RU2193779C2 - Method and preparation for treating the cases of flat cell cancer - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: preparation contains effective quantity of or molecules of antibody bindable on WFGNRWHEGYR amino acid sequence as active ingredient. Method involves administering the preparation for treating the cases of flat cell cancer. EFFECT: enhanced effectiveness of treatment. 8 cl, 5 dwg, 6 tbl

Description

 The invention relates to a method for the treatment of squamous cell carcinoma based on the expression of the variable exon v6 of the CD44 gene, and also to a means for implementing such a method.

и др., 1991). В то время как наименьшая изоформа CD44 стандартная форма CD44s повсеместно выражается в ряду различных тканей, в том числе эпителиальных клеток, определенные варианты сплайсинга CD44 (CD44v) выражаются только на подгруппе эпителиальных клеток. Изоформы CD44 так вырабатываются через альтернативный сплайсинг, что последовательности 10 экзонов (v1-v10) в CD44s полностью вырезаются, однако при больших вариантах могут существовать в различных комбинациях (Screaton и др., 1992; Heider и др., 1993; Hofmann и др., 1991). Варианты различаются тем, что в определенное положение внеклеточной части белка встраиваются различные аминокислотные последовательности. Такие варианты можно было обнаружить в различных опухолевых клетках человека и в ткани опухоли человека. Так, недавно была исследована экспрессия вариантов CD44 в ходе колоноректального канцерогенеза (Heider и др., 1993). Экспрессия вариантов CD44 недостаточна в нормальном эпителии толстой кишки человека, и лишь слабая экспрессия обнаруживается в пролиферирующих клетках крипт. На поздних стадиях прогрессирования опухоли, например в случае аденокарциномы, выражаются все злокачественные перерожденные варианты CD44. Далее, недавно была показана экспрессия сплай-синговых вариантов CD44 в активированных лимфоцитах, а также в случае неходжкинских лимфом (Koopman и др., 1993).Recently it has been shown that the expression of glycoprotein variants of the surface of CD44 is necessary and sufficient to cause the so-called spontaneous metastatic behavior not only in the non-metastatic rat pancreatic adenocarcinoma cell line, but also in the non-metastatic rat fibrosarcoma cell line (

et al., 1991). While the smallest isoform of CD44, the standard form of CD44s is universally expressed in a number of different tissues, including epithelial cells, certain splicing variants of CD44 (CD44v) are expressed only on a subgroup of epithelial cells. CD44 isoforms are so produced through alternative splicing that sequences of 10 exons (v1-v10) in CD44s are completely excised, however, with large variants, they can exist in various combinations (Screaton et al., 1992; Heider et al., 1993; Hofmann et al. , 1991). Variants differ in that different amino acid sequences are inserted into a specific 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 CD44 variants during colonorectal carcinogenesis has recently been investigated (Heider et al., 1993). The expression of CD44 variants is insufficient in the normal epithelium of the human colon, and only weak expression is found in proliferating crypt cells. In the late stages of tumor progression, for example in the case of adenocarcinoma, all malignant degenerate variants of CD44 are expressed. Further, the expression of splice variants of CD44 in activated lymphocytes, as well as in the case of non-Hodgkin lymphomas, has also been shown recently (Koopman et al., 1993).

 There are various approaches that are suitable for the differential expression of the variant exon of the CD44 gene in tumors and normal tissues for diagnostic and treatment methods (international patent applications 94/02633, 94/12631, 95/00658, 95/00851, European patent 0531300).

 The expression of variant CD44 molecules in squamous cell carcinoma was also investigated. Salmi et al. (1993) found, using the Var3.1-specific antibody, a weakening of v6 expression in tumor cells compared to normal cells. Brooks et al. (1995) obtained heterogeneous staining for nasopharyngeal cancer using a specific v6 antibody 11.9. Only in 2 out of 12 cases, strong staining was achieved, while in most cases only weak focal expression of v6 could be detected immunohistologically.

 The objective of the presented invention was to develop new methods of treating squamous cell cancer, as well as to develop means for implementing such methods.

 This problem is solved by the proposed tool for the treatment of squamous cell carcinoma containing the active substance and certain final additives, due to the fact that as the active substance it contains an effective amount of an antibody or antibody molecule that (which) binds to the amino acid sequence WFGNRWHEGYR.

 This agent is used in a method for treating squamous cell cancer based on binding of an antibody molecule to an epitope encoded by the variable exon v6 of the CD44 gene by using an antibody or antibody molecule that recognizes the amino acid sequence WFGNRWHEGYR, which is an additional object of the invention.

 According to a preferred embodiment of the method for treating squamous cell carcinoma, the monoclonal antibody BIWA-1 (VFF-18), formed from a hybridoma cell line deposited under DSM ACC2174, or a derivative thereof, is used as an antibody or antibody molecule.

According to a further preferred embodiment of the method for treating squamous cell carcinoma, a monoclonal antibody, a Fab or F (ab ') ₂ immunoglobulin fragment, a recombinantly produced antibody, a recombinantly produced chimeric or humanized antibody, a bifunctional antibody or a single chain derivative of an antibody are used as an antibody or antibody molecule ( scFv).

