example, its fragments or recombinant and antibody molecules with a similar idiotype, can bind to radioactive isotopes, for example, 90u, 191), 186de, 188Be, 1535, b/u, 212Vi, 213Vi, 177u (Omtsaagi et al., 1993; IGepnaga et al., 1985; Mgiezepdogr et al. 1991; Muyrig et al., 1989; Magameois et al., 1995a, Chigsis to ZsPeipregd, 1994), with toxins (Mighena et al.,, 1991; Migetsa and Tpogre, 1991; Kheytap et al., 1993; Tpeceg et al., 1993 ), with cytostatics (Zspgarre et al., 1992), with prodrugs (U/apd et al., 1992; Zepieg et al., 1989), with substances that

Me, are photoactivated (Nettipao et al., 1993), with an antibody molecule with a different specificity or with radioactive compounds. The antibody molecule can further connect with a cytokine or other immunomodulatory polypeptide, for example, with tumor necrosis factor, lymphotoxin (Keivii et al., 1996) or interleukin-2 (VeskKeg" et al., 1996). Antibody molecules for insertion into the pre-targeting system can also be modified,

F 250, for example, with the help of streptavidin or biotin (soodm/ip, 1995).

It is useful to be able, in accordance with the method of diagnosis according to the invention, to examine samples taken from sick patients, for example, from biopsies, which establish the presence of sufficient data for the diagnosis of squamous cell cancer or a diagnosis is already made to characterize the tumor in sufficient detail. Detection of variant CO44 molecules containing the amino acid sequence encoded by the variable mb exon can be performed on the protein surface using antibodies or on the nucleic acid surface using

GF! specific probes of nucleic acids or primers for polymerase chain reaction (PCR). The invention therefore relates to antibody molecules and nucleic acids suitable as probes or seeds for such methods, and to the use of such antibodies and nucleic acids for the diagnosis and analysis of squamous cell carcinoma. For example, tissue sections can be examined immunohistochemically using antibodies by known methods. 60 Extracts obtained from tissue samples or fluids contained in the body can be further studied by other immunological methods using antibodies, for example, Western blotting, with the help of enzyme immunosorbent assays (EI IZA-test, Sanu and Kaukipaaiia, 1989), radioimmunoassays analyzes (KIA, Satsu and Mygrpu, 1989) or applied immunoassays. Research can be conducted qualitatively, semi-quantitatively or quantitatively.

Along with the diagnosis of ip migo, antibody molecules with specificity according to the invention are also suitable for the ip mimo diagnosis of squamous cell cancer. If the antibody molecule has a detectable label, it is possible to determine the label for diagnostic purposes, for example, for visual detection of a tumor ip mimo (acquisition of an image) or, for example, for radio-guided surgery. For the use of antibodies conjugated with radioactive isotopes for immunoscintigraphy (imaging) there are, for example, a number of protocols on the basis of which a specialist can carry out the invention (Vissagai et al., 1989; Keepap et al., 1987; Regkeeps and Ritt, 1992 ; SoiIsNneg et al., 1987; Tpotrzgop et al., 1984).

The data obtained by detecting and/or quantifying the expression of the variant epitope of mb СО44 can thus be used for diagnosis and prognosis. In this case, a combination with other prognostic parameters, for example, with the degree of tumor development, can be useful.

Antibody molecules with the specificity according to the invention and, if necessary, linked to the cytotoxic agent 7/0 Can be usefully used for the treatment of squamous cell cancer. In this case, the application can be carried out systemically or topically, for example, by intravenous (bolus or continuous infusion), intraperitoneal, intramuscular, subcutaneous or other injection/infusion. Protocols for the administration of conjugated or unconjugated antibodies (such as whole immunoglobulins, fragments, recombinant humanized molecules, or others) form the basis of the technique (MiezPipe et al., 1991; Iagwop et al., 1991; Mietsa et al.

Trogre, 1991; Mena et al., 1991; Vgei" and others, 1992, 1995; Rhevzv et al., 1989: Mueipeg et al., 1989; Snnaiya et al., 1989; Zeags et al., 1982). Therapeutic use can, for example, be carried out similarly to the use of antibody 71.1A5MI (Zeyeg et al., 1993). Unmodified monoclonal antibodies can be inserted directly into treatment when they exhibit relevant cytotoxic intrinsic effector function, for example, for complement-induced or antibody-induced cellular cytotoxicity (KievptioPeg et al., 1994). Suitable monoclonal antibodies for such use are a mouse antibody of the Id2a isotype or an antibody of the human IDOI type. Unmodified antibodies can further be used to induce an antitumor response characteristic of the patient by anti-idiotypic mechanisms (White et al., 1993;

Kpa?gaevii and others, 1994).

The best variant of the invention from the point of view of use for treatment consists in binding humanized immunoglobulin specific to mb or its E(ab')"-fragment to UU (Soyaagi et al., 1993; o

Mghiezepaogr et al., 1995), 191u (Magameuz et al., 1995a, 1995r; Oymueyy et al., 1995; Rgezvz et al., 1995; Trpotaz et al., in the collection: Satsu 1985, pp. 230-239) , 186rBe (Vgei et al., 1992, 1995) or with another appropriate radioactive isotope and enter for radioimmunotherapy of squamous cell cancer. For example, a VIMUA-I antibody or a humanized version of VIMUA-I, or a P(abB)-fragment of VIMUA-I or a humanized antibody can bind to 90u using a chelating linker such as isothiocyanate benzyl-diethylenetriaminepentaacetate (ITCB-DTPA), and a specific activity of 5-20mCi/mg, preferably 1OmCi/mg, should be achieved. This agent can then be administered to a patient with an antigen-positive tumor in a dosage of 0.1 to 1 mCi/kg body weight, preferably 0.3 to 0.5 mCi/kg body weight. If the antibody molecule is bound to 191, with a specific activity of 2MCi/mg, an appropriate dosage scheme is possible, for example, 2 x 150mCi over a period of 6 weeks. A specialist can set the maximum possible dosage using known methods (Magameois et al., 199582, 19955). If the total amount of protein intended for administration is from 2 to 5 mg, administration can be carried out in the form of a rapid intravenous bolus injection. With large amounts of protein, the infusion can be carried out in a convenient form for administration. In the case of monoclonal antibodies, it may be necessary for the agent to be “mixed with an excess (eg, 15-fold molar excess) of non-radioactive antibody before administration; in this case, it is better to administer as an intravenous infusion, for example, over 15 minutes. c The input can be repeated. Treatment can be combined with external radiation therapy. Further, treatment with" can be supported by a bone marrow transplant: this is especially necessary when, during treatment, a dose of more than 1.6 grays enters the bone marrow.

