SK284378B6 - Use of an antibody molecule which binds to the amino acid sequence WFGNRWHEGYR, for the preparation of a medicament for the treatment of squamous cell carcinomas - Google Patents
Use of an antibody molecule which binds to the amino acid sequence WFGNRWHEGYR, for the preparation of a medicament for the treatment of squamous cell carcinomas Download PDFInfo
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Abstract
Description
Vynález sa týka použitia protilátkovej molekuly, ktorá sa viaže na aminokyselinovú sekvenciu WFGNRWHEGYR na výrobu liečiva na liečenie karcinómov skvamóznych buniek, ktorý je založený na väzbe molekuly protilátky na epitop, ktorý je kódovaný variabilným exónom V6 génu CD44.The invention relates to the use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR for the manufacture of a medicament for the treatment of squamous cell carcinomas which is based on binding of the antibody molecule to an epitope encoded by the variable exon V6 of the CD44 gene.
Doterajší stav technikyBACKGROUND OF THE INVENTION
Nedávno sa preukázalo, že expresia variantov povrchových glykoproteínov CD44 je potrebná a postačujúca, aby sa vyvolalo takzvané spontánne metastázovanie nielen v nemetastázovanej bunkovej línii adenokarcinómu potkana, ale aj v nemetastázovanej fibrosarkómovej bunkovej línii potkana (Gunthert et al., 1991). Kým najmenšia izoforma CD44, štandardná forma CD44, je všeobecne exprimovaná v rade rozličných tkanív vrátane epitelových buniek, určité štcpnc varianty CD44 (CD44v) sú exprimované iba jednou podskupinou epitelových buniek. Uvedené izoformy CD44 sa vytvoria alternatívnym štepením tak, že sekvencie 10 exónov (vl až vlO) v génoch CD44 sa úplne vystrihnú, jednako však pri vyšších variantoch môžu nastávať rôzne kombinácie (Screaton et al., 1992; Heider et al., 1993; Hofmann et all., 1991). Varianty sa rozlišujú tým, že na určitom mieste mimobunkových častí proteínov sú vložené rozdielne aminokyselinové sekvencie. Také varianty možno dokázať v rôznych ľudských nádorových bunkách a ľudskom nádorovom tkanive. Tak sa nedávno zistila expresia variantov CD44 v priebehu kolorektálnej karcinogenézy (heider et al., 1993). Expresia variantov CD44 chýba v normálnom ľudskom kolónovom epiteli, a dokázateľná je iba slabá expresia v proliferovaných bunkách krýpt. V neskorších štádiách rozvoja nádoru, napríklad v adenokarcinómoch, všetky malígne odrody exprimujú varianty CD44. Ďalej sa nedávno preukázala expresia stepných variantov CD44 v aktivovaných lymfocytoch ako aj v neHodgkinových lymfómoch (Koopman et al., 1993).Recently, it has been shown that expression of CD44 surface glycoprotein variants is necessary and sufficient to induce so-called spontaneous metastasis not only in the non-metastasized rat adenocarcinoma cell line but also in the non-metastasized rat fibrosarcoma cell line, (1991). While the smallest CD44 isoform, the wild-type form of CD44, is generally expressed in a variety of tissues including epithelial cells, certain CD44 cleavage variants (CD44v) are expressed by only one subset of epithelial cells. Said CD44 isoforms are generated by alternative cleavage such that the 10 exon sequences (v1 to v10) in the CD44 genes are completely excised, but different combinations may occur with higher variants (Screaton et al., 1992; Heider et al., 1993; Hofmann et al., 1991). Variants are distinguished in that different amino acid sequences are inserted at a certain location of extracellular portions of the proteins. Such variants can be detected in various human tumor cells and human tumor tissue. Thus, expression of CD44 variants during colorectal carcinogenesis has recently been identified (heider et al., 1993). Expression of CD44 variants is absent in normal human colon epithelium, and only poor expression in proliferated crypt cells is detectable. In the later stages of tumor development, for example in adenocarcinomas, all malignant varieties express CD44 variants. Furthermore, the expression of CD44 steppe variants in activated lymphocytes as well as in non-Hodgkin's lymphomas has recently been demonstrated (Koopman et al., 1993).
Známe sú pokusy o využití rozdielov v expresii variantných exónov génov CD44 v nádoroch a v normálnych tkanivách na diagnostické a terapeutické účely (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP 0 531 300).Attempts have been made to exploit the differences in expression of variant exons of CD44 genes in tumors and normal tissues for diagnostic and therapeutic purposes (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP 0 531 300).
Expresia variantných CD44-molekúl v karcinómoch dlaždicového epitelu bola tiež už skúmaná. Sami et al. (1993) našiel pomocou v6-špecifickcj protilátky Var3.1 úbytok v6-expresie v nádorových bunkách v porovnaní s normálnymi bunkami. Brooks et a/.(l995) dosiahol pomocou v6-špecifickej protilátky 11.9 heterogénne vyfarbenie nosohltanového karcinómu. Iba v 2/12 prípadov sa dosiahlo silné vyfarbenie, zatiaľ čo vo väčšine prípadov sa mohol dosiahnuť iba slabý fokálny imunohistologický dôkaz.The expression of variant CD44 molecules in squamous epithelial carcinomas has also been investigated. Sami et al. (1993) found, by the v6-specific antibody Var3.1, a decrease in v6-expression in tumor cells compared to normal cells. Brooks et al. (1995) achieved a heterogeneous staining of nasopharyngeal carcinoma with the v6-specific antibody 11.9. Only in 2/12 cases strong staining was achieved, while in most cases only weak focal immunohistological evidence could be achieved.
Úlohou tohto vynálezu preto bol vývoj nového spôsobu diagnózy a terapie karcinómov dlaždicového epitelu, ako aj príprava prostriedkov na uskutočnenie daného spôsobu.It is therefore an object of the present invention to develop a new method for diagnosing and treating squamous cell carcinoma as well as to provide means for carrying out the method.
Podstata vynálezuSUMMARY OF THE INVENTION
Podstatou vynálezu je použitie protilátkovej molekuly, ktorá sa viaže na aminokyselinovú sekvenciu WFGNRWHEGYR na výrobu liečiva na liečenie karcinómov skvamóznych buniek, ktorý je založený na väzbe molekuly protilátky na epitop, ktorý je kódovaný variabilným exónom V6 génu CD44. Molekuly protilátky so zodpove dajúcou špecifickosťou sú vhodné najmä ako nosič, aby selektívne dosiahli karcinóm dlaždicového epitelu in vivo.The present invention provides the use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR for the manufacture of a medicament for the treatment of squamous cell carcinomas based on binding of the antibody molecule to an epitope encoded by the variable exon V6 of the CD44 gene. Antibody molecules of corresponding specificity are particularly useful as carriers to selectively achieve squamous cell carcinoma in vivo.
Výhodné sú také použitia, ktoré sa vyznačujú použitím molekuly protilátky, ktorá rozoznáva aminokyselinovú sekvenciu QWFGNRWHEGYRQT, výhodnejšie aminokyselinové sekvencie WFGNRWHEGYR. Výhodná je pritom najmä monoklonová protilátka BIWA-1 (kloň VFF-18), ktorá je odvodená z hybridomovej bunkovej línie, ktorá bola 7. 6. 1994 uložená v DSM (- Deutsche Sammlung flir Mikroorganismen und Zellkulturen GmbH, Mascheorder Weg lb, D-38 124 Braunschweig, BRD) pod úložným číslom DSM ACC2174, alebo deriváty tejto protilátky.Preferred are those uses that are characterized by the use of an antibody molecule that recognizes the amino acid sequence QWFGNRWHEGYRQT, more preferably the amino acid sequence WFGNRWHEGYR. Particularly preferred is the monoclonal antibody BIWA-1 (VFF-18 clone), which is derived from a hybridoma cell line which was deposited at DSM (Deutsche Sammlung flir Microorganismen und Zellkulturen GmbH, Mascheorder Weg 1b, D- 38,124 Braunschweig, BRD) under accession number DSM ACC2174, or derivatives thereof.
Ďalším predmetom vynálezu je aj farmaceutický prostriedok na liečenie uvedených chorôb.A further object of the invention is also a pharmaceutical composition for the treatment of said diseases.
Nukleová a aminokyselinová sekvencia variantných exónov v6 génov CD44 je známa (Screaton et. al., 1992; Tolg et al., 1993). Jestvovanie degenerovaných alebo všetkých variantov nie je z hľadiska uskutočnenia tohto vynálezu podstatné; také varianty sú vo vynáleze preto výslovne zahrnuté.The nucleic and amino acid sequences of variant exons of the v6 CD44 genes are known (Screaton et. Al., 1992; Tolg et al., 1993). The existence of degenerate or all variants is not essential to the practice of this invention; such variants are therefore expressly included in the invention.
Sekvencia exónu v6 ľudských génov CD44 je:The exon v6 sequence of the human CD44 genes is:
ΟΑΤΡδδΤΤΕΕΊ’ΑΊ’ΟΟΑΤΡδδΤΤΕΕΊ'ΑΊ'Ο
TC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ACC CAGTC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ACC CAG
KEQWFGNRWHEGYRQKEQWFGNRWHEGYRQ
AAG GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAAAAG GAA CAG TGG TTT GGC
TPREDSHSTTGTATPREDSHSTTGTA
ACA CCC AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G.ACA CCC AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G.
Vynález sa môže uskutočniť s polyklonovými alebo s mnoklonovými protilátkami, ktoré sú špecifické pre jeden epitop, kódovaný s exónom v6, najmä pre epitop v rozsahu aminokyselinovej sekvencie QWFGNRWHEGYRQT, najvýhodnejšie v rozsahu aminokyselinovej sekvencie WFGNRWHEGYR. Príprava protilátky proti známym aminokyselinovým sekvenciám sa môže vykonať spôsobmi, ktoré sú známe (Catty, 1989). Peptid s touto sekvenciou sa napríklad môže pripraviť synteticky a môže sa použiť v imunizačnom protokole ako antigén. Iným spôsobom možno prípravu uskutočniť tak, že sa pripraví fúzovaný proteín, ktorý obsahuje vyžadovanú aminokyselinovú sekvenciu, tak, že nukleová kyselina (syntetická alebo pripravená z vhodnej vzorky napríklad polymerázovou-reťazovou rekaciou(PCR)), kódujúca pre uvedenú sekvenciu, sa integruje do vektora expresie a fúzovaný proteín sa exprimuje v hostiteľskom organizme. Podľa potreby čistený, fúzovaný proteín sa potom môže použiť ako antigén alebo ako inzert špecifickej protilátky v imunizačnom protokole, alebo - v prípade monoklortovej protilátky - môže sa hybridom, ktorý včlenenenú špecifickú protilátku exprimuje, môže sa vhodným spôsobom selektovať. Takéto spôsoby sú v rámci doterajšieho stavu techniky známe. Heider et al. (1993, 1996a) a Koopman et al. (1993) opísali prípravu protilátky proti variantnému epitopu z CD44.The invention may be practiced with polyclonal or multiclonal antibodies that are specific for a single epitope encoded by exon v6, particularly for an epitope within the amino acid sequence QWFGNRWHEGYRQT, most preferably within the amino acid sequence WFGNRWHEGYR. Preparation of an antibody against known amino acid sequences can be accomplished by methods known (Catty, 1989). For example, a peptide with this sequence can be synthetically prepared and used as an antigen in an immunization protocol. Alternatively, the preparation can be carried out by preparing a fusion protein containing the desired amino acid sequence by integrating a nucleic acid (synthetic or prepared from a suitable sample, for example, by polymerase chain reaction (PCR)) encoding for said sequence, integrated into a vector expression and the fusion protein is expressed in the host organism. If desired, the purified, fusion protein can then be used as an antigen or as a specific antibody insert in an immunization protocol, or - in the case of a monocortical antibody - the hybrid that expresses the incorporated specific antibody can be appropriately selected. Such methods are known in the art. Heider et al. (1993, 1996a) and Koopman et al. (1993) described the preparation of an antibody against a variant epitope of CD44.