 Another preferred embodiment of the method for treating squamous cell cancer is that an antibody or antibody molecule is used that (which) is coupled to a radioactive isotope, photoactivated compound, radioactive compound, enzyme, fluorescent dye, biotin molecule, toxin, cytostatic, prodrug, molecule antibodies with a different specificity, cytokine or other immunomodulatory polypeptide.

 In particular, the presented invention relates to a method that is based on strong and homogeneous expression of v6 in squamous cell carcinoma, which unexpectedly could be firmly established, contrary to the theory known in the art. Antibody molecules with appropriate specificity are suitable, in particular, as a drug base, to selectively reach in vivo squamous cell carcinoma.

 A method is preferred, characterized in that an antibody molecule is used that recognizes the amino acid sequence QWFGNRWHEGYRQT, particularly preferably the amino acid sequence WFGNRWHEGYR. Particularly preferred is the BIWA-1 monoclonal antibody (clone VFF-18), which is formed from the hybridoma cell line deposited on 7. 6. 1994 under DSM ACC2174 in the German Collection of Microorganisms and Cell Culture (DSM) GmbH, Masheroder Veg 1b, D- 38124 Braunschweig, Germany (international patent application 95/33771), or a derivative of this antibody.

 Another aspect of the present invention relates to means for implementing the method according to the invention.

и др., 1993). Существование дегенерированных или аллельных вариантов не имеет значения для осуществления изобретения; поэтому подобные варианты определенно включаются. Последовательность экзона v6 человеческого гена CD44 представляет из себя:
Q A T P S S T T E E T A T Q
ТС CAG GCA ACT CCT AGT AGT АСА ACG GAA GAA АСА GCT ACC CAG
R E Q W F G N R W H E G Y R Q
AAG GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAA
T P R E D S H S T T G T A
АСА CCC AGA GAA GAC TCC CAT TCG АСА АСА GGG АСА GCT G.The nucleotide and amino acid sequence of the variant exon v6 of the CD44 gene is known (Screaton et al., 1992,

et al., 1993). The existence of degenerated or allelic variants is not important for the implementation of the invention; therefore, such options are definitely included. The sequence of exon v6 of the human CD44 gene is:
QATPSSTTEETATQ
TC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ACC CAG
REQWFGNRWHEGYRQ
AAG GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAA
TPREDSHSTTGTA
ACA CCC AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G.

 The invention can be carried out with polyclonal or monoclonal antibodies specific for the epitope that is encoded by exon v6, in particular the epitope within the amino acid sequence QWFGNRWHEGYRQT, particularly preferably within the amino acid sequence WFGNRWHEGYR. Obtaining antibodies against known amino acid sequences can be carried out by known methods (Catty, 1989). For example, a peptide with such a sequence can be synthetically prepared and introduced as an antigen into the immunization protocol. Another way involves obtaining a composite protein containing the desired amino acid sequence, in such a way that a nucleic acid (synthetic or, for example, one that can be obtained by polymerase chain reaction (PCR) from a suitable sample) that encodes this sequence is integrated into an expression vector and a fusion protein formed upon fusion is expressed in the host organism. If necessary, the purified composite protein can then be inserted as an antigen into the immunization protocol, and insertion-specific antibodies or, in the case of monoclonal antibodies, hybridomas that express insertion-specific antibodies are selected in an appropriate manner. Such methods are caused by the prior art. Heider et al. (1993, 1996a) and Koopman et al. (1993) describe the production of antibodies against variant CD44 epitopes.

и Seemann, 1991), биспецифичными или бифункциональными антителами (Weiner и др., 1993; Goodwin, 1989, Featherstone, 1996), одноцепочечным производным антитела (scFv, Johnson и Bird, 1991), полными или фрагментарными иммуноглобулинами (Coloma и др., 1992; Nesbit и др., 1992; Barbas и др., 1992) или полученными путем перемешивания цепей антителами (Winter и др., 1994). Гуманизированные антитела могут, например, быть получены путем трансплантации КОУ (европейский патент 0239409). Также могут быть модифицированы каркасные участки (европейский патент 0519596; международная заявка на патент 9007861). Для гуманизирования антител теперь можно применять такие способы, как ПЦР (см., например, европейский патент 0368684; европейский патент 0438310; международную заявку на патент 9207075), или компьютерное моделирование (см., например международную заявку на патент 9222653). Могут также быть получены и применены составные белки, например, составной белок из одноцепочечного производного антитела и токсина (Chaudhary и др., 1990; Friedman и др., 1993). Под широкое понятие "антитело" или "молекулы антител" должны подпадать, кроме поликлональных и моноклональных антител, все обсуждаемые в этом разделе соединения, а также другие соединения, которые структурно происходят от иммуноглобулинов и получаются известными для этого способами.Antibody molecules, which are derivatives of poly- or monoclonal antibodies, for example, Fab- or F (ab ') ₂ fragments of immunoglobulins, recombinantly produced single-chain derivative of an antibody (scFv), chimeric or humanized antibody, can also be used for the method according to the invention, as well as other molecules specifically bound to the epitope that are encoded by exon v6. From full immunoglobulin antibodies BIWA-1 (VFF-18) or another antibody can, for example, be obtained Fab - or F (ab ') ₂ fragments or other fragments (Kreitman et al., 1993). The specialist, in addition, is able to obtain recombinant specific for the v6 antibody molecules. In particular, he can, according to the analysis of the amino acid sequence of the BIWA-1 antibody (VFF-18) and / or using the hybridoma cell line that produces this antibody, especially from the genetic information contained in it, obtain recombinant antibody molecules of the same idiotype, such as , BIWA-1 (VFF-18), i.e., antibody molecules that have the same amino acid sequence as the BIWA-1 antibody (VFF-18) in the region of the antigen binding position (complementarity determining regions, KOC). Appropriate methods are determined by the prior art. Such recombinant antibody molecules may, for example, be humanized antibodies (Shin et al., 1989;