Antibody molecules according to the invention can also be used ex mimo for the purification of SOC4-positive preparations of cell lines and preparations of progenitor cells (immune purification). Radiation therapy or chemotherapy of squamous cell cancer can be supplemented by an autologous bone marrow transplant. The preparation of hematopoietic cell lines and progenitor cells used in this case should not contain tumor cells. This can be achieved by incubation with antibody molecules according to the invention, for example, antibody-toxin conjugates (MuKierizvi et al., 1994; German patent 196 48 209.7).

Ge») 20 Antibody molecules according to the invention can be further introduced in the form of a recombinant design in

T-cell receptor of T-lymphocytes. Such reprogrammed T-lymphocytes bind selectively to tumor cells that express an antigen, and show a cytotoxic effect, thus, they can be used for the treatment of squamous cell cancer (PCT/EPO6O0O4631; APepzsptia et al., 1996).

Fig.1. Determination of specificity for the epitope of the VIM/A-I antibody by binding to synthetic peptides derived from the sequence of human CO44U6b.

GF) The corresponding peptide CO44u6b of rats was tested with the antibody 1.1TAZMI. Binding was determined using

EPZA-test, and the peptides were immobilized on tablets for titration (for comparison, Neiaeg et al., about 1996B, fig.2). -: no binding, h/-: weak binding, t: strong binding.

Fig. 2: Immunohistochemical analyzes of squamous cell carcinoma of the larynx (a) and metastasis in the liver of esophageal cancer 60 (b) using the specific for CO44ub monoclonal antibody VIMUA-Y.

In both cases, it is possible to observe the reactivity of the antibody in relation to the membrane of tumor cells.

Initial magnification 40x, additional staining with hematoxylin.

Fig. 3: Comparison of antigen binding of different CO44ub specific monoclonal antibodies.

Binding of four different CO44ub6-specific monoclonal antibodies to human A-431 squamous cell carcinoma cells was measured using the EGISA test. Monoclonal antibody VIM/A-Y shows a higher affinity to tumor cells than other monoclonal antibodies.

Fig. 4: Refined mapping of the epitope of the monoclonal antibody VIMUA-Y.

Binding of VIMUA-II on different overlapping synthetic peptides spanning amino acids 18-32

Coded CO44u6 area was measured in the competitive EGISA-TEST. The minimal binding sequence (peptide mb(19-29)) is underlined.

Fig. 5: Biodistribution of labeled 125). VIMUA-I in hairless mice subjected to xenogeneic transplantation of A-431.

Accumulation of antibodies is established as 90 of the injected dose ID/g (the mean value of their standard error of measurement) after 4, 24, 48, 120 and 168 hours after the injection. 70 Example 1; Expression of СО44mb in squamous cell carcinoma

Cloth

In total, in 126 cases, immunohistochemically paraffinized tumor samples were analyzed using the monoclonal antibody VIMUA-I (clone MEE-18) for the expression of СО44m6. In 31 cases, the samples included primary squamous cell cancer (in 15 cases of the larynx, in 16 cases of the skin), in 91 cases included metastases in 75 lymph nodes (larynx, n-38; lung, n-27; esophagus, n-11; oral cavity , n-11; tonsils, n-4) and in 4 cases included liver metastases (esophagus).

Antibody

The common variant region of NRKII-type CO44mM (Noitanpg et al., 1991) was amplified by polymerase chain reaction (PCR) from kdDnKk of human keratinocytes. Both PCR primers 5-SABOSTOOSASSSAAAATOAASAAAATO-Z, positions 25-52, and 5-TOATAAOSAASOATTOASATTASASTTOOA-3, positions 1013-984 of the variant site of ISI C97, as described

Neutapp et al., contained an EsoKiI restriction site, which was used to directly clone the PCR product into the pPOEX-2T vector (Ztiy et al., 1988). The resulting construct (ROEH cСОr44u NRKII m3-m10) encodes a component protein with a molecular weight of about 7OkDa, consisting of Glutathione-Z-transferase Gα spiziozot |iaropisit and exons mo-mO of human CO44 (Fig. 1; Neideg and etc., 1993).

The constituent protein was expressed in E. soya and then subjected to purification by means of affinity chromatography on i9) glutathione-agarose (Ztiy et al., 1988).

Female Vair/c mice were immunized intraperitoneally with a component protein purified by affinity chromatography according to the following scheme: 1. immunization: 9dOmkg of the component protein in complete Freund's adjuvant 2. 1 C. immunization: BbbBOmkg of the component protein in complete Freund's adjuvant Immunizations were carried out with o with an interval of 4 weeks. 14 days after the last immunization, the animals were immunized again for three weeks! the following days, each time 1 omkg of the component protein in a phosphate-buffered saline solution (SPF). The day after that, the cells of the spleen of one animal with a high titer of antibodies were subjected to fusion with -

with murine myeloma cells RZ.Xb63-A98.653 using polyethylene glycol 4000. The hybridoma cells were then subjected to selection on plates for titration in a medium containing hypoxanthine, aminopterin and thymidine (GAT medium) (KoPieg and Miev(eip, 1975: Keagpeu and others, 1979).

Determination of the antibody titer in serum or hybridoma screening was performed using the EP IZA test. In such a test, tablets for titration were first coated with a component protein (glutathione-5-transferase-SХО44у3-10) or only glutathione-Z-transferase. Then, they were incubated with serial dilutions of serum samples or hybridomas, and specific antibodies were determined using peroxidase-conjugated antibodies against mouse immunoglobulin. Hybridomas reacting only with glutathione-5-transferase were rejected. Antibodies that remained were initially characterized in the EIG IZA test using domain-specific constituent proteins (exon m3Z, exon m5-mb, exon mubm/, exon m8-mI0) (Koortap et al., 1993). their immunohistochemical 5 reactivity was tested on sections of human skin.

Ge") VIMUA-I (MEB-18; see also the international patent application 95/33771 for its preparation and properties) was bound only to component proteins containing the domain encoded by the exon of ub. In order to further determine the epitope of the antibody, various synthetic peptides representing parts of av! mb domain (Fig. 1). A peptide of 14 amino acids, mbO, showed the strongest binding. Therefore, the epitope of VIMMA-I lies entirely or partially within the OMUEOMAMUMUNESUKOT sequence of the domain encoded by the mb exon. This sequence is homologous to the binding epitope of the antibody 1.1A5MI, which was used in the SL therapeutic rat model and which is specific for rat CO44m6 (Fig. 1).