V spôsobe podľa tohto vynálezu môžu sa jednako použiť tiež molekuly protilátky, ktoré sú odvodené of poly- alebo monoklonových protilátok, napríklad fragment Fab alebo fragmenty F(ab')2 imunoglobulínu, rekombinantne pripravená jednoreťazová protilátka (scFv), chimérová alebo humanizovaná protilátka, ako aj iné molekuly, ktoré sa špecificky viažu na epitop, ktorý je kódovaný exónom v6. Protilátka BIWA-1 (VFF-18) alebo iné protilátky, napríklad fragmenty Fab alebo F(ab')2 sa môžu získať z úplného imunoglobulínu (Kreitman et al., 1993). Popri uvedenom je odborník v danej oblasti schopný vytvoriť molekuly rekombinantnej v6-špecifickej protilátky. Podľa analýzy a minokyselinovej sekvencie protilátky BIWA-1 (VFF-18) a/alebo s použitím hybridómovej bunkovej línie, ktorá túto protilátku produkuje, najmä ktorá obsahuje genetickú informáciu, môže odborník pripraviť molekuly rekombinantnej protilátky s rovnakým idiotypom, ako je BIWA-1 (VFF18), to znamená molekuly protilátky, ktorá má v oblasti antigénového väzbového miesta (complementaritydetermining regions, CDR) rovnakú aminokyselinovú sekvenciu ako protilátka BIWA-1 (VFF-18). Vhodné spôsoby sú v rámci doterajšieho stavu techniky známe. Uvedené molekuly rekombinatnej protilátky môžu napríklad byť humanizovaná protilátka (Shin et al., 1989; Gussov et Seemann, 1991), bišpecifická alebo bifunkčnú protilátka (Weiner et al., 1993; Goodwin, 1989; Featherstone, 1996), jednoreťazová protilátka (scFv, Johnson et Bird, 1991), úplný alebo fragmentovaný imunoglobulín (Coloma et al., 1992; Nesbiot et al., 1992; Barbas et al., 1992), alebo pomocou “chain shuffling pripravená protilátka (Winter et al., 1994). Humanizovaná protilátka sa môže pripraviť napríklad CDR-štepmi (EP 0 239 400). Môžu sa tiež modifikovať rámcové oblasti (EP 0 519 596; WO 90 07 861). Na humanizovanie protilátok sa dnes používajú spôsoby, ako je PCR (pozri napríklad EP 0 368 684; EP 0 438 310; WO 92 07 075), alebo počítačové modelovanie (pozri napríklad WO 92 22 653). Ďalej sa môžu tiež pripraviť a použiť fúzované proteíny, napríklad jednoreťazovou protilátkou/toxínom fúzovaný proteín (Chaudhary et al., 1990; Friedman et al., 1993). Pod uvádzaný pojem „protilátka” a „molekula protilátky” patria všetky v tejto časti diskutované zlúčeniny bez polyklonových a monoklonových protilátok, ako aj ďalšie zlúčeniny, ktoré možno štruktúrne odvodiť od imunoglobulínu a sú pripraviteľné známymi spôsobmi.However, antibody molecules that are derived from poly- or monoclonal antibodies, for example a Fab fragment or F (ab ') 2 immunoglobulin fragments, a recombinantly produced single chain antibody (scFv), a chimeric or humanized antibody, such as as well as other molecules that specifically bind to an epitope that is encoded by exon v6. The BIWA-1 antibody (VFF-18) or other antibodies, for example Fab or F (ab ') 2 fragments, can be obtained from complete immunoglobulin (Kreitman et al., 1993). In addition, one of skill in the art is able to generate recombinant v6-specific antibody molecules. Based on the analysis and amino acid sequence of BIWA-1 (VFF-18) and / or using a hybridoma cell line that produces this antibody, particularly that contains genetic information, one of ordinary skill in the art can prepare recombinant antibody molecules of the same idiotype as BIWA-1 ( VFF18), i.e., an antibody molecule having the same amino acid sequence in the antigen binding site (CDR) region as the BIWA-1 antibody (VFF-18). Suitable methods are known in the art. For example, said recombinant antibody molecules may be a humanized antibody (Shin et al., 1989; Gussov et Seemann, 1991), a bispecific or bifunctional antibody (Weiner et al., 1993; Goodwin, 1989; Featherstone, 1996), single chain antibody (scFv, Johnson et Bird, 1991), complete or fragmented immunoglobulin (Coloma et al., 1992; Nesbiot et al., 1992; Barbas et al., 1992), or chain shuffling antibody prepared (Winter et al., 1994). For example, a humanized antibody can be prepared by CDR-grafting (EP 0 239 400). Framework regions can also be modified (EP 0 519 596; WO 90 07 861). Methods such as PCR (see for example EP 0 368 684; EP 0 438 310; WO 92 07 075) or computer modeling (see for example WO 92 22 653) are now used to humanize antibodies. Furthermore, fusion proteins can also be prepared and used, for example, a single chain antibody / toxin fusion protein (Chaudhary et al., 1990; Friedman et al., 1993). The terms "antibody" and "antibody molecule" include all compounds discussed herein without polyclonal and monoclonal antibodies, as well as other compounds that can be structurally derived from immunoglobulin and are obtainable by known methods.
Pri známom epitope (porovnaj obr. 1, obr. 4) protilátky BIWA-1 (VFF-18) je priemerný odborník v danej oblasti schopný rovnako pripraviť ekvivalentnú protilátku s rovnakou väzbovou špecifickosťou. Také protilátky sú preto rovnako zahrnuté v rozsahu tohto vynálezu.In the known epitope (cf. FIG. 1, FIG. 4) of BIWA-1 (VFF-18), one of ordinary skill in the art can equally prepare an equivalent antibody with the same binding specificity. Such antibodies are therefore also included within the scope of the invention.
Na diagnostické účely sa môžu molekuly protilátky, výhodne molekuly protilátky BIWA-1, jej fragmenty alebo molekuly rekombinantnej protilátky rovnakého idiotypu viazať s rádioaktívnymi izotopmi, ako je napríklad 125I, l3lI, Ί, 99mTc alebo rádioaktívnymi zlúčeninami (Larson et al., 1991; Thomas et al., 1989; Srivastava, 1988), enzýmami, ako je napríklad peroxidáza alebo alkalická fosfatáza (Catty a Raykundalia, 1989), s fluorescenčnými farbivami (Johnson, 1989), alebo s molekulami biotínu (Guesdon et al., 1979). Na terapeutické použitie sa môžu molekuly špecifickej v6- protilátky, výhodne molekuly protilátky BIWA-1 (VFF-18), alebo od VFF-18 odvodené molekuly protilátky, napríklad jej fragmenty alebo molekuly rekombinantnej protilátky s rovnakým idiotypom, viazať s rádioizotopmi, ako sú 90Y, 131I, 186Re, l53Sm, 67Cu, 212Bi, 213Bi, 177Lu (Quadri et al., 1993; Lenhard et al., 1985; Vriesendorp et al., 1991; Wilbur et al., 1989; Maraveyas et al., 1995, Jurcic et Scheinberg, 1994), toxínmi (Vitetta et al., 19091; Vitetta et Thorpe, 1991; Kreitman et al., 1993;, Theuer et al., 1993), cytostatikmi (Schrappe et al.,For diagnostic purposes may be an antibody molecule, preferably a molecule of the antibody BIWA-1, fragments thereof or recombinant antibody molecules with the same idiotype of binding to a radioactive isotope such as 125 I, l3l I Ί, 99mTc or radioactive compounds (Larson et al., 1991; Thomas et al., 1989; Srivastava, 1988), 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, the specific molecule can be N 6 antibody molecule, preferably BIWA-1 (VFF-18), and the VFF-18-derived antibody molecules, e.g., fragments or recombinant antibody molecules with the same idiotype binding with radioisotopes, such as 90 Y, 131 I, 186 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., 1995, Jurcic et Scheinberg, 1994), toxins (Vitetta et al., 19091; Vitetta et Thorpe, 1991; Kreitman et al., 1993 ;, Theuer et al., 1993), cytostatics (Schrappe et al. al.
1992) , proliečivami (Wang et al., 1992; Senter et al., 1989), s fotoaktivovateľnými látkami (Hemming et al.,1992), prodrugs (Wang et al., 1992; Senter et al., 1989), with photoactivatable agents (Hemming et al.,
1993) , s molekulou protilátky s inou špecifickosťou, alebo s rádioaktívnymi zlúčeninami. Molekula protilátky môže byť ďalej viazaná s cytokínom alebo iným imunomodulujúcim polypeptidom, napríklad s tumorovým nekrotickým faktorom, lymfotoxínom (Reisfeld et al., 1996), alebo s interleukínom-2 (Becker et al., 1996). Molekuly protilátky sa na použitie v systémoch s cieľovaním (“pretargeting systems”) môžu modifikovať tiež napríklad streptavidínom alebo biotinom (Goodwin, 1995).1993), with an antibody molecule of other specificity, or with radioactive compounds. The antibody molecule may further be coupled to a cytokine or other immunomodulating polypeptide, for example, a tumor necrosis factor, lymphotoxin (Reisfeld et al., 1996), or interleukin-2 (Becker et al., 1996). Antibody molecules can also be modified, for example, with streptavidin or biotin for use in pretargeting systems (Goodwin, 1995).
V diagnostických spôsoboch podľa tohto vynálezu sa môžu výhodne vyšetrovať vzorky odobraté pacientom, napríklad z biopsií, pri ktorých je podozrenie na karcinóm dlaždicového epitelu, alebo už s existujúcou diagnózou, alebo s nádorom, pri ktorých sa vyžaduje bližšie spresnenie. Dôkaz variantných molekúl CD44, obsahujúcich aminokyselinovú sekvenciu, kódovanú variabilným exónom v6, sa môže vykonať pomocou protilátky na proteínovej úrovni, alebo na úrovni nukleových kyselín, pomocou špecifických sond nukleových kyselín, alebo pomocou primárov na polymerázovú - reťazcovú reakciu (PCR).Vynález sa preto týka tiež molekúl protilátok a nukleových kyselín, ktoré sú vhodné na použitie v uvedených spôsoboch ako sondy alebo priméry, a použitia takých protilátok a nukleových kyselín na diagnózu a analýzu karcinómov dlaždicového epitelu. Imunohistochemicky s protilátkami sa napríklad môžu vyšetrovať rezy tkanív pomocou známych spôsobov. Extrakty získané zo vzoriek tkanív, alebo telové tekutiny sa ďalej môžu vyšetrovať ďalšími imunologickými spôsobmi s použitím protilátok, napríklad pijakovanim (westem blots), pri enzýmovej imunoadsorbentovej analýze (ELISA, Catty at Raykundalia, 1989), rádioimunoanalýze (RIA, Catty et Murphy, 1989), alebo v podobných imunoanalytických spôsoboch. Vyšetrovania sa môžu vykonávať kvalitatívne, semikvantitatívne alebo ako kvantitatívne analýzy.In the diagnostic methods of the present invention, samples taken from a patient can advantageously be examined, for example from biopsies in which squamous epithelial carcinoma is suspected, or with an existing diagnosis, or with a tumor in which further refinement is required. Detection of variant CD44 molecules containing the amino acid sequence encoded by the variable exon v6 can be performed using an antibody at the protein level or at the nucleic acid level, by specific nucleic acid probes, or by primers for polymerase chain reaction (PCR). it also relates to antibody and nucleic acid molecules that are suitable for use in said methods as probes or primers, and to the use of such antibodies and nucleic acids for diagnosis and analysis of squamous epithelial carcinomas. For example, tissue sections can be examined by immunohistochemistry with antibodies using known methods. Extracts obtained from tissue samples or body fluids may further be screened by other immunological methods using antibodies, for example, by western blotting, enzyme immunoadsorbent analysis (ELISA, Catty at Raykundalia, 1989), radioimmunoassay (RIA, Catty et Murphy, 1989) ), or similar immunoanalytical methods. Investigations may be performed qualitatively, semi-quantitatively or as quantitative analyzes.
Popri diagnostike in vitro sú molekuly protilátky so špecifickosťou podľa tohto vynálezu vhodné tiež na diagnostiku karcinómov dlaždicového epitelu in vivo. Ak bude molekula protilátky nosičom detegovateľnej značky, potom sa detekcia značky môže využívať na diagnostické účely, napríklad na zviditeľnenie nádoru in vivo (zobrazovanie Imaging), alebo napríklad pri rádiochirurgii (radioguided surgery). Pri použití rádioaktívnymi izotopmi konjugovanej protilátky na imunoscintilografiu sa napríklad získa celý rad záznamov, na základe ktorých odborník môže vyvodiť vyžadované závery (Siccardi et al., 1989; Keenan et al., 1987; Perkins et Pimm, 1992; Colcher et al., 1987; Thompson et al., 1984).In addition to in vitro diagnostics, antibody molecules with specificity of the invention are also useful for diagnosing squamous epithelial carcinomas in vivo. If the antibody molecule is a carrier of a detectable label, then the detection of the label can be used for diagnostic purposes, for example, to visualize the tumor in vivo (imaging imaging), or for example in radioguided surgery. For example, the use of radioactive isotopes of conjugated antibody for immunoscintilography yields a number of records from which one can draw the desired conclusions (Siccardi et al., 1989; Keenan et al., 1987; Perkins et Pimm, 1992; Colcher et al., 1987 Thompson et al., 1984).
Dôkaz a/alebo kvantifikácia zvýšenej hladiny expresie variantných CD44 epitopov v6 môže vyústiť do diagnózy a prognózy stavu. Výhodne sa môžu pritom využiť kombinácia s ďalšími prognostickými parametrami, napríklad s určením stupňa nádoru (mit dem Tumorgrad).Evidence and / or quantification of the increased expression level of variant CD44 v6 epitopes may result in diagnosis and prognosis of the condition. Combinations with other prognostic parameters can be advantageously used, for example in determining the degree of tumor (mit dem Tumorgrad).