and Seemann, 1991), bispecific or bifunctional antibodies (Weiner et al., 1993; Goodwin, 1989, Featherstone, 1996), single chain derivatives of an antibody (scFv, Johnson and Bird, 1991), complete or fragmented immunoglobulins (Coloma et al., 1992; Nesbit et al., 1992; Barbas et al., 1992) or obtained by mixing chains with antibodies (Winter et al., 1994). Humanized antibodies can, for example, be obtained by transplantation of KOU (European patent 0239409). Frame sections can also be modified (European patent 0519596; international patent application 9007861). Methods for PCR humanization can now be applied, such as PCR (see, for example, European patent 0368684; European patent 0438310; international patent application 9207075), or computer simulation (see, for example, international patent application 9222653). Compound proteins can also be prepared and used, for example, a compound protein from a single chain derivative of an antibody and a toxin (Chaudhary et al., 1990; Friedman et al., 1993). The broad concept of "antibody" or "antibody molecules" should include, in addition to polyclonal and monoclonal antibodies, all the compounds discussed in this section, as well as other compounds that are structurally derived from immunoglobulins and obtained by methods known for this.

 It is within the capabilities of a person skilled in the art to obtain information about an epitope (cf. FIG. 1, FIG. 4) of an equivalent BIWA-1 (VFF-1) antibody with the same binding specificity. Such antibodies are therefore also included in the invention.

The present invention can also be used in the diagnosis of antibody molecules, preferably BIWA-1 antibody molecules, fragments thereof or recombinant antibody molecules with a similar idiotype, can be combined, for example, with radioactive isotopes, such as, for example, ¹²⁵ J, ¹³¹ J, ¹¹¹ In, ^99m TC, or with radioactive compounds (Larson et al., 1991; Thomas et al., 1989; Srivastava, 1989), with enzymes, such as peroxidase or alkaline phosphatase (Catty and Raykundalia, 1989), with fluorescent dyes (Johnson, 1989) or with biotin molecules (Guesdon et al., 1979). For therapeutic use, v6-specific antibody molecules, preferably BIWA-1 antibody molecules (VFF-18) or VFF-18 derived antibody molecules, for example fragments thereof or recombinant antibody molecules with a similar idiotype, can bind to radioactive isotopes, for example, ⁹⁰ Y , ¹³¹ J, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ⁶⁷ Cu, ²¹² Bi, ²¹³ Bi, ¹⁷⁷ Lu (Quadri et al., 1993; Lenhard et al., 1985; Vriesendorp et al., 1991; Wilbur et al. 1989, Maraveyas et al., 1995a, Jurcic and Scheinberg, 1994), with toxins (Vitetta et al., 1991; Vitetta and Thogre, 1991; Kreitman et al., 1993; Theuer et al., 1993), with cytostatics (Schrappe et al., 1992), with prodrugs (Wang et al., 1992; Senter et al., 1989), with photoactivable substances (Hemming et al., 1993), with an antibody molecule with a different specificity, or with radioactive compounds. An antibody molecule may further bind to a cytokine or other immunomodulatory polypeptide, for example, tumor necrosis factor, lymphotoxin (Reisfeld et al., 1996) or interleukin-2 (Becker et al., 1996). Antibody molecules for insertion into the pre-targeting system can also be modified, for example, using streptavidin or biotin (Goodwin, 1995).

 It is useful that in accordance with this diagnostic method it is possible to study samples taken from patients, for example, from biopsies, which establish the presence of sufficient data to diagnose squamous cell carcinoma or are already diagnosed in order to characterize the tumor in sufficient detail. Detection of variant CD44 molecules containing the amino acid sequence encoded by the variable exon v6 can be performed on the surface of the protein using antibodies or on the surface of a nucleic acid using specific nucleic acid probes or primers for polymerase chain reaction (PCR). The invention therefore relates to antibody molecules and nucleic acids that are suitable as probes or primers 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 known for this purpose. Extracts obtained from tissue samples or body fluids can be further examined by other immunological methods using antibodies, such as Western blotting, using enzyme immunosorbent assays (ELISA test, Catty and Raykundalia, 1989), radioimmunoassays (RIA, Catty and Murphy, 1989) or applied immunoassays. Research can be conducted qualitatively, semi-quantitatively or quantitatively.

 Along with in vitro diagnostics, antibody molecules with specificity according to the invention are also suitable for in vivo diagnosis of squamous cell carcinoma. If the antibody molecule has a detectable label, label determination can be made for diagnostic purposes, for example, for visual detection of a tumor in vivo (imaging), or, for example, for radiosurgical surgery. For the use of antibodies conjugated with radioactive isotopes for immune scintigraphy (imaging), there are, for example, a number of protocols on the basis of which a specialist can carry out the invention (Siccardi and others, 1989; Keenan and others, 1987; Perkins and Pimm, 1992; Colcher et al., 1987; Thompson et al., 1984).