Immunohistochemistry

Before incubation with the primary antibody, paraffin sections (4 μm) were deparaffinized in a rotary device for histological studies (Roth, Germany) three times, each time for 10 minutes, and then rehydrated in higher alcohols. Sections were quickly washed with distilled water and then boiled in

F, in a microwave oven (Sharp K-6270 model) three times for 10 minutes at 600W in a 0.01M buffer containing citric acid - sodium citrate. After each incubation in the microwave, the sections were cooled for 20 minutes. After the last cooling, the medium was washed in SFR and preincubated with normal goat serum (1095 u bo SFR). After three washes in SFR, the sections were incubated for one hour with the primary antibody (VIMUA-I: 5μg/ml; mouse Id (corresponding isotype negative control) 5μg/ml in SFR/195, bovine serum albumin (BSA)). Normal human skin sections were used as a positive control for the color reaction; because keratinocytes express the CO44 isoform containing m3-m10. Endogenous peroxidases were blocked with 0.390 hydrogen peroxide in SFR, and the sections were incubated for 30 minutes with biotinylated secondary antibody 65 (anti-mouse Ids-E(aub)", DAKO Corporation). To develop the color, the sections were incubated for 30 minutes with horseradish peroxidase, which binds to biotin in the streptavidin-biotin-peroxidase complex (DAKO Corporation).

Then the sections were incubated for 5-10 minutes in 3,3-diamino-9-ethylcarbazole substrate (Sigma Immunochemicals), the reaction was stopped by adding water, and the sections were additionally stained with hematoxylin. Coloration was assessed using a Zeiss axioscopic light microscope, color intensity was quantified as follows: w-- strong expression; -, moderate expression; well, weak expression; -, no unambiguously detected expression or no detected expression. Only tumor cells with clear staining of the membrane were evaluated as positive. The percentage of positive tumor cells was estimated in each section and two groups were formed: focally positive tumors (less than 1095 tumor cells reacted with the antibody) and positive tumors (10 or more 956 tumor cells were positive). When less than 8095 7/0 tumor cells in the positive cells reacted with the antibody, the corresponding percentage value was obtained.

126 cases of squamous cell carcinoma of various origins were analyzed using the CO44ub6 monoclonal antibody VIMUA-I. Expression of CO44ub-containing isoforms was observed in all but one of the tumor samples. Most of the samples showed antigen expression on 80-10095 tumor cells, the staining was limited to the membrane of tumor cells. No reaction was observed in the case of stromal tissue, lymphocytes, muscle cells or endothelium.

To quantify the expression of CO44ub molecules on these tumor cells, sections of normal human skin were stained in parallel with tumor sections. Normal skin keratinocytes express high levels of isoforms

СО44 and are considered to be the most strongly expressing СО44уб6 among the normal cells described to date. Therefore, the staining of keratinocytes was taken as a standard and classified as "strong" (---5) in the applied evaluation system. In most of the studied tumor samples, the staining of tumor cells was the same, or even stronger, than the staining of skin keratinocytes, only in some cases weak staining of the tumor (3 cases of lymph node metastases) or moderate (2 primary cancers, 10 metastases) was observed. The staining reaction was completely homogeneous within a given tumor section, with most of the tumor cells in the section having the same intensity of staining. No significant differences between primary tumors and metastases were observed in the CO44mu6b expression sample. Detailed summarized results are shown in Table 1, examples are shown in Fig. 2. i) yu zo zveutsvv| 100000 marshland 0001 arable field 000000 primary field 0000 ryan | o cotton 0100000 pezhvina 0000oran pato 000000 first 0000ryan - zv btavvyao 0000000 pera 00000oryano 00 F avvovyayu 000000pervina 00000orano 5

Bimiyu 000000 original 00000oryan

Bmosai 00100000 firstborn 000005 van 0000000 firstborn 00000ryan | h 4 vza 0100000 privina 00000arano Z s lav va 001000000pervivao// tran . conspiracy 0000000pervyana 000000 g" ze; I; and 0000000first 00000ploughed 5

Zaamyaa 0000000 original 00000oryan lawsuit yav000000 peyavina 00000orano 5

Ф dzvvivvv 0000000primvlna 00000shkri sh mov 00000000 primavny 000000vvv 000000primvina 00000shjry 0100

F introduced 0010000 Present00,000 Shchrri B »Introduced 110000 00000 Wizard 1 led 010000 PERNICAL0000KRA SL WAV 00100000 Primary 00000 000 000 Yu. name) :

Movotev 01000000000000/Schyskri /// I Small 000000 0000 00000 Shchkri BUD 0100000 Primary 11000 PIA 1 PROC 0000000 PER 00000 SCHOKRA JBV Ved 10 Metestaves in Pvzhfa Universities of Cake 00002 laryngeal node zhk ooo! Metsteulmeezhniah wine 1 period with Meyasteu beer wine leni

І pogo oi Meyusnou lenation vne ee ch sobor oi Mesto piftlnn yetno and vobo (9 ol Metastao in lymphetnchniy volo tota 072 and zh. Mepkyaz in lifetchniya kl tran 80952 Matt lisenya vyvetno y

Meovone Mene lyotnnia'kii ve 1 le her and tic node (oootortani

And the star. building

And Mr. Veletnya lyn, but she has her sister Mensnou lyvevnny vne ee and govo4i95| | Metastasis in lymph and

Zob vi Metsteu Lithuania lin 000

Poet 2 Mezhsteu lydyanyi lyno 4

FR 1 Place! 22 M Meyasteu loved and fainted. Metsteulnetzhnyakh let o 7531192 Metastasis in the lymph node to her Sons of them; tata nodule (lungs | nn t Zooso Mensneu luvetnia vet leno about MB; 1 Metu lefetnnivxy leno her pen on NN

And the dawn of her terror ele teettia p menktyaz in the piuffeteny vu styaund | 2 7 TI Metastasis to a lymph node tin giant sy 16641 91 AI Metastasis to a lymph node| esophagus ove|va| |Metyastv in the lizhratic node of the stevoyud | 2 1mov2 1 Metastv in the lizhralic node stevokhdu language 1 Metastvz in the lizhralic street! stevoyud povo 110 Metastvz in the limeral node of steveyudu ptBo2/92. 10 Metastasis to Lizhralichy Street! Stevoydu poBo2 (821 Metastasis to pyzhphalic nodules stevohdu (20292 Metastasis to lymph node of the cavity bo3o 92 Metastasis to lymphatic tumor of the oral cavity Again o 13350921 Metastasis to lizhratic node of cavity horn 3358210 Metastasis to lymph node of cavities