Molekuly protilátky so špecifickosťou podľa tohto vynálezu, prípadne viazané s cytotoxickým činidlom sa môžu výhodne použiť na terapiu karcinómov dlaždicového epitelu. Použitie môže pritom byť systémové alebo topikálne, napríklad intravenózne (ako bolus alebo infúzia), intraperitoneálne, intramuskuláme, subkutánne a iné injekcie/infúzie. Protokoly podávania konjugovaných alebo nekonjugovaných protilátok (či už ako úplné imunoglobuliny, fragmenty, rekombinantné humanizovanc molekuly alebo iné) sú v súčasnom stave techniky známe (Mulshine et al., 1991; Larson et al., 1991; Vitetta et 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). Terapeutické použitie môže byť napríklad obdobné, ako sa používajú protilátky 1.1ASML (Seiter et al., 1993). Nemodifikovaná monoklonová protilátka sa môže terapeuticky použiť priamo, ak je pre cytotoxický účinok vhodná ako efektor, napríklad pre komplementáme-indukovanú alebo protilátkovo-indukovanú bunkovú cytotoxickosť ( Riethmíiller et al., 1994). Vhodnou monoklonovou proti látkou na uvedené použitie sú protilátka izotyp lgG2a myši a protilátka typu IgGl človeka. Nemodifíkované protilátky sa môžu ďalej použiť na indukciu vlastnej protinádorovej reakcie pacienta anti-idiotypovým mechanizmom (Baum et al. 1993; Khazaeli e/ al., 1994).Antibody molecules with specificity according to the invention, optionally bound to a cytotoxic agent, can be advantageously used for the treatment of squamous epithelial carcinomas. The use may be systemic or topical, for example intravenously (such as a bolus or infusion), intraperitoneally, intramuscularly, subcutaneously and other injections / infusions. Protocols for administering conjugated or unconjugated antibodies (whether as complete immunoglobulins, fragments, recombinant humanized molecules or the like) are known in the art (Mulshine et al., 1991; Larson et al., 1991; Vitetta et Thorpe, 1991; Vitetta et al. al., 1991; Breitz et al., 1992, 1995; Press et al., 1989; Weiner et al., 1989; Chatal et al., 1989; Sears et al., 1982). For example, the therapeutic use may be similar to that of the 1.1ASML antibodies (Seiter et al., 1993). The unmodified monoclonal antibody can be used therapeutically directly if it is suitable as an effector for a cytotoxic effect, for example, for complement-induced or antibody-induced cellular cytotoxicity (Riethmiller et al., 1994). Suitable monoclonal antibodies for use herein are the IgG2a mouse antibody and the human IgG1 type antibody. Unmodified antibodies can further be used to induce a patient's own anti-tumor response by an anti-idiotypic mechanism (Baum et al. 1993; Khazaeli et al., 1994).
Výhodná forma uskutočnenia terapeutického použitia záleží od toho, že sa spojí humanizovaný v6-špecifický imunoglobulín alebo jeho fragment F(ab')2 s 90Y (Quadri et al., 1993; Vriesedorp et al., 1995), 131I (Maraveyas et al., 1995a, 1995b; Juweid et al., 1995; Press et al., 1995; Thomas et al., in: Catty 1985, strany 230 až 239), 186Re (Breitz et al., 1992, 1995), alebo s iným vhodným rádioizotopom a použije sa na rádioimunoterapiu karcinómov dlaždicového epitelu. Napríklad sa môže spojiť protilátka B1WA-1, alebo humanizovaná verzia protilátky BIWA-1, alebo fragment F(ab')2 protilátky BIWA-1 alebo humanizovanej protilátky s 90Y s použitím chelátotvomcho linkera ako je ICTB-DTPA (izotiokyanátbenzyl-dietylentriaminopentaacetát), pričom sa má dosiahnuť špecifická aktivita 5 až 20 mCi.mg'1, výhodne 10 mCi.mg'1 Uvedená účinná látka sa potom podáva pacientovi s antigén-pozitívnym nádorom v dávkach od 0,1 do 1 mCi.kg'1 telesnej hmotnosti, výhodne 0,3 až 0,5 Mci.kg’1 telesnej hmotnosti. Ak je molekula protilátky viazaná s 131I, môže sa pri špecifickej aktivite 2 mCi.mg'1 dávkovať napríklad podľa schémy 2 x 150 mCi, s odstupom 6 týždňov. Odborník v tejto oblasti môže pomocou známych spôsobov stanoviť maximálne možné dávkovanie (Maraveyas et al., 1995a, 1995b). Ak sa má podať celkové množstvo proteínu od 2 do 5 mg, môže sa podávať vo forme rýchlej intravenóznej injekcie. Pri väčších množstvách podávaného proteínu môže byť vhodnou formou podávania infúzia. Pri podávaní monoklonových protilátok môže byť nevyhnutné, aby sa účinná látka pred podávaním zmiešala s nadbytkom (napríklad desaťnásobným molovým nadbytkom) nerádioaktívnej protilátky; v tomto prípade sa podávanie uskutoční lepšie vo forme intravenóznej infúzie, napríklad počas 15 minút. Použitie sa môže opakovať. Terapia sa môže kombinovať s terapiou vonkajším ožarovaním. Terapia sa ďalej môže podporiť transplantáciou kostnej drene; táto je potom nevyhnutná najmä vtedy, ak sa pri terapii dosiahla v kostnej dreni dávka 1,6 Gy.A preferred embodiment of therapeutic use consists in combining the humanized v6-specific immunoglobulin or its F (ab ') 2 fragment with 90 Y (Quadri et al., 1993; Vriesedorp et al., 1995), 131 I (Maraveyas et al. al., 1995a, 1995b; Juweid et al., 1995; Press et al., 1995; Thomas et al., in: Catty 1985, pages 230-239), 186 Re (Breitz et al., 1992, 1995), or with another suitable radioisotope and used for radioimmunotherapy of squamous cell carcinomas. For example, a B1WA-1 antibody, or a humanized version of BIWA-1, or a F (ab ') 2 fragment of BIWA-1 or humanized 90 Y antibody can be coupled using a chelating linker such as ICTB-DTPA. wherein a specific activity of 5 to 20 mCi.mg -1 is preferred, preferably 10 mCi.mg -1. The active ingredient is then administered to a patient with an antigen-positive tumor at doses of 0.1 to 1 mCi.kg -1 body weight, preferably 0.3 to 0.5 Mci.kg '1 body weight. If the antibody molecule is coupled to 131 L, it can be dosed at, for example, 2 x 150 mCi at a specific activity of 2 mCi.mg -1 , 6 weeks apart. One of ordinary skill in the art can determine the maximum possible dosage by known methods (Maraveyas et al., 1995a, 1995b). If a total amount of protein of 2 to 5 mg is to be administered, it may be given as a rapid intravenous injection. For larger amounts of protein administered, infusion may be a suitable form of administration. When administering monoclonal antibodies, it may be necessary for the active ingredient to be mixed with an excess (e.g., a 10-fold molar excess) of the non-radioactive antibody prior to administration; in this case, administration is preferably done as an intravenous infusion, for example over 15 minutes. The use may be repeated. The therapy may be combined with external radiation therapy. Therapy may further be supported by bone marrow transplantation; this is inevitable, in particular, when a dose of 1.6 Gy has been achieved in the bone marrow during therapy.
Molekuly protilátky podľa tohto vynálezu sa ďalej môžu použiť tiež ex vivo na čistenie CD34-pozitívnych základných aj priebežných bunkových preparátov (Immunopurging). Terapia ožarovaním a chemoterapia karcinómov dlaždicového epitelu sa môže podporiť autológnou transplantáciou kostnej drene. Pritom použité hematopoietické základné a priebežné bunkové preparáty musia byť bez nádorových buniek. Možno to dosiahnuť inkubáciou s molekulami protilátok z tohto vynálezu, napríklad inkubáciou s konjugátmi protilátka-toxín (Myklebust et al., 1994; DE P 196 48 209.7).Furthermore, the antibody molecules of the invention can also be used ex vivo for purification of CD34-positive baseline and continuous cell preparations (Immunopurging). Radiation therapy and chemotherapy of squamous cell carcinomas can be supported by autologous bone marrow transplantation. The hematopoietic baseline and continuous cell preparations used must be free of tumor cells. This can be achieved by incubation with the antibody molecules of the invention, for example by incubation with antibody-toxin conjugates (Myklebust et al., 1994; DE P 196 48 209.7).
Molekuly protilátky podľa tohto vynálezu sa môžu ďalej použiť vo forme rekombinantného konštruktu, do ktorého bol vložený bunkový T-receptor T-lymfocytov. Týmto spôsobom reprogramované T-lymfocyty sa selektívne viažu na nádorové bunky, exprimujúce antigén a vyvolávajú cytotoxický účinok; preto ich možno použiť v terapii karcinómov dlaždicového epitelu (PCT/EP 96 04 631; Altenschmidt et al., 1996).The antibody molecules of the invention may further be used in the form of a recombinant construct into which a T cell receptor of T cells has been introduced. In this way, reprogrammed T lymphocytes selectively bind to tumor cells expressing the antigen and induce a cytotoxic effect; therefore, they can be used in the therapy of squamous epithelial carcinomas (PCT / EP 96 04 631; Altenschmidt et al., 1996).
Prehľad obrázkov na výkresochBRIEF DESCRIPTION OF THE DRAWINGS
Obr. 1: Stanovenie epitopovej špecifickosti protilátky BIWA-1 väzbou na syntetický peptid, odvodený z CD44-v6 sekvencie človeka. Zodpovedajúci peptid CD44-v6 potkana bol skúšaný s protilátkou 1.1ASML. Väzba sa určovala pomocou ELISA (enzýmovou imunoadsorbentovou analýzou), pričom sa peptid imobilizoval na mikrotitračnej platni (porovnaj: Heider et al., 1996b, obr. 2; -: nijaká väzba; +/-: slabá väzba; +: silná väzba.Fig. 1: Determination of epitope specificity of BIWA-1 by binding to a synthetic peptide derived from a human CD44-v6 sequence. The corresponding rat CD44-v6 peptide was tested with antibody 1.1ASML. Binding was determined by ELISA (enzyme-linked immunoadsorbent analysis), wherein the peptide was immobilized on a microtiter plate (cf. Heider et al., 1996b, Fig. 2; -: no binding; +/-: weak binding; +: strong binding.
Obr. 2: Imunohistochemická analýza karcinómu dlaždicového epitelu hrtanu (a) a pečeňovej metastázy karcinómu pažeráka (b) s CD44-v6-špecifickou monoklonovou protilátkou BIWA-1. V obidvoch prípadoch možno vidieť reaktivitu protilátky s membránou nádorových buniek. Pôvodné zväčšenie bolo 40x, protivyfarbovanie hematoxylínom.Fig. 2: Immunohistochemical analysis of squamous cell carcinoma of the larynx (a) and liver metastasis of esophageal carcinoma (b) with the CD44-v6-specific monoclonal antibody BIWA-1. In both cases, the reactivity of the antibody to the tumor cell membrane can be seen. The original magnification was 40x, counter staining with hematoxylin.
Obr. 3: Porovnanie väzby antigénu rôznych CD44-VÔšpecifických monoklonových protilátok. Väzba štyroch rôznych CD44-v6-špecifických mAb na ľudské SCC A431-bunky sa merala pomocou ELISA. Monoklonová protilátka BIWA-1 má vyššiu afinitu pre nádorové bunky ako ostatné monoklonové protilátky.Fig. 3: Comparison of antigen binding of various CD44-specific monoclonal antibodies. Binding of four different CD44-v6-specific mAbs to human SCC A431 cells was measured by ELISA. The monoclonal antibody BIWA-1 has a higher affinity for tumor cells than other monoclonal antibodies.
Obr. 4: Podrobnejšie epitopové mapovanie mAb BIWA-1. Kompetitívnym meraním spôsobom ELISA sa merala väzba protilátky BIWA-1 na rôzne prekrývajúce sa syntetické peptidy, ktorých aminokyseliny 18 až 32 CD44v6 kódovanej oblasti sú zmenené. Minimálna väzbová sekvencia (peptid v6(19-29) je podčiarknutá.Fig. 4: More detailed epitope mapping of mAb BIWA-1. By competitive ELISA, the binding of BIWA-1 to various overlapping synthetic peptides whose amino acids 18 to 32 of the CD44v6 encoded region were altered was measured. The minimum binding sequence (v6 peptide (19-29)) is underlined.
Obr. 5: Biodistríbúcia I251-B1WA- 1 v A-431 xenotransplantovaných mláďatách myší. Je znázornená akumulácia ako % ID.g'1 (stredná hodnota + štandardná odchýlka) po 4, 24, 48, 120 a 168 hodinách po injekcii.Fig. 5: Biodistribution of I25 1-B1WA-1 in A-431 xenografted young offspring. The accumulation is shown as% ID.g -1 (mean + standard deviation) at 4, 24, 48, 120 and 168 hours after injection.