 Data obtained by detecting and / or quantifying the expression of the variant epitope v6 CD44 can thus be used to make a diagnosis and prognosis. In this case, a combination with other prognostic parameters, for example, with the degree of tumor development, may be useful.

и др., 1994). Подходящими моноклональными антителами для такого применения являются мышиное антитело изотипа IgG2a или антитело человеческого типа IgGl. Немодифицированные антитела могут далее использоваться для индукции противоопухолевой реакции, свойственной больному, по антиидиотипическому механизму (Baum и др. , 1993; Khazaeli и др., 1994).Antibody molecules with specificity according to the invention and, if necessary, associated with a cytotoxic agent can be used to treat squamous cell carcinoma. Moreover, 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 administering conjugated or unconjugated antibodies (such as complete immunoglobulins, fragments, recombinant humanized molecules, or others) form the basis of the technique (Mulshine et al., 1991; Larsonn et al., 1991; Vitetta and Thorpe, 1991; Vitetta et al., 1991; Breitz et al., 1992. 1995; Press et al., 1989; Weiner et al. 1989; Chatal et al. 1989; Sears et al. 1982). The therapeutic use can, for example, be carried out analogously to the use of ASML antibody 1.1 (Setter et al., 1993). Unmodified monoclonal antibodies can be inserted directly during treatment when they exhibit an internal effector function suitable for cytotoxic effects, for example, complement-induced or antibody-induced cellular cytotoxicity (

et al., 1994). Suitable monoclonal antibodies for such use are a murine IgG2a isotype antibody or a human IgGl type antibody. Unmodified antibodies can then be used to induce an antitumor response characteristic of the patient by the anti-idiotypic mechanism (Baum et al., 1993; Khazaeli et al., 1994).

A preferred embodiment of the invention from the point of view of use for treatment is that the humanized v6-specific immunoglobulin or its F (ab ') ₂ fragment is linked to ⁹⁰ Y (Quadri et al., 1993; Vriesendorp et al., 1995), ¹³¹ J (Maraveyas et al. 1995a, 1995b; Juweid et al. 1995; Press et al. 1995; Thomas et al., Compiled: Catty 1985.p. 230-239), ¹⁸⁶ Re (Breitz et al. ., 1992, 1995) or with another suitable radioactive isotope and administered for radioimmunotherapy of squamous cell carcinoma. For example, a BIWA-1 antibody, or a humanized version of BIWA-1, or an F (ab ') ₂ fragment of BIWA-1, or a humanized antibody can bind to ⁹⁰ Y using a chelating linker, such as isothiocyanatobenzyl-diethylenetriaminepentaacetate (ITCB-DTPA), moreover, a specific activity of 5-20 mCi / mg, preferably 10 mCi / mg, must be achieved. This agent can then be administered to a patient with an antigen-positive tumor in a dosage of from 0.1 to 1 mCi / kg body weight, preferably from 0.3 to 0.5 mCi / kg body weight. If the antibody molecule is bound to ¹³¹ J, with a specific activity of 2 mCi / mg, a suitable dosage schedule is possible, for example 2 • 150 mCi for a period of 6 weeks. One of ordinary skill in the art can determine the maximum possible dosage (Maraveyas et al., 1995a, 1995b). With a total amount of 2 to 5 mg of protein intended for administration, administration can be carried out in the form of a rapid intravenous bolus injection. With large amounts of protein, the infusion can be made in a form convenient for administration. In the case of monoclonal antibodies, it may be necessary for the agent to be mixed with an excess (for example, a 10-fold molar excess) of a non-radioactive antibody before administration; in this case, it is better to administer as an intravenous infusion, for example, over 15 minutes. The introduction may be repeated. Treatment can be combined with external radiation therapy. Further treatment can be supported by bone marrow transplantation; this is especially necessary when, during treatment, a dose of more than 1.6 gray is delivered to the bone marrow.

 The antibody molecules of the invention can also be used ex vivo to purify CD34-positive cell line preparations and precursor cell preparations (immune purification). Radiation therapy or chemotherapy for squamous cell carcinoma can be supported by an autologous bone marrow transplant. The preparation of hematopoietic cell lines and precursor cells used in this process should not contain tumor cells. This can be achieved by incubation with antibody molecules according to the invention, for example, antibody-toxin conjugates (Myklebust et al., 1994; German patent 19648209.7).

 The antibody molecules of the invention can then be introduced as a recombinant construct into a T cell receptor for T lymphocytes. Such reprogrammed T-lymphocytes bind selectively on antigen-expressing tumor cells and exhibit a cytotoxic effect, so they can be used to treat squamous cell carcinoma (PCT / EP 9604631; Altenschmidt et al., 1996).

Graphic materials
FIG. 1. Determination of specificity for the BIWA-1 antibody epitope by binding to synthetic peptides derived from the sequence of human CD44v6.

 The corresponding rat CD44v6 peptide was tested with ASML 1.1 antibody. Binding was determined using an ELISA test, with the peptides being immobilized on titration plates (for comparison, Heider et al., 1996b, FIG. 2). -: no binding, +/-: weak binding, +: strong binding.

 FIG. 2. Immunohistochemical analysis of squamous cell carcinoma of the larynx (a) and liver metastasis of the esophageal cancer (b) using a specific CD44v6 monoclinal antibody BIWA-1.