B3oya 921 Metastasis to the lymph node of the cavitary horn OZloZh 616402 1 Metastasis to the lymphatic node of the cavity

Pviva 8210 Metastasis to the lymphatic tumor of the cavity of the tetu oto and pva12 0921 Metastasis to the lizhratic node of the cavitary horn

Pvvzb 3210 Metastasis to the lymphatic ul torozhnyniteu pvvzv 9210 Metastases to the lizhratic node of the cavity horn 2 pvvzb 921 Metastasis to the lymphatic ul of the cavity (von v2 10 Metastasis to the pyzhralic nodes of the mydala (in v2 1 Metastasis to the pyzhphalic node of the mydala vviv|v2. |Metastasis to the lizhralichy street| myalgia povlo (v2 Metastasis in the lizhphatic universities of the mydala cm 5 az v4| 11000 Metastases of the esophagus

Tumor cells reacted positively with VIMUA-I 80-10095. In cases where a smaller number of tumor cells reacted with the antibody, the corresponding percentage is indicated. Yes)

Example 2: Expression of CO44ub in kidney cancer, prostate cancer and liver metastases in colon cancer/

The fabric of

19 cases of kidney cancer were analyzed (12 cases of clear cell cancer, 5 cases of chromophilic, 1 case of chromophobe, 1 oncocytoma), 16 cases of primary adenocarcinoma of the prostate and 19 cases of metastases

In the lymph nodes in colon cancer. |se)

Antibody

VIM/A-Y (see example 1).

Immunohistochemistry «

For research, see example 1.

In contrast to squamous cell carcinoma, in most studied cases of kidney and prostate cancer, no expression could be detected or only focal expression of CO44u6 isoforms was detected. In the case of expression, "» greater than focal, in prostate cancer, the staining was mainly diffuse cytoplasmic and weak " or heterogeneous compared to the staining of normal prostate epithelium. In 5095 studied liver metastases in colon cancer, expression of CO44u6 isoforms was higher than focal. Staining was weak to moderate in most cases, and in most cases less than 10,095 (22) tumor cells in the sample showed VIMUA-I staining. The results are summarized in table 2. - o (22) 11 eta | foul oituvna | recovery, secondary prostate cancer | 000opervina 00016183

Crayfish 00000100000 pevina 00009) 71111012

Colon and rectum metastasivpechnu 130 71771816 o Example 3: Characterization of specific antibodies to СО44u6 (name) Cell line

The human squamous cell carcinoma A-431 cell line (spontaneous epidermoid carcinoma of the vulva) was obtained from the 60 American Collection of Type Cultures (CoscQuei! MO) and cultivated according to the manufacturer's instructions.

The surface expression of isoforms containing CO44ub was determined in the analysis using the cell sorting unit with fluorescence excitation (HAS5 analysis), and the monoclonal antibody VIM/A-II was used, connected to fluorescein isothiocyanate (FITC).

Analysis of kinetic constants 65 Determination of the affinity and kinetics of the interaction of the monoclonal antibody and СО44уб6 was carried out using surface plasma resonance (SPR), and the ViAsoge 2000 system was used

(Pharmacy Bayesensor firm). A component protein including glutathione-Z-transferase and CO44, which contained the region encoded by exons y3-m10 (glutathione-Z-transferase/C044 y3-m10), was immobilized on the CM5 sensor chip, and the amine coupling method was used, respectively to the manufacturer's instructions.

Antibody in different concentrations (8-132nm) in buffer nvz POMM "M-(2-hydroxy)ethylpiperazine-M'-2-ethanesulfonic acid pH 4, 150MM sodium chloride, 34А4MM ethylenediaminetetraacetic acid /EDTK/, 0.059565 surface-active substance P20 ViAsoge) was injected through the antigen-specific surface at a flow rate of 5 μl/min. The interaction was calculated by the change in the signal

PPR Dissociation of the antibody was observed for 5 minutes in a flow of buffer (HB). The surface of the chip was regenerated with the help of a separate pulse in 15 μl of ZOmMM hydrochloric acid. Data analysis and calculation of kinetic constants were carried out with the help of VIA evaluation software (Pharmacy Bayesensor), version 2.1.

In this way, the antigen affinity of the monoclonal antibody VIMUA-I was compared! and other monoclonal antibodies specific for CO44ub6 (MER4, MER7, BVA-13 (id01, CFU systems, Abingdon, England)).

Kinetic constants and affinity constants of different antibodies were determined in each case in two independent /5 experiments. Table C shows the association rates (Ka), dissociation rates (Ka) and dissociation constants (Kia) for four monoclonal antibodies. All monoclonal antibodies had similar Ka and Ku, with the exception of BBA-13, which had three times lower Ka and MER7, which showed a significantly higher dissociation rate (factor 5) compared to other monoclonal antibodies. This results in reduced binding affinity for MEE7 and BVA-13 compared to MER4 and VIMUA-I. Antibody VIMUA-I has the lowest K y of all 20 studied antibodies. school 25 o

IC) 30 Antibody-protein interaction analysis using the EI IZA test. "at

A-431 cells expressing CO44m6b were cultured in 96-well plates (Falcon Microtest II,

Becton Dickinson, Lincoln Park, New Jersey) of 5x107 cells per well in the medium of VRMI 1640 with 1095 - amniotic calf serum (HCS) overnight at 37"C. After washing with SFR/O.0590 tween 20 "-- the cells were fixed with chilled to 0"C with ethyl alcohol for 71 minutes, then washed. 35 |ncubation with primary antibodies (MEE4, MEE?7, VIMUA-I, BVA-13, Ing/ml-bOOng/ml, in each case in x buffer, for testing: SFR/ 0.590 BSA/ 0.05965 tween 20) was carried out for 1 hour at room temperature and then washed three times. Her secondary antibody used was horseradish peroxidase-conjugated rabbit anti-mouse Id antibody (DAKO Corporation, Copenhagen, Denmark; 1:6000 dilution in assay buffer) (1 hour/ at room temperature). After three washings, it was stained with a 373 solution of thymol blue (Kirkegaard and Perry, Gaithersburg, USA). Extinction was measured using a Hewlett-Packard reader used in the EI IZA test. Figure 3 shows that the relative affinity of antibodies, as determined by ViAsoge analysis, is reflected in their interaction with tumor cells, with VIMUA-I clearly demonstrating the highest binding affinity.