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Príklad 1Example 1
Expresia CD44-v6 v karcinómoch dlaždicového epiteluExpression of CD44-v6 in squamous epithelial carcinomas
Tkanivotissue
Imunohistochemicky sa pomocou mAb BIWA-1 (kloň VFF-18) analyzovalo spolu 126 v parafíne zaliatych vzoriek nádorov na expresiu CD44-v6. Vzorky zahŕňali 31 prípadov primárnych karcinómov dlaždicového epitelu (15 prípadov z hrtanu, 16 prípadov kože), 91 prípadov metastáz lymfatických uzlín (hrtan, n = 38; pľúca, n = 27; pažerák, n = 11; ústna dutina, n = 11; tonzily, n = 4) a 4 prípady metastáz pečene.A total of 126 paraffin-embedded tumor samples for CD44-v6 expression were analyzed immunohistochemically using mAb BIWA-1 (VFF-18 clone). Samples included 31 cases of primary squamous cell carcinomas (15 cases of larynx, 16 skin cases), 91 cases of lymph node metastases (larynx, n = 38; lung, n = 27; esophagus, n = 11; oral cavity, n = 11; tonsils, n = 4) and 4 cases of liver metastasis.
Protilátkaantibody
Z ľudskej keratínocytovej-cDNA sa polymerázovoureťazcovou reakciou (PCR) z CD44v amplifikovala úplná variantná oblasť typu HPKII (Hofmann et al., 1991). Obidva PCR priméry 5 -CAGGCTGGGAGCCAAATGAAGAAAATG-ď v polohe 25 až 52, a 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-ď v polohe 1013 až 984 variantnej LCLC97 oblasti, ako opisuje Hofmann et al. obsahujú rozoznávacie miesto ScoRI, ktoré je nevyhnutné na to, aby sa klonoval PCR-produkt priamo do vektora pGEX-2T (Smith et al., 1988). Výsledný konštrukt (pGEX CD44v HPKII, v3-vl0) kóduje pre fúzovaný proteín o približne 70 kD, pozostávajúci z glutation-S-transferázy z Schistosoma japonicum a z exónov v3 až vlO humánnej CD44 (Obr. 1; Heider et al., 1993). Fúzovaný proteín bude exprimovaný v E. coli a následne afinitne čistený cez glutatiónovú agarózu (Smith et al., 1988).The complete HPKII-type variant region was amplified from human keratinocyte-cDNA by the polymerase chain reaction (PCR) of CD44v (Hofmann et al., 1991). Both PCR primers 5 -CAGGCTGGGAGCCAAATGAAGAAAATG-d at position 25 to 52, and 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-d at position 1013 to 984 of the variant LCLC97 region, as described by Hofmann et al. contain the ScoRI recognition site, which is necessary for the PCR product to be cloned directly into the pGEX-2T vector (Smith et al., 1988). The resulting construct (pGEX CD44 in HPKII, v3-v10) encodes for a fusion protein of approximately 70 kD, consisting of glutathione-S-transferase from Schistosoma japonicum and exons v3 to v10 of human CD44 (Fig. 1; Heider et al., 1993). The fusion protein will be expressed in E. coli and subsequently affinity purified via glutathione agarose (Smith et al., 1988).
Samičky myši Balb/c sa potom intraperitoneálne imunizovali s afinitne vyščisteným fúzovaným proteínom podľa schémy:Female Balb / c mice were then immunized intraperitoneally with affinity purified fusion protein according to the scheme:
1. imunizácia: 50 pg fúzovaného proteínu v úplnom Freundovom adjuvans;1. immunization: 50 µg of fusion protein in complete Freund's adjuvant;
2. a 3. imunizácia: 50 pg fúzovaného proteínu v neúplnom Freundovom adjuvans.2nd and 3rd immunizations: 50 µg fusion protein in incomplete Freund's adjuvant.
Imunizácie sa vykonávali vždy s odstupom 4 týždňov. 14 dní po poslednej imunizácii sa zvieratá ešte tri po sebe nasledujúce dni imunizovali s 10 pg fúzovaného proteínu v PBS. Na nasledujúci deň sa bunky sleziny zvieraťa s vysokým titrom protilátky fúzovali s bunkami myelómu myši P3.X63-Ag8.653 pomocou polyetylénglykolu 4000. Hybridómové bunky sa potom vyselektovali na mikrotitračnej platni v prostredí HAT (Kohler et Milstein, 1975; Keamey et al., 1979).Immunizations were performed every 4 weeks apart. 14 days after the last immunization, the animals were immunized with 10 µg of fusion protein in PBS for three consecutive days. On the next day, the spleen cells of the high antibody titer animal were fused with P3.X63-Ag8.653 mouse myeloma cells using polyethylene glycol 4000. The hybridoma cells were then selected on a microtiter plate in HAT medium (Kohler et Milstein, 1975; Keamey et al., 1979).
Stanovenie titrov protilátok v sére, prípadne vytriedenie prestarnutých hybridómov sa vykonalo pomocou ELISA. Pri tejto skúške sa na mikrotitračných platniach najprv vytvoril povlak fúzovaného proteínu (GST-CD44v3-vlO), alebo povlak iba z glutatión-S-transferázy. Následne sa inkubovali v sérii zriedení vzorky séra, respektíve vzorky prestarnutých hybridómov a stanovovala sa špecifická protilátka proti myšaciemu imunoglobulínu pomocou peroxidázy, konjugovanej s protilátkou. Hybridómy, ktoré reagujú iba s glutatión-S-transferázou, sa zvrhli. Ostávajúca protilátka sa najprv charakterizovala pomocou ELISA s doméno-špecifickými fúzovanými proteínmi (exon v3, exon v5+v6, exon v6+v7, exon v8-v10) (Koopman et al., 1993). Jej imunohistochemická reaktivita sa skúšala na rezoch ľudskej kože.Determination of serum antibody titers or screening of outdated hybridomas was performed by ELISA. In this assay, a fusion protein coating (GST-CD44v3-v10), or a coating of glutathione S-transferase only, was first formed on the microtiter plates. Subsequently, serum samples and samples of aged hybridomas were incubated in a series of dilutions and the specific anti-mouse immunoglobulin antibody was determined using antibody-conjugated peroxidase. Hybridomas that only react with glutathione-S-transferase have degenerated. The remaining antibody was first characterized by ELISA with domain-specific fusion proteins (exon v3, exon v5 + v6, exon v6 + v7, exon v8-v10) (Koopman et al., 1993). Its immunohistochemical reactivity was tested on sections of human skin.
BIWA-l (VFF-18; prípravu a vlastnosti pozri tiež v WO 95/33 771) sa viazala iba na fúzované proteíny, ktoré obsahovali domény, kódované exónom v6. Aby sa ďalej ohraničil epitóp protilátky, použili sa pri ELISA rôzne syntetické peptidy, predstavujúce časti vó-domény (obr. 1). Najsilnejšiu väzbu mal 14aminokyselinový peptid v6D. Preto epitóp protilátky BIWA-l leží čiastočne alebo celkom v sekvencii QWFGNRWHEGYROT domény, ktorá je kódovaná exónom v6. Táto sekvencia je homológna k väzbovému epitópu protilátky 1.1ASML, ktorá sa použila v terapeutickom modeli na potkanoch a ktorá je špecifická na CD44v6 potkana (obr. 1).BIWA-1 (VFF-18; see also WO 95/33771 for preparation and properties) only bound to fusion proteins that contained domains encoded by exon v6. In order to further limit the epitope of the antibody, various synthetic peptides representing portions of the δ-domain were used in the ELISA (Fig. 1). The 14 amino acid peptide v6D had the strongest binding. Therefore, the epitope of BIWA-1 lies partially or totally in the sequence of the QWFGNRWHEGYROT domain that is encoded by exon v6. This sequence is homologous to the binding epitope of antibody 1.1ASML, which was used in the rat therapeutic model and which is specific to the rat CD44v6 (Fig. 1).
Imunohistochémiaimmunohistochemistry
Pred inkubáciou s primárnou protilátkou sa parafínové rezy (4 μηι) odparafinovali v Rotihistole (Roth, BRD) trikrát vždy po 10 minút a potom v stúpajúcom alkoholovom rade rehydrovali. Potom sa rezy krátko opláchli destilovanou vodou a potom sa trikrát povarili v prostredí 0,01 M tlmivého roztoku Na-citranu, vždy počas dobu 10 minút, v mikrovlnnej rúre (Sharp, Model R-6270) pri 600 W. Po každej mikrovlnnej inkubácii sa rezy nechali 20 minút vychladnúť. Po poslednom povarení sa nosič opláchol v PBS v predinkuboval v normálnom kozom sére (10 %-né v PBS). Po troch premytiach v PBS sa rezy inkubovali s primárnou protilátkou (BIWA-l: 5 pg.mľ'; myšací IgG (izotyp zodpovedajúci negatívnej kontrole) 5 pg.mľ1; v PBS/1 % BSA) počas 1 hodiny. Ako pozitívna kontrola sa pri farebnej reakcii použili normálne ľudské kožné rezy, keďže keratínocyty exprimujú izoformu CD44, obsahujúcu v3vlO. Endogénne peroxidázy sa blokovali 0,3 %-ným roztokom H2O2 v PBS a rezy sa inkubovali s biotinylovanou sekundárnou protilátkou (proti-myšací IgG-F(ab )2, DÁKO Corp.) počas 30 minút. Na vyfarbenie sa rezy inkubovali 30 minút s chrenovou peroxidázou, ktorá bola viazaná na biotín ako streptavidínový-biotínový-peroxidázový komplex.Prior to incubation with the primary antibody, paraffin sections (4 μηι) were dewaxed in Rotihistol (Roth, BRD) three times for 10 minutes each and then rehydrated in an ascending alcohol row. The sections were then rinsed briefly with distilled water and then boiled three times in a 0.01 M Na-citrate buffer, for 10 minutes each, in a microwave oven (Sharp, Model R-6270) at 600 W. After each microwave incubation, the slices were allowed to cool for 20 minutes. After the last boiling, the carrier was rinsed in PBS in preincubation in normal goat serum (10% in PBS). After three washes in PBS, sections were incubated with primary antibody (BIWA-l: 5 pg.mľ ', mouse IgG (isotype matching negative control) 5 pg.mľ 1, in PBS / 1% BSA) for 1 hour. Normal human skin sections were used as a positive control in the color reaction since keratinocytes express the v3v10-containing CD44 isoform. Endogenous peroxidases were blocked with 0.3% H 2 O 2 in PBS and sections were incubated with biotinylated secondary antibody (anti-mouse IgG-F (ab) 2 , DAK Corp.) for 30 minutes. For staining, sections were incubated for 30 minutes with horseradish peroxidase, which was bound to biotin as a streptavidin-biotin-peroxidase complex.
Rezy sa potom inkubovali v 3,3-amino-9-etylkarbazolovom substráte (Sigma Immunochemicals) počas 5 až 10 minút, reakcia sa zastavila H2O a rezy sa proti vyfarbili hematoxylínom. Vyhodnotenie sfarbenia sa vykonalo sveteľným mikroskopom (Axioskop, Zeiss) a sfarbenie sa vyjadrilo nasledovne: +++, silná expresia; ++, mierna expresia; +, slabá expresia; -, nejednoznačná alebo žiadna detegovateľná expresia. Ako pozitívne sa vyhodnotili iba nádorové bunky s jasným sfarbením membrány. Percentuálny podiel pozitívnych nádorových buniek v každom reze sa určil hrubým odhadom tak, že sa bunky rozdelili do dvoch skupín: fokálne pozitívne nádory (menej ako 10 % nádorových buniek zreagovalo s protilátkou) a pozitívne nádory (10 alebo viac % pozitívnych nádorových buniek). Ak reagovalo s protilátkou menej ako 80 % nádorových buniek z pozitívnych buniek, potom sa vyjadruje zodpovedajúci podiel v percentách.Sections were then incubated in 3,3-amino-9-ethylcarbazole substrate (Sigma Immunochemicals) for 5-10 minutes, quenched with H 2 O, and sections were stained against hematoxylin. The color evaluation was performed with a light microscope (Axioskop, Zeiss) and the coloring was expressed as follows: +++, strong expression; ++, mild expression; +, poor expression; -, ambiguous or no detectable expression. Only tumor cells with clear membrane staining were evaluated as positive. The percentage of positive tumor cells in each section was determined by rough estimation by dividing the cells into two groups: focal positive tumors (less than 10% of tumor cells reacted with antibody) and positive tumors (10 or more% of positive tumor cells). If less than 80% of the positive tumor cells reacted with the antibody, then the corresponding percentage is expressed.
Pomocou CD44-v6-špecifickej monoklonovej protilátky BIWA-l sa analyzovalo 126 prípadov karcinómov dlaždicového epitelu rôzneho pôvodu. Vo všetkých okrem jednej vzorky nádoru, pozorovali sa izoformy s expresiou CD44-v6. Veľká časť vzoriek vykázala expresiu antigénu v 80 až 100 % nádorových bunkách, vyfarbenie bolo obmedzené na membránu nádorových buniek. V tkanivách stromy, lymfocytov, svalových buniek a endotelu sa nepozorovala žiadna reakcia.Using the CD44-v6-specific monoclonal antibody BIWA-1, 126 cases of squamous epithelial carcinomas of various origins were analyzed. In all but one tumor sample, isoforms expressing CD44-v6 were observed. A large proportion of the samples showed antigen expression in 80-100% of the tumor cells, staining was limited to the tumor cell membrane. No reaction was observed in the tissues of trees, lymphocytes, muscle cells and endothelium.