 In both cases, the reactivity of the antibody against the membrane of tumor cells can be observed. Initial 40x magnification, additional hematoxylin staining.

 FIG. 3. Comparison of antigen binding of various CD44v6 specific monoclonal antibodies.

 The binding of four different CD44v6 specific monoclonal antibodies on human squamous cell cancer A-431 cells was measured using an ELISA test. The BIWA-1 monoclonal antibody exhibits a higher affinity for tumor cells than other monoclonal antibodies.

 FIG. 4. Refined mapping of the epitope of a monoclonal antibody BIWA-1.

 Binding of BIWA-1 on various overlapping synthetic peptides spanning amino acids 18-32 of the encoded CD44v6 region was measured in a competitive ELISA test. The minimal binding sequence (peptide v6 (19-29)) was emphasized.

FIG. 5. Biodistribution of ¹²⁵ J-labeled BIWA-1 in hairless mice subjected to A-431 xenogenic transplantation.

 Antibody accumulation is set as% of injected dose (IG) / g (mean ± standard error of measurement) 4, 24, 48, 120 and 168 hours after injection.

Examples
Example 1. Expression of CD44v6 in squamous cell carcinoma
the cloth
A total of 126 cases were analyzed immunohistochemically paraffinized tumor samples using monoclonal antibody BIWA-1 (clone VFF-18) for expression of CD44v6. In 31 cases, the samples included primary squamous cell carcinoma (in 15 cases of the larynx, in 16 cases of the skin), in 91 cases, metastases in the 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 the NIRC type CD44v (Hofmarm et al., 1991) was amplified by polymerase chain reaction (PCR) from cDNA of human keratinocytes. Both primers were PCR 5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3 ', positions 25-52. and 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-3 '. positions 1013-984 of the variant region of LCLC97, as described by Hofmann et al., contained the EcoRI restriction enzyme recognition site, which was used to directly clone the PCR product into the pGEX-2T vector (Smith et al., 1988). The resulting construct (pGEX CD44v HPKII, v3-vl0) encodes a compound protein with a molecular weight of about 70 kDa consisting of Schistosoma japonicum glutathione S-transferase and human CD44 exons v3-v10 (Fig. 1; Heider et al., 1993 ) The composite protein was expressed in E. coli and then purified by glutathione agarose affinity chromatography (Smith et al., 1988).

Female Balb / c mice were immunized intraperitoneally with purified protein affinity chromatography as follows:
1. immunization: 90 μg of the compound protein in Freund's complete adjuvant;
2. and 3. immunization: 50 μg of the compound protein in Freund's complete adjuvant.

и Milstein, 1975; Keamey и др., 1979).Immunization was carried out at intervals of 4 weeks. 14 days after the last immunization, the animals were again immunized over the next three days, each time 10 μg of the composite protein in phosphate buffered saline (PBS). The next day after that, the spleen cells of one animal with a high titer of antibodies were fused with P3X63-Ag8.653 mouse myeloma cells using polyethylene glycol 4000. The hybridoma cells were then selected on titration plates in a medium containing hypoxanthine, aminopterin and thymidine ( GAT medium) (

and Milstein, 1975; Keamey et al. 1979).

 Serum antibody titer or hybridoma screening was performed using an ELISA test. In such a test, titration plates were first coated with a compound protein (glutathione-S-transferase — CD44v3-10) or only glutathione-S-transferase. Serum dilutions or hybridomas were then incubated with serial dilutions and specific antibodies were determined using peroxidase-conjugated anti-mouse immunoglobulin antibodies. Hybridomas that only reacted with glutathione S-transferase were discarded. The remaining antibodies were first characterized in an ELISA test using domain-specific composite proteins (exon v3, exon v5 + v6, exon v6 + v7, exon v8-v10) (Koopman et al., 1993). Their immunohistochemical reactivity was tested on sections of human skin.

 BIWA-1 (VFF-18; for preparation and properties, see also International Patent Application 95/33771) was bound only to compound proteins containing a domain encoded by exon v6. To further establish the boundary of the antibody epitope, various synthetic peptides that represent portions of the v6 domain were used in the ELISA test (Fig. 1). The 14 amino acid peptide, v6D, showed the strongest binding. Therefore, the BIWA-1 epitope lies wholly or partially within the sequence of the QWFGNRWHEGYRQT domain, which is encoded by exon v6. This sequence is homologous to the ASML antibody 1.1 epitope that was used in a therapeutic rat model and which is specific for rat CD44v6 (FIG. 1).