The domain of the protein encoded by exon mb of the CO44 gene consists of 45 amino acids (Fig. 4). To be precise

Ф epitope recognized by VIM/A-I, a series of synthetic peptides were used in EGISA-tests. Previous experiments indicate binding on the centrally located 14-mer (amino acid residues 18-31; Fig. 4, cf. also Fig. 1), and not on the peptide outside this area. Therefore, a second series of peptides was synthesized and tested in competitive EGI5ZA-tests (Fig. 4). The results show that the peptide 19-29 (M/EEMKUUNESUK) (He) 20 represents the minimal structure required for high-affinity binding. Elimination of the C-terminal arginine residue leads to a more than 100-fold weakened binding. сл Example 4: Biodistribution of radioiodine-labeled antibody to СО44уб6 in hairless mice with a xenograft

Xenograft model A-431 99 to eight-week-old female hairless mice VAI V/s pi/pi (VK Universal company, Rentoy, West Africa)

GF) injected subcutaneously into the left midline of 5x109 cultured A-431 cells (human epidermoid vulvar cancer). Animals after xenotransplantation bearing A-431 tumors were used for two weeks in biodistribution experiments (tumor mass: 40-50 ΩgH). in Introduction of radioactive iodine in VIMUA-Y

Protein C - the purified monoclonal body VIMMA-I (mouse IDS1) was bound on streptavidin, and a heterobifunctional cross-linker, succinimidyl ether of 4-(M-maleimidomethyl)cyclohexane-i-carboxylic acid was used. The lysyl residues of streptavidin were bound to the reduced cysteinyl residues of the antibody, which were formed during pre-treatment of the antibody with dithiothreitol. c5 The resulting conjugates (1:1) (29095) were further purified using ion exchange chromatography. For experiments on the biodistribution of VIM/A-I streptavidin was labeled with 729) on the primary amino group of lysine, and p-iodophenyl reagent (PIF, NEN DuPont, Wilmington, Germany) was used to introduce the label, according to the method

ММШХиг и и и. (1989). Depletion of VIM/A-I with streptavidin or 7125 did not alter the immunoreactivity or pharmacokinetics of the antibody in mice.

Experiments on biodistribution

Hairless mice that underwent xenotransplantation with human A-431 tumors were injected intravenously through the lateral tail vein with 5-7 μCi 729I per BOmkg of the monoclonal antibody VIM/A-Y (specific activity 0.1-0.14 mCi/mg). The study of biodistribution as a function of time was carried out in groups of 3 animals (n-3) 4, 24, 48, 120 and 168 hours after injection. At selected time points, mice were anesthetized, exsanguinated through the retroorbital plexus, and killed by decapitation. Nine organs and tissues were collected and minced, including blood, tail, lung, liver, spleen, stomach, kidney, intestine and tumor. The radioactivity in the tissues was calculated in comparison with the standard of the injected antibody preparation in a gamma scintillation counter (Packard Instrument Company, Meriden, Connecticut), and the energy window of the counter was set at 25-8OkeV for 7292. The percentage of injected dose/g of tissue was calculated (95 ID/g).

Previous experiments have shown that VIM/A-I does not cross-react with mouse antigen СО44у6. Table 4 and Fig. 5 show the absorption of radioactivity in tumors and normal tissue. In the case of iodinated VIMUA-Y, a rapid uptake by the tumor was observed (7.695 IU/g at 4 hours after injection), which increased to more than 1895 IU/g at 48 hours and then remained constant until 120 hours. Seven days after the injection (168 hours), the tumor contained another 15,395 IU/g of tissue. Tumor/tissue ratios were calculated at individual time points and are shown in Table 4. 24 hours after injection, the tumor-tissue ratio was 0.48 and rose to 3.16 on the seventh day. Absorption in normal tissue was low most likely caused by the background value of the blood pool in the biopsy tissue. In the selective targeting of ip mimo xenografts of human squamous cell carcinoma in hairless mice, it is shown using labeled 125; VIMUA-Y, that c 29 Monoclonal antibody has a high potential as a targeting carrier for diagnostic and therapeutic d) use in patients with squamous cell cancer. from F

F

- with Fo « e : s Average values (p-3); standard deviations "795 z" Example 5: Different expression of СО44уб6 for a large number of human tumors In another study, a total of 544 tumor samples were studied immunohistochemically using the monoclonal antibody VIMUA-Y (clone МЕР-18) for the expression of СО44у6. The samples were either paraffinized, or immediately after surgical removal, they were frozen in liquid nitrogen and stored until use at -70°C. The following tumors were analyzed: basal cell carcinoma (n-16), adenocarcinoma (AK) of the breast (n-55), colon cancer (p-83), squamous cell carcinoma (SCC) of the head and neck (p-125), lung cancer - (p-120), AK of the prostate (p-34), kidney cancer (p-27). SCC of the skin (p- 15) and AK of the stomach (p-69). Tissues were obtained through routine surgical intervention or by taking biopsies, obtaining normal tissue was accompanied by obtaining tumor samples. An immunohistochemical study was performed, as in example 1.

Ge") 20 Table 5 presents an overview of the immunohistochemical analysis of 397 different tumor samples using the monoclonal antibody VIMUA-Y. 99 Type Total p |Positive cases of AK of the mammary gland with metastases in the spinal cord 5 | 61100 because AK lung primary tumor 35 15 43

Purlevno 000 primary tumor 81905 metastases in lizhfetin you 1800

AK of the stomach primary tumor 22 | 15 68 o

Pi ns NI VE and NOYA

SCLC: small cell lung cancer

No or only slight reactivity was observed in small cell lung cancer, kidney cancer, and prostate cancer. All other investigated types of tumors expressed CO44ub-containing isoforms in different amounts. Most of the studied AK of the mammary glands showed reactivity with VIM/A-1, and the studied types

RCC (larynx, lungs and esophagus) expressed СО44у6б in 10,095 cases.

A total of 185 cases of RCC of various types and different classifications were studied for reactivity with VIMUA-I. These were 67 cases of primary RCC (larynx, p-15; oral cavity, p-16; oropharynx, p-3; skin, p-15), 77 samples of lymph node metastases (larynx, p-12; lung, p- 27; esophagus, n-11; oral cavity, n--b; oropharynx, n-7; laryngopharynx, n-10; tonsil, n-4) and From the sample from metastases in the liver (esophagus). Table 6 shows the results of the immunohistochemical analysis of all the examined samples of RCC. cm 5" o type Total n Negative Focal pos. Positive.

Laryngoscope ML 1OG 01000 10 100.