Na kvantifikáciu expresie CD44-v6 molekúl uvedených nádorových buniek sa paralelne s nádorovými rezmi vyfarbovali rezy normálnej ľudskej kože. Normálne kožné keratínocyty exprimujú vysokú hladinu izoforiem CD44 a počítajú sa k nim CD44-v6, najviac exprimujúce normálne bunky, aké boli doteraz opísané. Z toho dôvodu sa keratínocytové vyfarbenie považuje za porovnávacie a v tomto vyhodnocovacom systéme sa označuje ako “silné” (+++)· Vo väčšine skúmaných vzoriek nádorov bolo sfarbenie nádorových buniek porovnateľné alebo aj silnejšie ako sfarbenie kožných keratínocytov; iba v málo prípadoch bolo sfarbenie nádorov slabé (3 prípady metastázovaných lymfatických uzlín), alebo stredné (2 primárne karcinómy, 10 metastáz). Farebná reakcia bola v rezoch daných nádorov veľmi homogénna, pričom väčšina nádorových buniek rezov mala rovnakú intenzitu sfarbenia. Medzi primárnymi nádormi a metastázami neboli v expresii CD44-v6 pozorované nijaké významné rozdiely. Podrobné zhrnutie výsledkov je v tabuľke 1, príklady sú znázornené na obr. 2.To quantify the expression of the CD44-v6 molecules of said tumor cells, normal human skin sections were stained in parallel to the tumor sections. Normal skin keratinocytes express a high level of CD44 isoforms and include CD44-v6, the most expressing normal cells as previously described. Therefore, keratinocyte staining is considered to be comparative and is referred to as "strong" (+++) in this scoring system. · In most tumor samples examined, tumor cell staining was comparable or even stronger than skin keratinocyte staining; In very few cases, tumor staining was poor (3 cases of metastasized lymph nodes) or moderate (2 primary cancers, 10 metastases). The color reaction was very homogeneous in the sections of the tumors, with most of the tumor cells having the same staining intensity. No significant differences in CD44-v6 expression were observed between primary tumors and metastases. A detailed summary of the results is given in Table 1, examples of which are shown in FIG. Second
Tabuľka 1 Expresia CD44-v6 v karcinómoch dlaždicového epiteluTable 1 Expression of CD44-v6 in squamous epithelial carcinomas
Tabuľka 2 Expresia CD44-v6 v adenokarcinóme prostaty, karcinóme obličiek a v pečeňových metastázach kolorektálneho karcinómuTable 2 Expression of CD44-v6 in prostate adenocarcinoma, renal carcinoma, and liver metastases of colorectal carcinoma
*80 až 100 % nádorových buniek zreagovaných pozitívne s BIWA-1. V prípadoch, keď zreagovalo s protilátkou menej nádorových buniek, uvádza sa zodpovedajúce percento.* 80-100% of tumor cells reacted positively with BIWA-1. Where fewer tumor cells have reacted with the antibody, the corresponding percentage is reported.
Príklad 2Example 2
Expresia CD44-v6 v bunkách karcinómu obličiek, karcinómu prostaty a pečeňových metastáz karcinómu hrubého črevaExpression of CD44-v6 in kidney carcinoma, prostate carcinoma, and liver metastasis of colon carcinoma cells
Tkanivotissue
Analyzovalo sa i 9 prípadov buniek karcinómu obličiek (12 prípadov čisto bunkové, 5 prípadov chromofilné, 1 prípad chromofóbne, 1 onkocytový), 16 primárnych adenokarcinómov prostaty a 19 prípadov metastáz lymfatických uzlín od karcinómu prostaty, ako aj 30 prípadov pečeňových metastáz karcinómu hrubého čreva.9 cases of kidney cancer cells were analyzed (12 cases purely cellular, 5 cases chromophilic, 1 case chromophobic, 1 oncocytic), 16 primary prostate adenocarcinomas and 19 cases of prostate cancer lymph node metastases, as well as 30 cases of liver metastases of colon cancer.
Protilátkaantibody
BIWA-1 (pozri príklad 1)BIWA-1 (see Example 1)
Imunohistochémiaimmunohistochemistry
Uskutočnenie rovnako ako v príklade 1.Embodiment as in Example 1.
Na rozdiel od karcinómov dlaždicového epitelu sa vo väčšine analyzovaných prípadov buniek karcinómu obličiek a karcinómu prostaty nezaznamenala nijaká alebo iba fokálna expresia izoforiem CD44-v6. V prípade karcinómu prostaty, keď bola expresia pri porovnaní s vyfarbením normálneho epitelu prostaty rozsiahlejšia ako fokálna, bolo sfarbenie prevážne difúzne cytoplazmatické a slabé, prípadne heterogénne. V 50 %-ách hodnotených metastáz pečene karcinómu hrubého čreva sa zistila viac ako fokálna expresia izoforiem CD44-v6. Vo väčšine prípadov bolo sfarbenie slabé až stredné, pričom sfarbenie s BIWA1 najčastejšie malo menej ako 100 % nádorových buniek vzorky. Súhrn výsledkov je uvedený v tabuľke 2.In contrast to squamous epithelial carcinomas, in most cases of renal and prostate cancer cells analyzed, there was no or only focal expression of the CD44-v6 isoforms. In prostate cancer, when expression was more extensive than focal compared to normal prostate epithelial staining, staining was predominantly diffuse cytoplasmic and weak, possibly heterogeneous. More than focal expression of CD44-v6 isoforms was found in 50% of the colon carcinoma liver metastases evaluated. In most cases, staining was mild to moderate, with less than 100% of the tumor cells staining most often with BIWA1. A summary of the results is given in Table 2.
Príklad 3Example 3
Charakterizovanie CD44-V6 špecifických protilátokCharacterization of CD44-V6 specific antibodies
Bunková líniaCell line
Z americkej zbierky (Američan Type Culture Collection, Rockwell MD, USA) sa prevzala ľudská bunková SCC-línia A431 (spontánny epidermoidný karcinóm vulvy) a pestovala sa podľa návodu výrobcu. Povrchová expresia izoforiem, obsahujúcich CD44-vó bola stanovovaná pomocou FACS-analýzy, pričom sa použil FITC-linker mAb BIWA-1.A human SCC-line A431 (spontaneous epidermoid vulvar carcinoma) was taken from the American Collection (American Type Culture Collection, Rockwell MD, USA) and grown according to the manufacturer's instructions. Surface expression of CD44-containing isoforms was determined by FACS analysis using the FITC-linker mAb BIWA-1.
Analýza kinetických konštántAnalysis of kinetic constants
Stanovenie afinity a kinetiky vzájomného pôsobenia monoklonovej protilátky a CD44-v6 sa vykonalo pomocou povrchovej plazmonovej rezonancie (Surface Plasmon Resonance, SPR), pričom sa použil systém BIAcore 2000 (Pharmacia Biosensor). Na senzorový čip CM5 sa imobilizoval glutatión-S-transferáza-CD44-fúzovaný proteín, ktorý obsahuje oblasť kódovanú exónmi v3 až vlO (GST/CD44 v3-vl0), pričom sa použil postup amínového spojenia podľa návodu výrobcu. Protilátka sa v rôznych koncentráciách (8 až 132 nM) v HBS (10 mM Hepes pH 7,4, 150 mM chloridu sodného, 3,4 mM EDTA, 0,05 % BIAcore tenzid P 20) injektovala rýchlosťou 5 pl.min'1 na antigén-špecifický povrch. Vzájomné pôsobenie sa vyjadrilo ako zmena SPR-signálu. Disociácia protilátky sa pozorovala v toku tlmivého roztoku (HBS). Povrch čipu sa regeneroval samostatným pulzom 15 μΐ 30 mM roztoku kyseliny chlorovodíkovej. Analýza získaných údajov a výpočet kinetických konštánt sa vykonal pomocou programového vybavenia BIA Evaluation Software (verzia 2.1, Pharmacia Biosensor).The affinity and kinetics of the interaction of the monoclonal antibody and CD44-v6 were determined by Surface Plasmon Resonance (SPR) using the BIAcore 2000 system (Pharmacia Biosensor). Glutathione-S-transferase-CD44-fusion protein containing the region encoded by exons v3 to v10 (GST / CD44 v3-v10) was immobilized to the CM5 sensor chip using the amine coupling procedure according to the manufacturer's instructions. The antibody at various concentrations (8-132 nM) in HBS (10 mM Hepes pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% BIAcore surfactant P 20) was injected at 5 pl.min '1 on an antigen-specific surface. The interaction was expressed as a change in SPR-signal. Dissociation of the antibody was observed in buffer flow (HBS). The chip surface was regenerated by a single pulse of 15 μΐ of a 30 mM hydrochloric acid solution. Analysis of the data obtained and calculation of kinetic constants was performed using BIA Evaluation Software (version 2.1, Pharmacia Biosensor).
Uvedeným spôsobom sa porovnala antigénová afinita protilátky BIWA-1 s ďalšími CD44-v6-špecifickými monoklonovými protilátkami (VFF4, VFF7, BBA-13 (IgGl, R&D Systems, Abingdon, UK). Kinetické a afinitné konštanty rôznych protilátok sa stanovili vždy v dvoch nezávislých pokusoch. V tabuľke 3 sa uvádzajú hodnoty stupňa asociácie (ka), stupňa disociácie (kd) a disociačné konštanty (Kd) pre 3 mAb. Všetky mAb vykázali blízke ka a kd s výnimkou BBA-13, ktorá mala trikrát nižšií ka a VFF7, ktorá mala významne vyšší disociačný stupeň (5-násobok) v porovnaní s hodnotami ostatných mAb. Z toho vyplýva nižšia väzbová aktivita pre VFF7 a BBA-13 v porovnaní s VFF4 a BIWA-1. BIWA-1 mala najnižšie Kd zo všetkých sledovaných protilátok.The antigenic affinity of the BIWA-1 antibody was compared to other CD44-v6-specific monoclonal antibodies (VFF4, VFF7, BBA-13 (IgG1, R&D Systems, Abingdon, UK).) The kinetic and affinity constants of different antibodies were determined in two independent Table 3 shows the degree of association (k a ), degree of dissociation (k d ), and dissociation constants (K d ) for 3 mAbs, all mAbs showing near k and if d except for BBA-13, which was three times lower. k a and VFF7, which had a significantly higher dissociation grade (5-fold) compared to other mAbs, resulting in lower binding activity for VFF7 and BBA-13 compared to VFF4 and BIWA-1. d from all antibodies of interest.
Tabuľka 3: Kinetické a afinitné konštanty rôznych CD44v6-špecifických mono-klonových protilátok (mAb)Table 3: Kinetic and affinity constants of various CD44v6-specific monoclonal antibodies (mAbs)
Analýza vzájomného účinku protilátky a proteínu pomocou enzýmovej imunoadsorbentovej analýzy (ELISA)Analysis of antibody-protein interaction by enzyme-linked immunoadsorbent analysis (ELISA)
Bunky A-431, exprimujúce CD44-v6 sa kultivovali v 96 jamkovej platni (Falcon Microtest III, Becton Dickinson, Lincoln Park, NJ, USA) pri počte 5.104 na každú jamku, v prostredí RPMI 1640 s 10 % fetálneho teľacieho séra, cez noc pri teplote 37 °C. Po premytí s PBS/0,05%-ným roztokom Twccnu 20 sa bunky 1 minútu fixovali etanolom, vychladeným ľadom a potom sa preparáty opláchli. Nasledovala inkubácia s primárnymi protilátkami (VFF4, VFF7, BIWA-1, BBA-13, 1 ng.mľ1 až 600 ng.mľ1, vždy v skúšobnom tlmivom roztoku: PBS /0,5 % BSA/ 0,05 % Tween 20) počas 1 hodiny pri teplote miestnosti. Po inkubácii nasledoval trojnásobný oplach. Ako sekundárna protilátka sa použila králičia-protimyšacia-IgG-chrenová peroxidázová konjugovaná protilátka (DÁKO Corporation, Kopenhagen, Dánsko; zriedenie 1:6 000 v skúšobnom tlmivom roztoku) (1 hodina pri teplote miestnosti). Po trojnásobnom oplachu sa vyvolalo sfarbenie s TMB-roztokom (Kirkegaard+Perry, Gaithersburg, USA). Absorbancia sa merala pomocou zariadenia ELISA-Reader (Hewlett-Packard).A-431 cells expressing CD44-v6 were cultured in a 96 well plate (Falcon Microtest III, Becton Dickinson, Lincoln Park, NJ, USA) at 5 x 10 4 per well, in RPMI 1640 medium with 10% fetal calf serum, via overnight at 37 ° C. After washing with PBS / 0.05% Twccn 20, the cells were fixed for 1 minute with ice-cold ethanol and then the preparations were rinsed. This was followed by incubation with primary antibodies (VFF4, VFF7, BIWA-1, BBA-13, 1 ng / ml 1 to 600 ng / ml ) , each in assay buffer: PBS / 0.5% BSA / 0.05% Tween 20 ) for 1 hour at room temperature. The incubation was followed by a triple rinse. A rabbit-anti-mouse-IgG-horseradish peroxidase conjugated antibody (DAKKO Corporation, Kopenhagen, Denmark; dilution 1: 6,000 in assay buffer) was used as a secondary antibody (1 hour at room temperature). After washing three times, staining with TMB solution (Kirkegaard + Perry, Gaithersburg, USA) was induced. Absorbance was measured using an ELISA-Reader (Hewlett-Packard).