Immunohistochemistry
Before incubation with the primary antibody, paraffin sections (4 μm) were dewaxed in a rotary histological instrument (Roth, Germany) three times, each 10 minutes, and then rehydrated in higher alcohols. Sections were quickly washed with distilled water and then boiled in a microwave oven (Sharp model R-6270) three times for 10 minutes at 600 W in a 0.01 M buffer containing citric acid - sodium citrate. After each incubation in the microwave, the sections were cooled for 20 minutes. After the last cooling, the carrier was washed in PBS and preincubated with normal goat serum (10% in PBS). After three washes in PBS, sections were incubated for one hour with the primary antibody (BIWA-1: 5 μg / ml; mouse IgG (isotype-negative control) 5 μg / ml in PBS / 1% bovine serum albumin / BSA /). Normal human skin sections were used as a positive control for the color reaction, since keratinocytes express the CD44 isoform containing v3-v10. Endogenous peroxidases are blocked by 0.3% hydrogen peroxide in PBS, and sections were incubated for 30 minutes with a biotinylated secondary antibody (anti-mouse IgG-F (ab ') ₂ , DACO Corporation). For color development, sections were incubated for 30 minutes with horseradish peroxidase, which was bound on biotin to the streptavidin-biotin-peroxidase complex (DACO Corporation). Then the sections were incubated for 5-10 minutes in a 3,3-diamino-9-ethylcarbazole substrate (Sigma Immunocemicals company), the reaction was stopped by the addition of water, and the sections were further stained with hematoxylin. Evaluation of staining was carried out using an Zeiss axioscopic light microscope, staining intensity was quantified as follows: "+++" - strong expression; "++" - moderate expression; "+" weak expression; "-" - unambiguously undetectable expression or lack of detectable expression. Only tumor cells with clear staining of the membrane were evaluated as positive. The percentage of positive tumor cells was approximately estimated in each section and two groups were formed: focal positive tumors (less than 10% of the tumor cells reacted with the antibody) and positive tumors (10 or more% of tumor cells were positive). When less than 80% of the tumor cells in positive cells reacted with the antibody, the corresponding percentage was obtained.

 Using a specific CD44v6 monoclonal antibody BIWA-1, 126 cases of squamous cell carcinoma of various origins were analyzed. Expression of CD44v6-containing isoforms was observed in all but one of the tumor samples. Most of the samples showed antigen expression on 80-100% of tumor cells, 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 CD44v6 molecules on these tumor cells, sections of normal human skin were stained in parallel with sections of tumors. Normal skin keratinocytes express a high level of CD44 isoforms and are considered the strongest CD44v6-expressing normal cells that have been described to date. Therefore, the staining of keratinocytes was taken as a standard and classified as “strong” (+++) in the applied assessment system. In most of the studied tumor samples, the color of the tumor cells was comparable to or even stronger than the color of the keratinocytes of the skin, only in a few cases there was a weak color of the tumor (3 cases of metastases to the lymph nodes) or moderate (2 primary cancers, 10 metastases). The staining reaction was absolutely homogeneous within a particular tumor section, with most of the tumor cell sections having the same color intensity. In the expression sample CD44v6, no significant differences were observed between the primary tumors and metastases. Detailed summarized results are given in table. 1, examples are presented in FIG. 2.

 80-100% of tumor cells reacted positively with BIWA-1. In cases where a smaller number of tumor cells reacted with the antibody, the corresponding percentage is indicated.

 Example 2. Expression of CD44v6 in kidney cancer, prostate cancer and liver metastases in colon cancer.

the cloth
We analyzed 19 cases of kidney cancer (12 cases of clear cell cancer, 5 cases of chromophilic, 1 case of chromophobic, 1 oncocytoma), 16 cases of primary prostate adenocarcinoma and 19 cases of lymph node metastases in colon cancer.

 Antibody BIWA-1 (see example 1).

Immunohistochemistry
For the study, see example 1.

 In contrast to squamous cell carcinoma, in most of the studied cases of kidney and prostate cancer, no expression could be detected or only focal expression of CD44v6 isoforms was detected. In the case of expression greater than focal in prostate cancer, staining was predominantly diffuse cytoplasmic and weak or heterogeneous compared with the staining of normal prostate epithelium. In 50% of the studied liver metastases with colon cancer, greater than focal expression of CD44v6 isoforms was detected. Staining in most cases was mild to moderate, with in most cases less than 100% of the tumor cells in the sample showed staining with BIWA-1. The results are summarized in table. 2.

Example 3. Characterization of specific for CD44v6 antibodies
Cell line
The human squamous cell carcinoma A-431 cell line (spontaneous epidermoid vulvar cancer) was obtained from the American Type Culture Collection (Rockwell MD) and cultured as directed by the manufacturer. The surface expression of CD44v6-containing isoforms was determined by analysis using a fluorescence stimulating cell sorting apparatus (FACS analysis) using the BIWA-1 monoclonal antibody coupled to fluorescein isothiocyanate (FITC).

Kinetic Constant Analysis
The affinity and kinetics of the interaction of the monoclonal antibody and CD44v6 were determined using surface plasma resonance (SPR), using the BIAcore 2000 system (Pharmacy Bayesensor). A composite protein including glutathione S-transferase and CD44, which contained the site encoded by exons v3-v10 (glutathione-S-transferase / CD44 v3-v10), was immobilized on a CM5 sensor chip, and the amine coupling method was used as directed manufacturer. Antibody in various concentrations (8-132nM) in HBS buffer (10 mM N- (2-hydroxy) ethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, 150 mM sodium chloride, 3.4 mM ethylenediaminetetraacetic acid / EDTA (0.05% surfactant P20 BIAcore) was injected through an antigen-specific surface at a flow rate of 5 μl / min. The interaction was calculated by the change in the SPR signal. Antibody dissociation was observed for 5 minutes in a stream of buffer (HBS). The surface of the chip was regenerated using a separate pulse in 15 μl of 30 mm hydrochloric acid. Data analysis and calculation of kinetic constants was performed using BIA estimation software (Pharmacy Bayesensor), version 2.1.