Rotolota (pp. with 0/0 o | o |z to o 7 ml 7 10101010 7100 o

Larynx (pp) 15001010 10 5 ob) o ml zo 113 oo 29 v

Legenya pyv 12 oo vve -- zv MmlyuI 27 010 11 4 26 9 t stavoyud po 000001005019 95 mlya mo|0 000 100. mp/ z ooo o z yuyu penyu ti le ooo ot yuyu. h 4 mi; 6000 06 05, Z s sha (n) 50000) 005 oo. Mydalya o Mle 40100004 |100 nya o Usyuyup | 85111111

Focal position: "1095 tumor cells are positive; MLV: metastases in lymph nodes; PO: primary b tumor; MP: metastases in the liver.

The expression of CO44ub-containing isoforms was detected in all but three tumor samples (one case of the larynx, 2 cases of the lung). Most samples showed antigen expression per 80-10,095 tumor cells within a single section, with staining mostly concentrated on the membrane of tumor cells. The most strongly stained homogeneous sample was observed in the case of cancer of the larynx, esophagus and me) of the larynx, and most of the tumor cells in the section had the same intensity of staining. with Literature:

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PROTOCOL OF SEQUENCES

(1) GENERAL DATA: « (1) APPLICANT: v s (A) NAME: Böhringer Ingelheim International GmbH,

AND (B) STREET: Rheinstrasse and? (C) CITY: Ingelheim. A) NAME: Research Center Karlsruhe GmbH o (B) STREET: PO Box 3640 (C) CITY: Karlsruhe

Me, (E) COUNTRY: Germany s (R) ZIP CODE: 76021 (A) LAST NAME: Haider, Karl-Heinz (B) STREET: Hervikusgasse 4/3/21 (C) CITY: Vienna (E) COUNTRY: Austria ( F, (R) POSTAL CODE: 1120 ka (A) SURNAME: Adolf, Gunther (B) STREET: Stiftgasse 15-17/10 vo (C) CITY: Vienna (E) COUNTRY: Austria (R) POSTAL CODE: 1070 ( A) LAST NAME: Ostermann, Elinborg (B) STREET: Mauerbachstr. 56/6 65 (C) CITY: Vienna (E) COUNTRY: Austria

(P) POSTAL CODE: 1140 (A) LAST NAME: Parcelt, Erik (B) STREET: Hans-Buchmüller-Gasse 8 (C) CITY: Purkexdorf (E) COUNTRY: Austria (P) POSTAL CODE: 3002 (A) LAST NAME: Sproll, Marlize (B) STREET: Schwenkgasse Z 70 (C) CITY: Vienna (E) COUNTRY: Austria (R) POSTAL CODE: 1120 (i) TITLE OF THE INVENTION:

The method of diagnosis and treatment of squamous cell cancer (her) NUMBER OF SEQUENCES: 16 (m) COMPUTER FORM: (A) INFORMATION MEDIA: Floppy disk (B) COMPUTER: IVM RO-compatible (C) OPERATING SYSTEM: RO-BO5/ M5-005 (B) SOFTWARE: Raigepiip Keyeaze 41.0,

Megviop Ya1.30 (ERA) (2) SEQUENCE DATA 5EO IJUO MO: 1: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 129 bp (B) TYPE: Nucleotide (C) FORM: both i) (B) TOPOLOGY: both (y) TYPE OF MOLECULE: genome-DNA (yX) CHARACTERS: u zo (A) NAME/CODE: exon (B) POSITION: 1..129 x, (B) OTHER DATA: o /rgodisi- "СО44 "

Labrei: ub -- /poie- "SepVapk daya raze assezviop Mo. I 05411" "o /syaip- ( ГП) (иХ) SIGNS: (A) NAZVAZHOD: SO (B) POSITION: 3..128 " (СХ) DATA ON PUBLICATION: in s (A) AUTHORS: Zsgeviop, OK

And Veii, MU and? Waskwop, OS

SogpeiIv, EV

Sepy, and b vei, U (B) TITLE: Sepotis vigisishge oi OMA epsodipd (pe IMtrposuie potipd geseriog CO44 hemeaiv ai Ivavi 12 - anegpaiimeiu zriisey ehopz o (C) JOURNAL: Rgos. Maii. Asad. Zei. O.5.A (r) VOLUME: 89

Me. (R) PAGES: 12160-12164 sp (Sb) DATE: December 1992 (KU ESSENTIAL RESIDUES IN THE SEQUENCE OF ZEO IO MO: 1: from 1 to 129 (СХ) PUBLICATION DATA: 5B (Н) REGISTRATION NUMBER: ОЕ 195 45 472.3 (I) APPLICATION DATE: December 6, 1995 (F, (Х) PUBLICATION DATA: ka (Н) REGISTRATION NUMBER: ОЕ 196 15 074.4 (I) APPLICATION DATE: April 17, 1996 60 (xi) SEQUENCE DESCRIPTION: ES IO MO: 1.: b5

TS SAB OSA AST SST AST AST ASA ASb SAM bAA ASA SST ACC SAb AAb 47 bip Aza TpgoRgo beg be TVg o TVg bio biz Teg oAL1ia Tpgobip bu 1 Z 10 15

SAA Sdo tob TtTtT obo AAS ASA T7ob SAT SAb bbA TAT 007 SAA ASA SSlo 95 sich bip Ter Re Siu Azp o ago Thr Niz b19 biu Tut tue bil TV bhe 20 25 zo

ABA SAA SAS OTOS SAT TS ASA ASA bob ASA OST b 129

At sila Avr bek Ni bak tls Svt biu Tykh A1a

Y 35 40 (2) SEQUENCE DATA 5EO IO MO: 2: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (6) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) DESCRIPTION SEQUENCES: 5EO IYUO MO: 2 bip Via TVi Rgo beg Tah Tph bi bi Taho Aia tlkobip Buv bi 1 5 10 15 sip Ter Rpe biu Azp Agu Ter Ni5 bij b1iu Tuk Agu Sip tTVh Rgo AhF i 20 25 30 bij ABr bek Ni bek TNI TBg biu TPh Aza 35 40 (2) SEQUENCE DATA 5EO IO MO: 3: s () SEQUENCE CHARACTERISTICS: o (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) FORM: single strand (6) TOPOLOGY: linear iv) (its) TYPE OF MOLECULE: peptide (x) PUBLICATION DATA: and (H) REGISTRATION NUMBER: ОЕ 195 45 472.3 | "c) () APPLICATION FILING DATE: December 6, 1995 - (x) PUBLICATION DATA: (H) REGISTRATION NUMBER: ОЕ 196 15 074.4 (se) (I) APPLICATION FILING DATE: April 17, 1996 (xi) SEQUENCE DESCRIPTION: 5EO IO MO: 3:

EE TIR Re b1u y Ag9 TIR o Nib5 bi t TUK Agu 51 " (2) DATA ON THE SEQUENCE 5EO IO MO: 4: - s () CHARACTERISTICS OF THE SEQUENCE: a (A) LENGTH: 27 base pairs "" (B) SPECIES: Nucleotide (C) FORM: single strand (6) TOPOLOGY: linear (o) (i) TYPE OF MOLECULE: other nucleic acids - (A) DESCRIPTION: /dess - "YEAR rgiteg"" (x) DATA ON PUBLICATION: («c ) (H) REGISTRATION NUMBER: OE 195 45 472.3

Fu 50 (17 APPLICATION DATE: 06 December 1995 (x) PUBLICATION DATA: sl (Н) REGISTRATION NUMBER: ОЕ 196 15 074.4 (И) APPLICATION DATE: 17 April 1996 (xi) SEQUENCE DESCRIPTION: 5EO IO MO: 4: 95 (2) SEQUENCE DATA 5EO IO MO: 5: (F, () SEQUENCE CHARACTERISTICS: ka (A) LENGTH: 30 base pairs (B) TYPE: Nucleotide in (C) FORM: single strand (6) TOPOLOGY: linear (i) TYPE OF MOLECULE: 5opviide Mysivipzatsyge (A) DESCRIPTION: /dezs - "ROK rgiteg"" (x) DATA ON PUBLICATION: 65 INU REGISTRATION NUMBER: ОЕ 195 45 472.3 () APPLICATION SUBMISSION DATE: December 6, 1995

(x) PUBLICATION DATA: (H) REGISTRATION NUMBER: ОЕ 196 15 074.4 (I) APPLICATION DATE: April 17, 1996 (xi) SEQUENCE DESCRIPTION: 5EO IO MO: 5:

TSATAASSAA SOATTOASAT TASSATTOSA 30 (2) SEQUENCE DATA- 5EO AND MO: 6: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) FORM: single strand. (6) TOPOLOGY: linear (its) TYPE OF MOLECULE: peptide (x) PUBLICATION DATA: 19 (H) REGISTRATION NUMBER: ОЕ 195 45 472.3 () APPLICATION DATE: 06 December 1995 (x) PUBLICATION DATA: (H) REGISTRATION NUMBER: OE 196 15 074.4 (I) APPLICATION DATE: April 17, 1996 (xi) SEQUENCE DESCRIPTION: 5EO IO MO: 6;

TIvr Rpe Ssiu A5p Ato Tgr Kiz b15 b1u Tuk Ago s 1 5 10 (2) SEQUENCE DATA 5EO IO MO: 7: сч () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 43 amino acids and) (B) TYPE: amino acid (C ) FORM: single strand (6) TOPOLOGY: linear u z o (its) TYPE OF MOLECULE: peptide (x) PUBLICATION DATA: x, (H) REGISTRATION NUMBER: ОЕ 195 45 472.3 o () APPLICATION DATE: 06 December 1995 ( x) DATA ON THE PUBLICATION: -- (H) REGISTRATION NUMBER: ОЕ 196 15 074.4 «о (I) APPLICATION DATE: April 17, 1996 (xi) SEQUENCE DESCRIPTION: 5EO IO MO: 7: big Aia Tak Rgo 5ei Zeg Tpg TV bio bi TpgoA1ia tTlk bil puz b10 1 5 10 15 « bip Ttr Rpe S1iu Azp Agud Tur Ni bich b1iu Tug Ahd bip tTagoRgo As - s 20 25 33 i bij ABr Beg Niz Beh TVg Tg Siu tbg Aia dia a 35 40 (2) DATA FOR THE SEQUENCE 5EO IO MO: 8: () CHARACTERISTICS OF THE SEQUENCE:

Ge» (A) LENGTH: 11 amino acids (B) TYPE: amino acid - (C) FORM: single strand o (6) TOPOLOGY: linear (its) TYPE OF MOLECULE: peptide

Me. (х) DATA ON PUBLICATION: sp (Н) REGISTRATION NUMBER: ОЕ 195 454723 () DATE OF APPLICATION SUBMISSION: December 06, 1995 (x) DATA ON PUBLICATION: (Н) REGISTRATION NUMBER: ОЕ 196 150744 (И) DATE OF APPLICATION SUBMISSION: 17 April 1996 (F, (xi) DESCRIPTION OF THE SEQUENCE: 5EO IO MO: 8: ko bek bek TpPh Tph biy bish TBh Aza Tvhobip Tu5 1 Z 10 60 (2) DATA ON THE SEQUENCE 5EO IO MO: 9: () CHARACTERISTICS OF THE SEQUENCE: ( A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) SHAPE: single thread" b5 (6) TOPOLOGY: linear

(her) TYPE OF MOLECULE: peptide (x) PUBLICATION DATA: (H) REGISTRATION NUMBER: ОЕ 195 45 472.3 () APPLICATION DATE: 06 December 1995 (x) PUBLICATION DATA: (Н) REGISTRATION NUMBER: ОЕ 196 15 074.4 (I) APPLICATION DATE: April 17, 1996 (xi) SEQUENCE DESCRIPTION: 5EO 13 MO: 9: 70 sib biz tpg dia TU Sip Pu5 Si bip Tgo 1 Z 10 (2) DATA ON SEQUENCE 5EO IO MO: 10: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids t (B) TYPE: amino acid (C) FORM: single strand (6) TOPOLOGY: - linear (its) TYPE OF MOLECULE: peptide (x) PUBLICATION DATA: (H) REGISTRATION NUMBER : ОЕ 195 45 472.3 () APPLICATION DATE: 06 December 1995 (х) PUBLICATION DATA: (Н) REGISTRATION NUMBER: ОЕ 196 15 074.4 (И) APPLICATION DATE: 17 April 1996 сх (xi) SEQUENCE DESCRIPTION: 5EO IХО MO: 10: Go)