Obr. 3 ukazuje, že pomerné afinity protilátky tak, ako boli stanovené analýzou BIAcore, reflektujú vzájomný účinok s nádorovými bunkami, pričom BIWA-1 celkom jasne má najvyššiu väzbovú afinitu.Fig. 3 shows that the relative affinities of the antibody, as determined by BIAcore analysis, reflect the interaction with tumor cells, with BIWA-1 quite clearly having the highest binding affinity.
Proteínová doména, ktorá je kódovaná CD44-exónom v6 pozostáva z 45 aminokyselín (obr. 4). Na presnejšie určenie epitopu, ktorý je u BIWA-1 známy, sa pri analýze ELISA použila séria syntetických peptidov. Predbežné pokusy poskytovali väzbu na centrálne umiestnený 14-mér (aminokyselinové zvyšky 18 až 31; obr. 4; porovnaj tiež s obr. 1), ale nie na peptidy mimo tejto oblasti. Preto sa syntetizovala druhá séria peptidov a peptidy sa skúšali kompetitívnou enzýmovou imunoadsorbentovou analýzou ELISA (obr. 4). Výsledky ukazujú, že najmenšiu štruktúru, ktorá je potrebná na vysokoafinitnú väzbu predstavuje peptid 19 až 29 (WFGNRWHEGYR). Eliminácia C-koncového arginínového zvyšku spôsobila viac ako 100- násobne slabšiu väzbu.The protein domain that is encoded by the CD44-exon v6 consists of 45 amino acids (Fig. 4). A series of synthetic peptides was used for ELISA analysis to more accurately determine the epitope known to BIWA-1. Preliminary experiments provided binding to the centrally located 14-mer (amino acid residues 18 to 31; Figure 4; cf. also Figure 1), but not to peptides outside this region. Therefore, a second series of peptides was synthesized and the peptides tested by competitive enzyme-linked immunoadsorbent ELISA (Fig. 4). The results show that peptide 19-29 (WFGNRWHEGYR) is the smallest structure required for high affinity binding. Elimination of the C-terminal arginine residue caused more than 100-fold weaker binding.
Príklad 4Example 4
Biodistribúcia rádiojódovanej CD44-v6-protilátky v mlád’atách myší s xenotransplantátmiBiodistribution of radioiodinated CD44-v6-antibody in juvenile xenograft mice
A-431 -Xenotransplantátový modelA-431 - Xenograft model
Osem týždňov staré samičky BALB/c nu/nu myší (B & K Universal, Renton, WA, USA) boli subkutánne injketované v ľavobočnej strednej línii kultivovanými 5.106 bunkami A-431 (humánneho epidermoidného karcinómu vulvy). Xenotransplantované zvieratá s A-431 nádormi sa v priebehu 2 týždňov použili na biodistrubučné pokusy (hmotnosť nádorov: 40 - 50 mg).Eight week old female BALB / c nu / nu mice (B&K Universal, Renton, WA, USA) were injected subcutaneously in the left midline with cultured 5 x 10 6 A-431 cells (human epidermoid vulva carcinoma). Xenotransplanted animals with A-431 tumors were used for biodistribution experiments over 2 weeks (tumor weight: 40-50 mg).
Rádiojódovanie protilátky BIWA-1BIODI-1 radio-iodination
Proteín G-čistenej monoklonovej protilátky BIWA-1 (IgGl myši) sa viazal so streptavidínom, pričom sa použil heterobifunkčný sieťujúci linker sukcinimidyl 4-(Nmaleinimido-metyl)cyklohexan-1 -karboxylát. Streptavidínlysylový zvyšok sa viazal na redukovaný protilátkacysteínylový zvyšok, ktorý sa pripravil vopred spracovaním protilátky s ditiotreitolom. Získaný 1 : 1 konjugát (> 90 %) sa ďalej čistil ionexovou chromatografiou. Na biodistribučné analýzy sa BIWA-l/SA značkoval cez primárny amín lyzínu. Na značenie sa použilo p-jódfenylové značkovacie činidlo (PIP; NEN Dupont, Wilmington, DE, USA) a použil sa spôsob, opísaný Willburom et al. (1989). Značenie protilátky BIWA-1 s SA avi s 125I nezmenilo jej imunoreaktivitu alebo farmakokinetiku protilátky v myšiach.The G-purified monoclonal antibody BIWA-1 (IgG1 mouse) protein was coupled to streptavidin using a heterobifunctional crosslinking linker of succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate. The streptavidin-lysyl residue was bound to a reduced antibody acysteinyl residue, which was prepared by pretreating the antibody with dithiothreitol. The obtained 1: 1 conjugate (> 90%) was further purified by ion exchange chromatography. For biodistribution analyzes, BIWA-1 / SA was labeled via the primary amine of lysine. A p-iodophenyl labeling reagent (PIP; NEN Dupont, Wilmington, DE, USA) was used for labeling and the method described by Willbur et al. (1989). Labeling of BIWA-1 with 125 I SA avi did not alter its immunoreactivity or antibody pharmacokinetics in mice.
Biodistribučné pokusyBiodistribution experiments
Mladé myši, ktoré boli xenotransplantované ľudskými nádormi A-431, sa injektovali s 5 až 7 pCi l25I na 50 pg monoklonovej protilátky BIWA-1 (špecifická aktivita 0,1 až 0,14 mCi.mg·') intravenózne do laterálnej žily chvosta. Štúdium časovej závislosti biodistribúcie sa vykonalo v čase 4, 24,48, 120 a 168 hodín po injekcii na skupinách troch zvierat (n = 3 ). Vo zvolenom čase sa myši odvážili, vykrvácali cez retro-orbitálny plexus a dislokáciou krku utratili. Odobralo sa deväť orgánov a tkanív, ktoré sa odvážili: krv, chvost, pľúca, pečeň, slezina, žalúdok, obličky, črevo a nádor. Merala sa rádioaktivita tkanív od injektovaného preparátu protilátky a porovnávala sa so štandardom. Meranie sa vykonávali gama-scintilátorom(Packard Inštrument Company, Meriden, CT, USA), pričom energiové okno sa nastavilo pre l25I na 25 až 80 keV. Vypočítali sa percentá z injektovanej dávky na g tkaniva (% ID.g1).Young mice xenografted with human A-431 tumors were injected with 5-7 pCi 125 I per 50 pg of BIWA-1 monoclonal antibody (specific activity 0.1 to 0.14 mCi.mg · -1) intravenously into the lateral tail vein. . Time biodistribution studies were performed at 4, 24, 48, 120 and 168 hours post-injection in groups of three animals (n = 3). At the selected time, the mice were weighed, bled through the retro-orbital plexus and sacrificed by neck dislocation. Nine organs and tissues were collected and weighed: blood, tail, lung, liver, spleen, stomach, kidney, intestine and tumor. Tissue radioactivity from the injected antibody preparation was measured and compared to a standard. Measurements were performed with a gamma scintillator (Packard Instrument Company, Meriden, CT, USA), with the energy window set at 125 to 25 to 80 keV for 125 I. Percentage of injected dose per g tissue (% ID.g 1 ) was calculated.
Predbežné pokusy ukázali, že BIWA-1 nereaguje krížovo s CD44-v6-antigénom myši. Tabuľka 4 a obr. 5 ukazujú záznam rádioaktivity v nádoroch a v normálnom tkanive. Jódovaná BIWA-1 ukazuje na rýchly príjem nádorom (7,6 % injektovanej dávky po 4 hodinách od injekcie), ktorý stúpne na viac ako 18 % ID.g’1 po 48 hodinách a potom do 120 hodín ostáva stály. Sedem dní po injekcii (168 hodín) nádor obsahoval ešte 15,3 % ID.g'1 tkaniva. Pre jednotlivé doby sa vypočítali pomery nádoru ku tkanivu, ktoré sú uvedené v tabuľke 4. Po 24 hodinách po injekcii bol pomer nádor: krv 0,48 a vzrástol na 3,16 v siedmom dni. Príjem v normálnom tkanive bol nízky a najpravdepodobnejšie bol spôsobený krvným pozadím v tkanivových biopreparátoch. Selektívne in vivo cielenie l25I-značenej BIWA-1 do humánnych SCCxenotransplantátov u myší ukazuje, že táto monoklonová protilátka má vysoký potenciál ako cielený nosič na diagnostické a terapeutické použitia u SCC-pacientov.Preliminary experiments have shown that BIWA-1 does not cross-react with mouse CD44-v6-antigen. Table 4 and FIG. 5 show the record of radioactivity in tumors and normal tissue. Iodinated BIWA-1 indicates rapid tumor uptake (7.6% injected dose 4 hours after injection), which rises to more than 18% ID.g -1 after 48 hours and then remains stable within 120 hours. Seven days after injection (168 hours), the tumor still contained 15.3% ID.g -1 tissue. Tumor to tissue ratios are calculated for each time as shown in Table 4. At 24 hours post injection, the tumor: blood ratio was 0.48 and increased to 3.16 on the seventh day. Intake in normal tissue was low and most likely was due to blood background in tissue biopreparations. Selective in vivo targeting of 125 I-labeled BIWA-1 to human SCC xenografts in mice shows that this monoclonal antibody has a high potential as a targeted carrier for diagnostic and therapeutic uses in SCC patients.
Tabuľka 4: Pomery 12SI - BIWA-1 v nádore : tkanive u myší s nádorom (A-431) v rôznych časoch po injekciiTable 4: Tumor: 12S I - BIWA-1 ratios: tissue in tumor-bearing mice (A-431) at different times after injection
“stredná hodnota (n = 3); štandardné odchýlky boli < 7 %.“Mean value (n = 3); standard deviations were <7%.
Príklad 5Example 5
Rozdielne expresie protilátky CD44-v6 u veľkého počtu ľudských nádorovDifferent expression of CD44-v6 in a large number of human tumors
V ďalšom vyšetrovaní bolo imunohistochemicky hodnotených spolu 544 vzoriek nádorov na expresiu CD44-v6 s použitím monoklonovej protilátky BIWA-1 (kloň VFF18). Vzorky sa najprv odparafinovali alebo sa ihneď po chirurgickom odobratí zmrazili v kvapalnom dusíku a do ich použitia sa uchovávali pri -70 °C. Analyzovali sa nasledovné nádory: Basalyóm (n = 16), prsný adenokarcinóm (AC) (n = 55), AC čreva (n = 83), karcinóm dlaždicového epitelu (SCC) hlavy a krku ( n = 125), pľúcny karcinóm (n = 120), AC prostaty (n = 34), karcinóm obličkových buniek (n = 27), SCC kože (n = 12) a AC žalúdka (n = 69). Tkanivá sa získali rutinným chirurgickým spôsobom alebo biopsiou, normálne tkanivo bolo dodané spoločne so vzorkou nádoru. Imunohistochemické vyšetrenie sa vykonalo rovnakým spôsobom, aký sa uvádza v príklade 1.In another investigation, a total of 544 tumor samples for CD44-v6 expression were immunohistochemically evaluated using monoclonal antibody BIWA-1 (VFF18 clone). Samples were first dewaxed or frozen in liquid nitrogen immediately after surgery and stored at -70 ° C until use. The following tumors were analyzed: Basalyoma (n = 16), breast adenocarcinoma (AC) (n = 55), AC intestine (n = 83), squamous epithelial carcinoma (SCC) of the head and neck (n = 125), lung carcinoma (n = 120), AC prostate (n = 34), renal cell carcinoma (n = 27), SCC skin (n = 12) and AC stomach (n = 69). Tissues were obtained by routine surgery or biopsy, normal tissue was delivered with a tumor sample. The immunohistochemistry was performed in the same manner as in Example 1.
Tabuľka 5 obsahuje prehľad imunohistochemíckých analýz 397 vzoriek rôznych nádorov s použitím monoklonovej protilátky BIWA-1.Table 5 provides an overview of immunohistochemical analyzes of 397 different tumor samples using the monoclonal antibody BIWA-1.
AC: adenokarcinóm; RCC: karcinóm buniek obličiek; SCLC: malobunková rakovina pľúc; SCC: karcinóm dlaždicového epitelu.AC: adenocarcinoma; RCC: renal cell carcinoma; SCLC: small cell lung cancer; SCC: squamous cell carcinoma.