In this way, the affinity for the BIWA-1 monoclonal antibody antigen and other CD44v6-specific monoclonal antibodies (VFF4, VFF7, BBA-13 (IgGl, R&D systems, Abindon, England)) was compared. Kinetic and affinity constants of different antibodies were determined in each case in two independent experiments. In the table. Figure 3 shows the values of association rates (k _a ), dissociation rates (k _d ) and dissociation constants (K _d ) for four monoclonal antibodies. All monoclonal antibodies had similar k _a and k _d with the exception of BBA-13, which was 3 times lower than k _a , and VFF7, which showed a significantly higher dissociation rate (factor 5) compared to other monoclonal antibodies. This results in reduced binding affinity for VFF7 and BBA-13 compared to VFF4 and BIWA-1. The BIWA-1 antibody has the lowest K _d of all antibodies tested.

Antibody-protein interaction analysis using an ELISA test
CD44v6-expressing A-431 cells were cultured in 96-well plates (Falcon Microtest III, Becton Dickinson, Lincoln Park, NJ) 5 × 10 ⁴ cells per cell in RPMI 1640 medium with 10% amniotic calf serum (OST) in overnight at 37 ^o C. After washing with PBS / 0.05% Tween 20, the cells were fixed chilled to 0 ^o With ethanol for 1 minute, and then washed. Incubation with primary antibodies (VFF4, VFF7, BIWA-1, BBA-13, 1 ng / ml - 600 ng / ml, in each case in test buffer: PBS / 0.5% BSA / 0.05% Tween 20) carried out for 1 hour at room temperature and then washed three times. A rabbit horseradish peroxidase conjugated anti-mouse IgG antibody (DACO Corporation, Copenhagen, Denmark; dilution 1: 6000 in test buffer) was used as a secondary antibody (1 hour at room temperature). After three washes, it was stained with a solution of thymol blue (Kierkegaard + Perry, Gaithersburg, USA). Extinction was measured using a Hewlett-Packard reader. used in the ELISA test.

 In FIG. Figure 3 shows that the relative affinity of antibodies, as determined by BIAcore analysis, is reflected in their interaction with tumor cells, with BIWA-1 clearly demonstrating the highest binding affinity.

 The protein domain encoded by exon v6 of the CD44 gene consists of 45 amino acids (Fig. 4). To accurately determine the epitope that BIWA-1 recognizes, a series of synthetic peptides were used in ELISA tests. Preliminary experiments indicate binding on the center-located 14 - measure (amino acid residues 18-31; Fig. 4, cf. also Fig. 1), and not on the peptide outside this region. Therefore, a second series of peptides was synthesized and studied in competitive ELISA tests (Fig. 4). The results show that peptide 19-29 (WFGNRWHEGYR) represents the minimum structure required for high affinity binding. Elimination of the C-terminal arginine residue leads to more than 100-fold weak binding.

Example 4. Biodistribution of radioactive iodine-labeled anti-CD44v6 antibody in xenograft hairless mice
Xenograft Model A-431
Eight-week-old female Balb / c nu / nu hairless mice (B&C Universal, Renton, West Africa) were injected subcutaneously into the left-sided midline of 5 x 10 ⁶ cultured A-431 cells (human epidermoid vulvar cancer). Animals after xenograft with A-431 tumors were used for two weeks in biodistribution experiments (tumor weight 40-50 mg).

The introduction of radioactive iodine in BIWA-1
Protein G, a purified BIWA-1 monoclonal body (mouse IgGl), was bound on streptavidin, using a heterobifunctional crosslinker, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester. Streptavidin lysing residues were bound on the restored cysteinyl residues of the antibody, which were obtained by pretreatment of the antibody with dithiothreitis. The resulting conjugates (1: 1) (> 90%) were further purified by ion exchange chromatography. For BIWA-1 / streptavidin biodistribution experiments, ¹²⁵ J was labeled with the primary amino group of lysine, and p-iodophenyl reagent (PIF, NEN Dupont, Wilmington, Germany) was used for labeling, following the method of Willbur et al. (1989). Labeling BIWA-1 with streptavidin or ¹²⁵ J did not alter the immunoreactivity or pharmacokinetics of the antibody in mice.

Biodistribution experiments
Hairless mice that underwent x-ray transplantation with human A-431 tumors were injected intravenously through the lateral tail vein of 5-7 μCi ¹²⁵ J per 50 μg of BIWA-1 monoclonal antibody (specific activity 0.1-0.14 mCi / mg). The study of biodistribution depending on time was carried out in groups of 3 animals (n = 3) at 4, 24, 48, 120 and 168 hours after injection. At selected time points, the mice were euthanized, bled through the retroorbital plexus and killed by decapitation. Nine organs and tissues were collected and crushed, including blood, tail, lung, liver, spleen, stomach, kidneys, intestines, and tumor. Tissue radioactivity was calculated compared to the standard of the injected antibody preparation in a gamma-scintillation counter (Packard Instrement Company, Meriden, Connecticut), and the energy window of the counter was set at 25-80 keV for ¹²⁵ J. The percentage of injected dose / g of tissue was calculated (% ID / g).

