Method for diagnosis and therapy of Hodgkin's lymphomas

Details Method for diagnosis and therapy of Hodgkin's lymphomas Method for diagnosis and therapy of Hodgkin's lymphomas

1999-12-21

2002-04-16

Huff, Sheela (Department: 1642)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S006120, C435S007230, C435S188000, C530S387300, C530S388850

Reexamination Certificate

active

06372441

ABSTRACT:

The present invention relates to processes for diagnosing and treating Hodgkin's lymphomas (lymphogranulomatosis) based on the expression of the variant exon v10 of the gene CD44 as the molecular target, agents for these processes and the use of these agents.
The highly glycosylated cell surface protein CD44 is involved in the interaction between cells and the extracellular matrix such as migration and activation of leukocytes in inflammation and immune monitoring, precursor formation of leukocytic and myeloid cells in bone marrow and also in the development of lymphoid organs and the interaction of cells with the extracellular matrix (Lesley et al., 1993, Günthert 1993, Pals et al., 1993, Mackay et al., 1994). The human CD44 gene is made up of at least 19 exons, of which at least 12 which code for the extracellular region are alternatively spliced (Screaton et al., 1992). The CD44 gene is transcribed in a number of normal tissues and carcinomas (Fox et al., 1994). Whereas the standard CD44 molecule (CD44s) is ubiquitously found expressed in epithelial and mesenchymal tissues, the various isoforms produced by alternative RNA splicing are found in very limited distribution (Heider et al., 1993). Some of the variant isoforms are involved in the activation of lymphocytes and occur in conjunction with metastasisation (Mackay et al., 1994, Günthert et al., 1991, Rudy et al., 1993, Koopman et al., 1993). Although the expression of variant CD44 has been shown to have a direct biological role in metastasis formation in carcinoma of the pancreas in rats (Günthert et al., 1991, Seiter et al., 1993), its role in human tumours is as yet unknown.
Various reports have been published showing that certain alternatively spliced forms of CD44 were expressed in human metastatic tumours (Heider et al., 1993 and 1996, Fox et al., 1994, Friedrichs et al., 1995, Kaufmann et al., 1995, Salles et al., 1993, Stauder et al., 1995, Koopman et al. , 1993, Tanabe et al., 1993) Studies of the expression of CD44 in non-Hodgkin's lymphomas (NHL) concentrated on analysing the so-called lymphocyte homing receptor CD44H or CD44s (Horst et al., 1990a, Horst et al., 1990b, Jalkanen et al., 1991, Möller et al., 1992). Whereas some authors (Horst et al., 1990a, Jalkanen et al., 1991, Picker et al., 1988, Pals et al., 1989, Fujiwara et al., 1993) found a correlation between increased CD44s expression and unfavourable prognosis, other authors (Terpe et al., 1994) could not confirm these findings. Recently, upregulation of CD44v3 and CD44v6 isoforms was found in NHL with unfavourable pathological status (Koopman et al., 1993, Terpe et al , 1994, Salles et al., 1993, Stauder et al., 1995), whilst variant specific CD44-mAbs were used (Mackay et al., 1994, Koopman et al., 1993, Fox et al., 1993).
Various approaches have been developed for making use of the differential expression of variant exons of the CD44 gene in tumours and normal tissues f or diagnostic and therapeutic purposes (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP 0531300).
The aim of the present invention was to develop new methods of diagnosing and treating Hodgkin's lymphomas (lymphogranulomatosis) and preparing agents for such processes.
This aim is achieved by means of the present invention. It relates to processes for diagnosing and treating Hodgkin's lymphomas (lymphogranulomatosis) which are based on the expression of the variant exon v10 of the CD44 gene as a molecular marker or target. Antibody molecules of corresponding specificity are particularly suitable as vehicles for selectively reaching Hodgkin's lymphomas in vivo.
Preferred processes are characterised in that an antibody molecule is used which binds specifically to the amino acid sequence SEQ ID NO. 2 (see Sequence Listing).