Pri malobunkových karcinómoch pľúc, karcinómoch buniek obličiek a AC prostaty sa nepozorovala nijaká alebo len obmedzená reaktivita. Všetky ostatné vyšetrované nádorové typy exprimujú izoformy obsahujúce CD44-V6 v rôznej miere. Väčšina vyšetrovaných prsných AC mala reaktivitu s BIWA-1 a hodnotené SCC (hrtan, pľúca a pažerák) exprimujú CD44-v6 v 100 %-tách všetkých prípadov.No or limited reactivity was observed in small cell lung carcinomas, renal cell carcinomas and AC prostate. All other tumor types examined express isoforms containing CD44-V6 to varying degrees. Most of the investigated breast ACs had reactivity with BIWA-1 and the SCCs evaluated (larynx, lung and esophagus) express CD44-v6 in 100% of all cases.
Na reaktivitu s protilátkou BIWA-1 sa vyšetrilo spolu 185 prípadov SCC rôzneho typu a klasifikácie. Z toho bolo 67 prípadov primárneho SCC (hrtan, n = 15: ústna dutina, n = 16; ústny hltan, n = 3; koža, n= 15), 77 vzoriek boli metastázy lymfatických uzlín (hrtan, n = 12; pľúca, n = 27; pažerák n = 11; ústna dutina, n = 6; ústny hltan, n = 7; podhltana, n= 10; nosných mandlí, n = 4) a 3 vzorky metastáz v pečeni (z nádoru pažeráka). Prehľad imunohistochemických analýz všetkých vyšetrovaných SCC-vzoriek sa uvádza v tabuľke 6.A total of 185 cases of SCC of different types and classifications were screened for reactivity with BIWA-1. Of these, 67 cases were primary SCC (larynx, n = 15: oral cavity, n = 16; oral pharynx, n = 3; skin, n = 15), 77 samples were lymph node metastases (larynx, n = 12; lung, n = 27; esophagus n = 11; oral cavity, n = 6; oral pharynx, n = 7; pharynx, n = 10; tonsils, n = 4) and 3 liver metastasis samples (from esophageal tumor). An overview of immunohistochemical analyzes of all SCC samples examined is given in Table 6.
Tabuľka 6Table 6
Expresia CD44-v6 v karcinómoch dlaždicového epiteluExpression of CD44-v6 in squamous epithelial carcinomas
Tabuľka 5Table 5
Expresia CD44-v6 v ľudských nádorochExpression of CD44-v6 in human tumors
*: 10 % alebo viac pozitívnych nádorových buniek*: 10% or more positive tumor cells
( Pozn.: fekálne pozitívne: < 10 % nádorových vzoriek pozitívnych; LNM: metastázy lymfatických uzlín; PT: primárny nádor; LM: metastázy pečene.)(Note: faecal positive: <10% positive tumor samples; LNM: lymph node metastasis; PT: primary tumor; LM: liver metastasis.)
Expresia izoforiem obsahujúcich CD44-v6 bola nájdená vo všetkých prípadoch okrem troch (jeden prípad hrtana, 2 prípady pľúc). Väčšina vzoriek mala expresiu antigénu v 80 až 100 % nádorových bunkách v celom reze, pričom sfarbenie bolo skoncentrované najmä na membrány nádorových buniek. Najsilnejší homogénne vyfarbený vzor sa našiel v nádore hrtana, pažeráka a podhltana, pričom väčšina nádorových buniek rezov mala rovnakú intenzitu sfarbenia.Expression of isoforms containing CD44-v6 was found in all but three cases (one case of larynx, 2 cases of lung). Most of the samples had antigen expression in 80-100% of the tumor cells in the whole section, with the staining being mainly concentrated on the tumor cell membranes. The strongest homogeneously stained pattern was found in the larynx, esophagus, and pharynx tumor, with most tumor cell sections having the same staining intensity.
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Sekvenčný protokolSequence protocol
1. Všeobecné údaje (1) Prihlasovateľ:1. General data (1) Applicant:
(A) Meno: Boehringer Ingelheim Intemational GmbH (B) Ulica: Rheinstrasse (C) Miesto: Ingelheim (E) Krajina: Nemecko (F) Poštové smerovacie číslo: 55 216 (G) Telefón: +49 - (0) - 6132 - 772 770 (H) Fax: +49 - (0) - 6132 - 774 377 (A) Meno: Forschungzcntrum Karlsruhe GmbH (B) Ulica: poštová schránka 3 640 (C) Miesto: Karlsruhe (E) Krajina: Nemecko (F) Poštové smerovacie číslo: 76 021 (A) Meno: Heider, Karl-Heinz (B) Ulica: Hervicusgasse 4/3/21 (C) Miesto: Wien (E) Krajina: Rakúsko (F) Poštové smerovacie číslo: 1 120 (A) Meno: Adolf, Guenther (B) Ulica: Stiftgasse 15 -17/10 (C) Miesto:Wien (E) Krajina: Rakúsko (F) Poštové smerovacie číslo: 1 070 (A) Meno: Ostermann, Elinborg (B) Ulica: Mauerbachstrasse 56/6 (C) Miesto: Wien (E) Krajina: Rakúsko (F) Poštové smerovacie číslo: 1 140 (A) Meno:Patzelt, Erik (B) Ulica: Hans-Buchmueller-Gasse 8 (C) Miesto: Purkersdorf (E) Krajina: Rakúsko (F) Poštové smerovacie číslo: 3 002 (A) Meno: Sproll, Marlies (B) Ulica: Schwenkgasse 3 (C) Miesto: Wien (E) Krajina: Rakúsko (F) Poštové smerovacie číslo: 1 120 (ii) Predmet vynálezu: Spôsob diagnózy a terapie karcinómov dlaždicového epitelu (iii) Počet sekvencii: 16 (iv) Počítačom čitateľný rámec:(A) Name: Boehringer Ingelheim Intemational GmbH (B) Street: Rheinstrasse (C) Place: Ingelheim (E) Country: Germany (F) Postcode: 55 216 (G) Telephone: +49 - (0) - 6132 - 772 770 (H) Fax: +49 - (0) - 6132 - 774 377 (A) Name: Forschungzcntrum Karlsruhe GmbH (B) Street: Mailbox 3 640 (C) Location: Karlsruhe (E) Country: Germany (F) Postcode: 76 021 (A) Name: Heider, Karl-Heinz (B) Street: Hervicusgasse 4/3/21 (C) Location: Wien (E) Country: Austria (F) Postcode: 1 120 (A) ) Name: Adolf, Guenther (B) Street: Stiftgasse 15 -17/10 (C) Place: Wien (E) Country: Austria (F) Postcode: 1 070 (A) Name: Ostermann, Elinborg (B) Street : Mauerbachstrasse 56/6 (C) Place: Wien (E) Country: Austria (F) Postcode: 1 140 (A) Name: Patzelt, Erik (B) Street: Hans-Buchmueller-Gasse 8 (C) Location: Purkersdorf (E) Country: Austria (F) Postcode: 3 002 (A) Name: Sproll, Marlies (B) Street: Sch wenkgasse 3 (C) Place: Wien (E) Country: Austria (F) Postal code: 1 120 (ii) Subject of the invention: Method of diagnosis and therapy of squamous cell carcinoma (iii) Number of sequences: 16 (iv) Computer-readable frame:
(A) Nosič údajov: pružný disk (B) Počítač: IBM PC kompatibilný (C) Operačný systém: PC - DOS / MS -DOS (D) Softvér: Patentln Release #1.0, Verzia #1.30 (EPA)(A) Data carrier: Flexible disk (B) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS -DOS (D) Software: Patentln Release # 1.0, Version # 1.30 (EPA)
2. Údaje o SEQ ID NO 1:2. SEQ ID NO 1 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 129 párov báz (B) Druh: nukleotid (C) Tvar závitovky: dvojitý (D) Topológia: dvojitá (ii) Druh molekúl: genomická DNA (ix) Označenie:(A) Length: 129 base pairs (B) Species: nucleotide (C) Worm shape: double (D) Topology: double (ii) Molecule type: genomic DNA (ix) Designation:
(A) Meno/kľúč: exón (B) Rozsah 1 .. 129 (D) Osobitné údaje: /product= “CD44” /label= v6 /note= “GenBank data base accesion No.(A) Name / Key: exon (B) Range 1 .. 129 (D) Special Data: / product = “CD44” / label = v6 / note = “GenBank data base accesion No.
L05411) /citation= ([1]) (ix) Označenie:L05411) / citation = ([1]) (ix)
(A) Meno/kľúč: CDS (B) Rozsah: 3.. 128 (x) Údaje o zverejnení:(A) Name / Key: CDS (B) Range: 3 .. 128 (x) Publication Details:
(A) Autori: Screaton, GR(A) Authors: Screaton, GR
Beli, MVBeli, MV
Jakson, DGJakson, DG
Comelis, FBComelis, FB
Gerth, UGerth, U.
Beli, JI (B) Názov: Genómová štruktúra DNA, kódujúca lymfocytový receptor CD44 odhaľuje najmenej 12 alternatívne štepených exónov (angl.).Beli, JI (B) Title: The genomic structure of DNA encoding the CD44 lymphocyte receptor reveals at least 12 alternatively digested exons.
(C) Časopis: Proc. Natl. Acad. Sci. U.S.A.(C) Magazine: Proc. Natl. Acad. Sci. U.S.A.
(D) Diel: 89 (F) Strany: 12 160-12 164 (G) Dátum: december 1992 (K) Významné zvyšky v SEQ ID NO: 1 : od 1 do 129 (x) Údaje o zverejnení:(D) Part: 89 (F) Pages: 12 160-12 164 (G) Date: December 1992 (K) Significant residues in SEQ ID NO: 1: from 1 to 129 (x) Publication Details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-1996 (xi) Opis sekvencie: SEQ ID NO: 1:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 1:
TC GAG GCA ACT CCT AGl AGT ACA ACG GAA GAA ACA GCT ACC CAG AA.G ΉTC GAG GCA ACT CCT AGl AGT ACA ACG GAA GAA ACA GCT ACC CAG AA.G Ή
Gin Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin LysGin Ala Thr Pro Ser Ser Thr Glu Glu Thr Ala Thr Gin Lys
GAA GAG TGG TTT GGC A?.C AGA TGG CAT GAG GGA TM CGC CAA ACA C.CC 95 Glu Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr ProGAA GAG GG GGT TT GGC G? GG GG GG GGA TM CGC GCA GCC CAA ACA C.CC 95 Glu Gin Trp
25 3025 30
AGA GAA GAG TCC CAT TCG ACA ACA GGG ACA GCT GAGA GAA GAG TCC CAT TCG
Arg Glu Asp Ser His Ser Thr Thr Gly Thr AlaArg Glu Asp Ser Ser Ser Thr Gly Thr Ala
4040
129129
2. Údaje o SEQ ID NO 2:2. SEQ ID NO 2 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 42 aminokyselín (B) Druh: aminokyselina (D) Topológia: lineárna (xi) Opis sekvencie: SEQ ID NO: 1:(A) Length: 42 amino acids (B) Species: amino acid (D) Topology: linear (xi) Sequence description: SEQ ID NO: 1:
2. Údaje o SEQ ID NO 3:2. SEQ ID NO 3 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 14 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 14 amino acids (B) Species: amino acid (C) Worm shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-april-1996 (xi) Opis sekvencie: SEQ ID NO: 3:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 3:
2. Údaje o SEQ ID NO 4:2. SEQ ID NO 4 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 27 párov báz (B) Druh: nukleotid (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: osobitná nukleová kyselina (A) Opis: /dese = “PCR primér” (x) Údaje o zverejnení:(A) Length: 27 base pairs (B) Species: nucleotide (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: special nucleic acid (A) Description: / dese = “PCR primer” (x) ) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-1996 (xi) Opis sekvencie: SEQ ID NO: 4:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 4:
CAGGCTGGGA GCCAAATGAA GAAAATG 27CAGGCTGGGA GCCAAATGAA GAAAATG 27
2. Údaje o SEQ IDNO5:2. SEQ IDNO5 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 30 párov báz (B) Druh: nukleotid (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: osobitná nukleová kyselina (A) Opis: /dese = “PCR primér” (x) Údaje o zverejnení:(A) Length: 30 base pairs (B) Species: nucleotide (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: special nucleic acid (A) Description: / dese = “PCR primer” (x) ) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-l 996 (xi) Opis sekvencie: SEQ ID NO: 5:(H) File number: DE 196 15 074.4 (I) Date of administration: 17-April-1 996 (xi) Description of sequence: SEQ ID NO: 5:
TGATAAGGAA CGATTGA CAT TAGAGTTGGA 3 0TGATAGGAA CGATTGA CAT TAGAGTTGGA 3 0
2. Údaje o SEQ ID NO 6:2. SEQ ID NO 6 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 11 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-1996 (xi) Opis sekvencie: SEQ ID NO: 6:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 6:
Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg 1 5 10Trp Phe Gly Asn Arg
2. Údaje o SEQ ID NO 7:2. SEQ ID NO 7 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 43 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 43 amino acids (B) Species: amino acid (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-l 996 (xi) Opis sekvencie: SEQ ID NO: 7:(H) File number: DE 196 15 074.4 (I) Date of administration: 17-April-1 996 (xi) Description of sequence: SEQ ID NO: 7:
2. Údaje o SEQ ID NO 8:2. SEQ ID NO 8 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 11 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-1996 (xi) Opis sekvencie: SEQ ID NO: 8:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 8:
Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys 1 5 10Ser Ser Thr Thr Glu Thr Ala Thr Gin Lys 1 5 10
2. Údaje o SEQ ID NO 9:2. SEQ ID NO 9 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 10 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 10 amino acids (B) Species: amino acid (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení.(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication details.