TI dia Tk bip was bii bip Ter Rbe b1u Azp 1 5 I 10 (2) SEQUENCE DATA 5EO IO MO: 11: iv) () CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids y-o (B) TYPE: amino acid (ав) (С) FORM: single thread - (6) TOPOLOGY: linear (its) TYPE OF MOLECULE: peptide (Ce) (х) PUBLICATION DATA: (Н) REGISTRATION NUMBER: ОЕ 195 45-472.3" () SUBMISSION DATE APPLICATIONS: December 06, 1995 (x) PUBLICATION DATA: (Н) REGISTRATION NUMBER: ОЕ 196 15 074.4 - с (I) APPLICATION FILING DATE: April 17, 1996 a (xi) SEQUENCE DESCRIPTION: 5EO IЮО MO: 11: "» sip Ter Rre biu Azy Aku TgroNiz bi) biu Tug Agu Sip TVg 1 5 10 (2) SEQUENCE DATA 5EO AND MO: 12: (22) () BEOSHOEMYA KEMMABISNEM: - (A) LENGTH: 11 amino acids (B) TYPE: amino acid (ав) (С) ZTKAMOEOKM: single strand bu 50 (6) TOPOLOGY: linear (its) TYPE OF MOLECULE: peptide sl (x) PUBLICATION DATA: (Н) REGISTRATION NUMBER: ОЕ 195 45 472.3 () APPLICATION SUBMISSION DATE: December 6, 1995 (x) PUBLICATION DATA: (Н) REGISTRATION NUMBER: ОЕ 196 15 074.4 о (I) APPLICATION SUBMISSION DATE И: 17-А-РК-1996 ko (хи) DESCRIPTION OF THE SEQUENCE: 5EO IYUO MO: 12:

Azp Ak Ter Niv bij s1u Tug Aga bip tah Rgo 60 1 5 10 (2) SEQUENCE DATA 5EO IO MO: 13: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid bo (C) FORM : a separate thread

(6) TOPOLOGY: linear (its) TYPE OF MOLECULE: peptide (x) PUBLICATION DATA: (H) REGISTRATION NUMBER: ОЕ 195 45 472.3 () APPLICATION DATE: 06 December 1995 (x) PUBLICATION DATA: (H) REGISTRATION NUMBER: OE 196 15 074.4 (I) APPLICATION DATE: April 17, 1996 70 (xi) SEQUENCE DESCRIPTION: 5EO IO MO: 13: si biu Tukhk Akad bip TP o Rgo Ahud bi) Azr beg 1 5 10 (2) DATA TO SEQUENCES 5EO IO MO: 14: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) FORM: single strand (6) TOPOLOGY: linear (its) TYPE OF MOLECULE: peptide (x) DATA ABOUT PUBLICATION: (Н) REGISTRATION NUMBER: ОЕ 195 45 472.3 () DATE OF APPLICATION SUBMISSION: December 06, 1995 (х) PUBLICATION DATA: (Н) REGISTRATION NUMBER: ОЕ 196 15 074.4 с (1) DATE OF APPLICATION SUBMISSION: April 17, 1996 ) (xi) DESCRIPTION OF THE SEQUENCE: 5EO IYUO MO: 14: o oStvh Vgs Aga bis Ar beg No 5ei tyag S1u 1 5 10

Io) zo (2) SEQUENCE DATA 5EO IO MO: 15: () SEQUENCE CHARACTERISTICS: x, (A) LENGTH: 42 amino acids o (B) TYPE: amino acid (C) FORM: single strand -- (6) TOPOLOGY: linear "o (her) TYPE OF MOLECULE: peptide (x) PUBLICATION DATA: (H) REGISTRATION NUMBER: ОЕ 195 45 472.3 () APPLICATION DATE: 06 December 1995 " (x) PUBLICATION DATA: s (H) REGISTRATION NUMBER: PE 196 15 074.4. (I) APPLICATION FILING DATE: April 17, 1996 and (xi) SEQUENCE DESCRIPTION: ZEO AND MO: 15:

Tgr Aza ABr Rgo Azp o b5et Trg TVt bij biz Aza A1a Tpgobip tsuz bij 1 5 10 15 y (is) Tuz Ter Rpe bis Abp bi Tkgrobip b1u Guz AzporRgo Rgo Tak Rho 5eh 20 25 30 - bij ABr Beg Niz Ma1i Tag b1its Siu So TVh av) 35 40

Ge» 50 (2) SEQUENCE DATA BE AND O MO: 16: () SEQUENCE CHARACTERISTICS: sl (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) FORM: single strand (6) TOPOLOGY: linear

HF) (her) TYPE OF MOLECULE: Peptide (x) PUBLICATION DATA: o (H) REGISTRATION NUMBER: ОЕ 195 45 472.3 () APPLICATION DATE: 06 December 1995 60 (x) PUBLICATION DATA: (H) REGISTRATION NUMBER: ОЕ 196 15 074.4 (И) -DATE OF APPLICATION SUBMISSION: April 17, 1996 (xi) SEQUENCE DESCRIPTION: 5EO IYUO MO: 16: b5

Claims (8)

The formula of the invention 1. Means for the treatment of squamous cell cancer containing an active compound, which is characterized by the fact that as an active compound it contains an effective amount of an antibody or an antibody molecule that binds to the amino acid sequence MEZO MAKUUNESUK. 2. The tool according to claim 1, which differs in that as an antibody or an antibody molecule it contains a monoclonal antibody VIM/A-1 (MEE-18), which is obtained from a hybridoma cell line deposited under MOZM ACC2174, or a derivative of this antibody. 3. Means according to one of claims 1 or 2, which is characterized by the fact that as an antibody or antibody molecule it contains a 70 monoclonal antibody, a Par- or P(ab')"-fragment of an immunoglobulin, a recombinantly obtained antibody, a recombinantly obtained chimeric or humanized antibody , bifunctional antibody or single-chain derived antibody (sgm). 4. Means according to one of claims 1-3, which is characterized by the fact that it contains an antibody or an antibody molecule that binds to a radioactive isotope, a photoactivated compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule , toxin, cytostatic, prodrug, antibody molecule with a different specificity, cytokine or other immunomodulatory polypeptide. 5. A method of treating squamous cell cancer based on the binding of an antibody molecule to an epitope encoded by the variable exon mb of the CO44 gene, which is characterized by the use of an antibody or an antibody molecule that recognizes the amino acid sequence MMEOMACUMUNESUK. b. The method according to claim 5, which differs in that as an antibody or antibody molecule, the monoclonal antibody VIMUA-1 (MEE-18) is used, which is obtained from the hybridoma cell line deposited under MOZM ACC2174, or a derivative of this antibody. 7. The method according to one of claims 5 or b, which is characterized by the fact that a monoclonal antibody, Bar- and E(ar)"-fragment of immunoglobulin, recombinantly obtained C antibody, recombinantly obtained chimeric or humanized antibody, bifunctional antibody is used as an antibody or antibody molecule antibody or (5) single-chain derivative antibody (sgm). 8. The method according to one of claims 5-7, which is characterized by the fact that an antibody or an antibody molecule is used, which is combined with a radioactive isotope, a photoactivated compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytostatic , prodrugs, molecule M) antibodies with a different specificity, a cytokine or another immunomodulatory polypeptide. so "c) "- (Se) - and? (o) - («c) (o) sl ime) 60 b5