Preliminary experiments showed that BIWA-1 does not cross react with the murine antigen CD44v6. In the table. 4 and in FIG. 5 shows the absorption of radioactivity in tumors and normal tissue. In the case of iodinated BIWA-1, rapid tumor uptake was observed (7.6% ID / g by 4 hours after injection), which increased to over 18% ID / g by 48 hours and then remained constant up to 120 hours. 7 days after injection (168 hours), the tumor contained another 15.3% ID / g of tissue. Proportions of the tumor: tissue was calculated at individual time points and are given in table. 4. 24 hours after the injection, the proportion of the tumor: blood was 0.48 and rose to 3.16 on the seventh day. Absorption in normal tissue was low and most likely caused by the background value of the blood pool in the biopsy tissue. Using in vivo selective targeting of human squamous cell carcinoma xenografts to hairless mice, it was shown using ¹²⁵ J BIWA-1 labeled that the monoclonal antibody has high potential as a targeting carrier for diagnostic and therapeutic use in patients with squamous cell carcinoma.

Example 5. Different expression of CD44v6 for a large number of human tumors
In another study, a total of 544 tumor samples were studied immunohistochemically using the BIWA-1 monoclonal antibody (clone VFF-18) for expression of CD44v6. Samples either
they were paraffinized, or immediately after surgical collection they were frozen in liquid nitrogen and stored until use at -70 ^o С. The following tumors were analyzed: basal cell tumors (n = 16), breast adenocarcinomas (AK) (n = 55), colon AK (n = 83), squamous cell carcinoma (RCC) of the head and neck (n = 125), lung cancer (n = 120), prostate AK (n = 34), kidney cancer (n = 27), skin RCC (n = 15) and AK stomach (n = 69). Tissues were obtained by routine surgery or by biopsy; normal tissue was associated with tumor samples. Immunohistochemical studies were performed as in example 1.

 In the table. 5 presents a review regarding immunohistochemical analysis of 397 different tumor samples using BIWA-1 monoclonal antibody.

 In small cell lung cancer, kidney cancer, and prostate AK, no or only slight reactivity was observed. All other investigated tumor types were expressed in various amounts containing CD44v6 isoforms. Most of the studied breast AKs showed reactivity with BIWA-1, and the investigated types of RCC (larynx, lung, and esophagus) expressed CD44v6 in 100% of cases.

 A total of 185 cases of RCC of various types and different classification for reactivity with BIWA-1 were investigated. These were 67 cases of primary RCC (larynx, n = 15; oral cavity, n = 16; oropharynx, n = 3; skin, n = 15), 77 samples of lymph node metastases (larynx, n = 12; lung, n = 27; esophagus, n = 11; oral cavity, n = 6; oropharynx, n = 7; laryngopharynx, n = 10; tonsil, n = 4) and 3 samples from metastases to the liver (esophagus). In the table. 6 shows the results of immunohistochemical analysis of all investigated RCC samples.

 Expression of CD44v6-containing isoforms was found in all but three tumor samples (one case of the larynx, 2 cases of the lung). Most samples showed antigen expression on 80-100% of tumor cells within one single section, with the stain predominantly concentrated on the membrane of the tumor cells. The most stained homogeneous sample was observed in the case of cancer of the larynx, esophagus and larynx, and most of the tumor cells of the slice had the same color intensity. The sequence protocol is presented at the end of the description.

Claims (8)

 1. An agent for treating squamous cell carcinoma containing an active substance and certain final additives, characterized in that as the active substance it contains an effective amount of an antibody or antibody molecule that (which) binds to the amino acid sequence WFGNRWHEGYR.  2. The tool according to p. 1, characterized in that it as an antibody or antibody molecule contains a monoclonal antibody BIWA-1 (VFF-18), formed from a hybridoma cell line deposited under DSM ACC2174, or a derivative of this antibody. 3. The tool according to one of p. 1 or 2, characterized in that it as an antibody or antibody molecule contains a monoclonal antibody, Fab or F (ab ') ₂ fragment of immunoglobulin, recombinantly obtained antibody, recombinantly obtained chimeric or humanized antibody , a bifunctional antibody or a single chain derivative of an antibody (scFv).  4. The tool according to one of paragraphs. 1 to 3, characterized in that it contains an antibody or antibody molecule that (which) binds to a radioactive isotope, a photoactivated compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytostatic agent, a prodrug, an antibody molecule with another specificity, cytokine or other immunomodulatory polypeptide.  5. A method for treating squamous cell carcinoma based on binding of an antibody molecule to an epitope encoded by the variable exon v6 of the CD44 gene, characterized in that an antibody or antibody molecule is used that recognizes the amino acid sequence WFGNRWHEGYR.  6. The method according to p. 5, characterized in that the monoclonal antibody BIWA-1 (VFF-18) formed from the hybridoma cell line deposited under DSM ACC2174 or a derivative of this antibody is used as an antibody or antibody molecule. 7. The method according to one of paragraphs. 5 and 6, characterized in that a monoclonal antibody, a Fab or F (ab ') ₂ fragment of an immunoglobulin, a recombinantly produced antibody, a recombinantly produced chimeric or humanized antibody, a bifunctional antibody or a single chain derivative of an antibody (scFv) are used as an antibody or antibody molecule )  8. The method according to one of paragraphs. 5 to 7, characterized in that the use of an antibody or antibody molecule, which (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 agent, a prodrug, an antibody molecule with another specificity, a cytokine or another immunomodulatory polypeptide.

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