Other aspects of the present invention are the use of antibody molecules of this kind in the processes according to the invention and agents for performing these processes.
The invention further relates to the use of an antibody molecule which is specific to an epitope within the amino acid sequence which is coded by the variable exon v10 of the CD44 gene, for preparing a pharmaceutical composition for the diagnosis and/or treatment of tumoral diseases. The tumoral disease in question is preferably Hodgkin's lymphoma (lymphogranulomatosis).
The invention further relates to an antibody molecule which is specific to an epitope within the amino acid sequence which is coded by the variable exon v10 of the CD44 gene for pharmaceutical use. Preferably, an antibody molecule of this kind is characterised in that it binds to SEQ ID NO. 2. It may be, in particular, a monoclonal antibody, an Fab- or F(ab′)
2
-fragment of an immunoglobulin, a recombinantly produced antibody, a recombinantly produced chimeric or humanised antibody or single chain antibody (scFv). Preferably, an antibody molecule of this kind is linked to a radioactive isotope, a radioactive compound, an enzyme, a toxin, a cytostatic, a prodrug, a cytokine or some other immunomodulatory polypeptide.
The nucleic and amino acid sequence of the variant exon v10 of the CD44 gene is known (Screaton et al., 1992, Tölg et al., 1993). These sequences are shown in the Sequence Listing (SEQ ID NO. 1 and 2). The existence of degenerate or allelic variants is unimportant to the performance of the invention; such variants are therefore expressly included.
The invention may be carried out with polyclonal or monoclonal antibodies specific to an epitope which is coded by the exon v10. The preparation of antibodies to known amino acid sequences can be carried out using methods known per se (Catty, 1989). For example, a peptide of this sequence may be prepared synthetically and used as an antigen in-an immunisation procedure. Another method is to prepare a fusion protein which contains the desired amino acid sequence, by integrating a nucleic acid (which may be prepared synthetically or, for example, by polymerase chain reaction (PCR) from a suitable probe) which codes for this sequence, into an expression vector and expressing the fusion protein in a host organism. The fusion protein, optionally purified, can then be used as an antigen in an immunisation procedure and insert-specific antibodies or, in the case of monoclonal antibodies, hybridomas which express insert-specific antibodies, are selected by suitable methods. Such methods are known in the art. Heider et al. (1993, 1996) and Koopman et al. (1993) describe the preparation of antibodies against variant epitopes of CD44.
However, for the process according to the invention, it is also possible to use antibody molecules derived from poly- or monoclonal antibodies, e.g. Fab- or F(ab′)
2
-fragments of immunoglobulins, recombinantly produced single chain antibodies (scFv), chimeric or humanised antibodies and other molecules which bind specifically to epitopes coded by exon v10. From a complete immunoglobulin it is possible for example to produce Fab-or F(ab′)
2
-fragments or other fragments (Kreitman et al., 1993). The skilled person is also capable of producing recombinant v10-specific antibody molecules. Corresponding methods are known in the art. Recombinant antibody molecules of this kind may, for example, be humanised antibodies (Shin et al., 1989; Gussow and Seemann, 1991), bispecific antibodies (Weiner et al., 1993; Goodwin, 1989), single chain antibodies (scFv, Johnson and Bird, 1991), complete or fragmentary immunoglobulins (Coloma et al., 1992; Nesbit et al., 1992; Barbas et al., 1992), or antibodies produced by chain shuffling (Winter et al., 1994). Humanised antibodies may be produced for example by CDR grafting (EP 0239400). Framework regions may also be modified (EP 0519596). In order to humanise antibodies, nowadays it is possible to use methods such as PCR (cf. for example EP 0368684; EP 0438310; WO 9207075) or computer modelling (cf. for example WO 9222653). It is also possible to prepare and use fusion proteins such as single chain antibody/toxin fusion proteins (Chaudhary et al., 1990; Friedman et al.,

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