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-1996 (xi) Opis sekvencie: SEQ ID NO: 9:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 9:
Glu Glu Thr Ala Thr Gin Lys Glu Gin Trp I 5 10Glu Glu Thr Ala Thr Gin Lys
2. Údaje o SEQ ID NO 10:2. SEQ ID NO 10 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 11 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-1996 (xi) Opis sekvencie: SEQ ID NO: 10:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 10:
Thr Ala Thr Gin Lys Glu Gin Trp Phe Gly Asdn 1 5 10Thr Ala Thr Gin Lys
2. Údaje o SEQ IDNO 11:2. SEQ IDNO 11 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 14 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 14 amino acids (B) Species: amino acid (C) Worm shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-1996 (xi) Opis sekvencie: SEQ ID NO: 11:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 11:
Gin Trp Phe Gly Asn Arg Tip His Glu Gly Tyr Arg Gin Ihr 1 5 10Gin Trp Phe
2. Údaje o SEQ ID NO 12:2. SEQ ID NO 12 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 11 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-deccmber-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-deccmber-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-1996 (xi) Opis sekvencie: SEQ ID NO: 12:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 12:
Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro 1 5 10Asn Arg Trp His Glu Gly Thr Arg Gin Thr Pro 1 5 10
2. Údaje o SEQ IDNO 13:2. SEQ IDNO 13 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 11 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-l996 (xi) Opis sekvencie: SEQ ID NO: 13:(H) File number: DE 196 15 074.4 (I) Date of administration: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 13:
Glu Gly Tyr Arg Gin Thr Pro Arg Glu Asp Ser 1 5 10Glu Gly Tyr Arg
2. Údaje o SEQ IDNO 14:2. SEQ IDNO 14 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 10 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 10 amino acids (B) Species: amino acid (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-april-1996 (xi) Opis sekvencie: SEQ ID NO: 14:(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 14:
Thr Pro Arg Glu Asp Ser His Ser Thr Gly 1 5 10Thr Pro Arg Glu Ser Ser Ser Thr Gly 1 5 10
2. Údaje o SEQ IDNO 15:2. SEQ IDNO 15 data:
(i) Označenie sekvencie:(i) Sequence designation:
(A) Dĺžka: 42 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 42 amino acids (B) Species: Amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-l996 (xi) Opis sekvencie: SEQ ID NO: 15:(H) File number: DE 196 15 074.4 (I) Date of administration: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 15:
2. Údaje o SEQ IDN0 16:2. Data of SEQ ID NO 16:
(i) Označenie sekvencie:(i) Sequence designation:
(A) DÍžka: 14 aminokyselín (B) Druh: aminokyselina (C) Tvar závitovky: jednovláknová (D) Topológia: lineárna (ii) Druh molekúl: peptid (x) Údaje o zverejnení:(A) Length: 14 amino acids (B) Species: Amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:
(H) Číslo spisu: DE 195 45 472.3 (I) Deň podania: 06-december-1995 (x) Údaje o zverejnení:(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:
(H) Číslo spisu: DE 196 15 074.4 (I) Deň podania: 17-apríl-l996 (xi) Opis sekvencie: SEQ ID NO: 16:(H) File number: DE 196 15 074.4 (I) Date of administration: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 16:
Claims (4)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19545472A DE19545472A1 (en) | 1995-12-06 | 1995-12-06 | Diagnosis of squamous epithelial cell carcinoma |
| DE19615074 | 1996-04-17 | ||
| PCT/EP1996/005448 WO1997021104A1 (en) | 1995-12-06 | 1996-12-05 | Method of diagnosing and treating epithelioma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| SK73698A3 SK73698A3 (en) | 1999-01-11 |
| SK284378B6 true SK284378B6 (en) | 2005-02-04 |
Family
ID=26020988
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SK736-98A SK284378B6 (en) | 1995-12-06 | 1996-12-05 | Use of an antibody molecule which binds to the amino acid sequence WFGNRWHEGYR, for the preparation of a medicament for the treatment of squamous cell carcinomas |
Country Status (26)
| Country | Link |
|---|---|
| EP (1) | EP0865609B1 (en) |
| JP (1) | JP2000502067A (en) |
| KR (1) | KR100473824B1 (en) |
| CN (1) | CN1151377C (en) |
| AR (1) | AR004360A1 (en) |
| AT (1) | ATE235056T1 (en) |
| AU (1) | AU726704B2 (en) |
| BG (1) | BG62985B1 (en) |
| BR (1) | BR9611901A (en) |
| CA (1) | CA2239709A1 (en) |
| CO (1) | CO4520233A1 (en) |
| CZ (1) | CZ174198A3 (en) |
| DE (1) | DE59610248D1 (en) |
| DK (1) | DK0865609T3 (en) |
| EE (1) | EE03783B1 (en) |
| ES (1) | ES2190484T3 (en) |
| HK (1) | HK1011560A1 (en) |
| MX (1) | MX9804459A (en) |
| NO (1) | NO319903B1 (en) |
| NZ (1) | NZ324314A (en) |
| PL (1) | PL184521B1 (en) |
| PT (1) | PT865609E (en) |
| SK (1) | SK284378B6 (en) |
| TR (1) | TR199801027T2 (en) |
| UY (1) | UY24389A1 (en) |
| WO (1) | WO1997021104A1 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7189397B2 (en) * | 1999-10-08 | 2007-03-13 | Arius Research Inc. | Cytotoxicity mediation of cells evidencing surface expression of CD44 |
| US20050100542A1 (en) * | 1999-10-08 | 2005-05-12 | Young David S. | Cytotoxicity mediation of cells evidencing surface expression of CD44 |
| US6972324B2 (en) | 2001-05-18 | 2005-12-06 | Boehringer Ingelheim Pharmaceuticals, Inc. | Antibodies specific for CD44v6 |
| EP1258255A1 (en) * | 2001-05-18 | 2002-11-20 | Boehringer Ingelheim International GmbH | Conjugates of an antibody to CD44 and a maytansinoid |
| US20030103985A1 (en) | 2001-05-18 | 2003-06-05 | Boehringer Ingelheim International Gmbh | Cytotoxic CD44 antibody immunoconjugates |
| WO2002094879A1 (en) * | 2001-05-18 | 2002-11-28 | Boehringer Ingelheim International Gmbh | Antibodies specific for cd44v6 |
| EP1391213A1 (en) * | 2002-08-21 | 2004-02-25 | Boehringer Ingelheim International GmbH | Compositions and methods for treating cancer using maytansinoid CD44 antibody immunoconjugates and chemotherapeutic agents |
| EP1417974A1 (en) * | 2002-11-08 | 2004-05-12 | Boehringer Ingelheim International GmbH | Compositions and methods for treating cancer using cytotoxic CD44 antibody immunoconjugates and radiotherapy |
| PL2531527T3 (en) * | 2010-02-04 | 2014-08-29 | Univ Miami | A monoclonal antibody to cd44 for use in the treatment of head and neck squamous cell carcinoma |
| US9218450B2 (en) * | 2012-11-29 | 2015-12-22 | Roche Molecular Systems, Inc. | Accurate and fast mapping of reads to genome |
| WO2019161528A1 (en) * | 2018-02-22 | 2019-08-29 | Abmart Shanghai Co., Ltd. | Therapeutic antibody and uses thereof |
| WO2023227644A2 (en) | 2022-05-25 | 2023-11-30 | Akiram Therapeutics Ab | Binding protein |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69406664T2 (en) * | 1993-06-18 | 1998-06-04 | Biotie Therapies Oy | COMPOSITIONS AND DIAGNOSTIC METHODS USING MONOCLONAL ANTIBODY AGAINST CD44V6 |
| EP0767378A1 (en) * | 1993-06-22 | 1997-04-09 | BOEHRINGER INGELHEIM INTERNATIONAL GmbH | Method for the diagnosis and analysis of stomach carcinoma |
| DE4326573A1 (en) * | 1993-08-07 | 1995-02-23 | Boehringer Ingelheim Int | Polypeptides encoded by exon v5 of the CD44 gene as targets for immunotherapy and immunoscintigraphy of tumors |
| UA58482C2 (en) * | 1994-06-08 | 2003-08-15 | Бьорінгер Інгельхайм Інтернаціональ Гмбх | MONOCLONAL ANTIBODY VFF-18 AGAINST CD44v6 AND ITS FRAGMENTS |
-
1996
- 1996-12-03 UY UY24389A patent/UY24389A1/en not_active IP Right Cessation
- 1996-12-04 AR ARP960105480A patent/AR004360A1/en unknown
- 1996-12-05 AT AT96942362T patent/ATE235056T1/en not_active IP Right Cessation
- 1996-12-05 JP JP9520993A patent/JP2000502067A/en not_active Ceased
- 1996-12-05 EE EE9800164A patent/EE03783B1/en not_active IP Right Cessation
- 1996-12-05 BR BR9611901A patent/BR9611901A/en not_active Application Discontinuation
- 1996-12-05 WO PCT/EP1996/005448 patent/WO1997021104A1/en not_active Application Discontinuation
- 1996-12-05 EP EP96942362A patent/EP0865609B1/en not_active Expired - Lifetime
- 1996-12-05 NZ NZ324314A patent/NZ324314A/en unknown
- 1996-12-05 SK SK736-98A patent/SK284378B6/en unknown
- 1996-12-05 DK DK96942362T patent/DK0865609T3/en active
- 1996-12-05 PT PT96942362T patent/PT865609E/en unknown
- 1996-12-05 KR KR10-1998-0704242A patent/KR100473824B1/en not_active IP Right Cessation
- 1996-12-05 CZ CZ981741A patent/CZ174198A3/en unknown
- 1996-12-05 ES ES96942362T patent/ES2190484T3/en not_active Expired - Lifetime
- 1996-12-05 PL PL96327066A patent/PL184521B1/en not_active IP Right Cessation
- 1996-12-05 CA CA002239709A patent/CA2239709A1/en not_active Abandoned
- 1996-12-05 TR TR1998/01027T patent/TR199801027T2/en unknown
- 1996-12-05 AU AU11773/97A patent/AU726704B2/en not_active Ceased
- 1996-12-05 CO CO96063967A patent/CO4520233A1/en unknown
- 1996-12-05 CN CNB961992484A patent/CN1151377C/en not_active Expired - Fee Related
- 1996-12-05 DE DE59610248T patent/DE59610248D1/en not_active Expired - Fee Related
-
1998
- 1998-06-04 MX MX9804459A patent/MX9804459A/en not_active IP Right Cessation
- 1998-06-05 NO NO19982588A patent/NO319903B1/en unknown
- 1998-06-05 BG BG102513A patent/BG62985B1/en unknown
- 1998-12-07 HK HK98112910A patent/HK1011560A1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0865609B1 (en) | 2003-03-19 |
| NO319903B1 (en) | 2005-09-26 |
| AU726704B2 (en) | 2000-11-16 |
| PT865609E (en) | 2003-08-29 |
| BR9611901A (en) | 1999-03-02 |
| AU1177397A (en) | 1997-06-27 |
| TR199801027T2 (en) | 1998-10-21 |
| UY24389A1 (en) | 2001-10-25 |
| EE03783B1 (en) | 2002-06-17 |
| CN1151377C (en) | 2004-05-26 |
| EP0865609A1 (en) | 1998-09-23 |
| CZ174198A3 (en) | 1999-02-17 |
| CO4520233A1 (en) | 1997-10-15 |
| MX9804459A (en) | 1998-09-30 |
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| KR19990071952A (en) | 1999-09-27 |
| SK73698A3 (en) | 1999-01-11 |
| WO1997021104A1 (en) | 1997-06-12 |
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| BG62985B1 (en) | 2000-12-29 |
| NZ324314A (en) | 2000-02-28 |
| NO982588D0 (en) | 1998-06-05 |
| PL327066A1 (en) | 1998-11-23 |
| CN1207811A (en) | 1999-02-10 |
| KR100473824B1 (en) | 2005-09-30 |
| DK0865609T3 (en) | 2003-06-23 |
| JP2000502067A (en) | 2000-02-22 |
| HK1011560A1 (en) | 1999-07-16 |
| AR004360A1 (en) | 1998-11-04 |
| ES2190484T3 (en) | 2003-08-01 |
| DE59610248D1 (en) | 2003-04-24 |
| CA2239709A1 (en) | 1997-06-12 |
| PL184521B1 (en) | 2002-11-29 |
| ATE235056T1 (en) | 2003-04-15 |
| NO982588L (en) | 1998-08-05 |
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