Primers and methods for the detection of disseminated tumor...

Details Primers and methods for the detection of disseminated tumor... Primers and methods for the detection of disseminated tumor...

1999-12-17

2002-11-05

Jones, W. Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S242000, C435S069100, C435S069300, C435S173300, C435S320100, C435S235100, C435S091100, C536S023500, C530S330000

Reexamination Certificate

active

06475727

ABSTRACT:

The present invention relates to primers that specifically hybridize to nucleic acid molecules complementary to the messenger RNA transcribed from genes encoding MAGE tumor-specific antigens or a part thereof or to a complementary strand thereof as well as to diagnostic compositions comprising said antigens. The present invention further relates to methods for detecting disseminated tumor cells employing the primers of the invention as well as methods for preparing a tumor adjuvant vaccine.
Immunotherapeutic approaches have previously been applied to patients with minimal residual disease and could reduce mortality and recurrence associated with distant metastases. Since early occult dissemination of tumor cells is already present in about half of the cancer patients, efficient treatment strategies have to be developed for eliminating residual tumor cells. Many immunocytochemical and PCR-based assays using histogenetic differentiation markers exclusively allow the detection of micrometastatic cells that worsen the prognosis after local tumorectomy without identifying antigens for adjuvant immunotherapy. Limitations of RT-PCR analyses in oncological disorders resulted in the tissue-specific rather than tumor-specific expression of marker genes. Detection of disseminated tumor cells is thus restricted to a few, certain tumor types deriving from similar histological origin. Moreover, tumor cells are only detectable in front of special tissue background, so that most published approaches depend on samples taken from mesenchymal compartments. The presence of ubiquitous transcriptional factors and minimal activation of promotor sequences in background cells may cause illegitimate transcription and reduce specificity and sensitivity due to competing background signals. Furthermore, most assays measure only a single marker gene not considering inter- and intra-individual tumor cell heterogenity.
Several tumor rejection antigens have recently been characterized that are presented on major histocompatibility complex (MHC) class I molecules to autologous cytolytic T-lymphocytes (CTL). The MAGE genes belong to a family of 12 closely related genes with an overall homology of 64-85% and are classified as “cancer-testis-antigens”.
The MAGE genes have been employed in the prior art to use primers specific for MAGE 1 to 4, 6 and 12 for the detection of a variety of tumors (WO 95/23874). The specific primers as well as the regions they prime to in the MAGE genes are, however, inappropriate for the diagnosis of disseminated tumor cells in human samples. WO 96/29430 discloses genetic markers for the detection of melanomas and breast cancer cells including occult cancer cells or metastatic cells derived therefrom. In said application, it is further described that a number of markers including MAGE-3 may be employed to detect such tumors. However, methods disclosed in this application are not expected to be suited for the detection of disseminated tumor cells derived from human malignacies of many different histological origins.
Since, in particular, the early reliable detection of disseminated tumor cells in a patient enables the early treatment and/or vaccination in order to prevent recurrence of cancerous growth, the technical problem underlying the present invention was to improve the sensitivity of the prior art methods for detecting such disseminated tumor cells in patients with maligancies of many different histological origins. The solution to said problem is achieved by the embodiments characterized in the claims.
Accordingly, the present invention relates to a primer specifically hybridizing to a nucleic acid molecule complementary to the messenger RNA transcribed from a gene encoding a MAGE tumor-specific antigen or a part thereof or to a complementary strand thereof, said primer being selected from the group of
(i) primers having the same 3′ portion as one of the following groups of primers:
(a)
5′-gtagagttcggccgaaggaac-3′
(SEQ ID NO: 1)

5′-caggagctgggcaatgaagac-3′
(SEQ ID NO: 2)

5′-cattgaaggagaagatctgcct-3′
(SEQ ID NO: 3)

5′-gagtagaagaggaagaagcggt-3′
(SEQ ID NO: 4)

5′-gaagccggcccaggctcg-3′
(SEQ ID NO: 5)

5′-gatgactctggtcagggcaa-3′
(SEQ ID NO: 6)

5′-caccaaggagaagatctgcct-3′
(SEQ ID NO: 7)

5′-tcctcagtagtaggagcctgt-3′
(SEQ ID NO: 8)

5′-tccgtgaggaggcaaggttc-3′
(SEQ ID NO: 9)

5′-atcggattgactccagagagta-3′
(SEQ ID NO: 10)

(b)
5′-tagagttcggccgaaggaac-3′
(SEQ ID NO: 11)

5′-ctgggcaatgaagacccaca-3′
(SEQ ID NO: 12)

5′-cattgaaggagaagatctgcct-3′
(SEQ ID NO: 13)

5′-caggcttgcagtgctgactc-3′
(SEQ ID NO: 14)

5′-ggctcggtgaggaggcaag-3′
(SEQ ID NO: 15)

5′-gatgactctggtcagggcaa-3′
(SEQ ID NO: 16)

5′-caccaaggagaagatctgcct-3′
(SEQ ID NO: 17)

5′-caggcttgcagtgctgactct-3′
(SEQ ID NO: 18)

5′-tccgtgaggaggcaaggttc-3′
(SEQ ID NO: 19)

5′-gagcctgcgcacccaccaa-3′
(SEQ ID NO: 20)
 and;
(ii) primers that overlap with a primer sequence depicted in (a) or (b).
The term “overlap” in this context means the overlap of at least one nucleotide.
Accordingly, said primers can hybridize to RNA, preferably mRNA or to DNA, preferably cDNA.
In accordance with the present invention, it was found that the primers having the above-recited 3′ ends are particularly useful in detecting disseminated tumor cells of many different histological origins at a sensitivity level that was unknown from the prior art. Additionally, primers that overlap with the above-recited nucleic acid sequences and that hybridize to MAGE genes have been found useful for the detection of disseminated tumor cells in human samples. Preferably, said primers overlap with the nucleic acid sequences depicted under (a), above. It is further preferred that the primers of the invention have a length of between 15 and 25 nucleotides. With the exception of primers that recognize MAGE-3 and -6, each of said primers specifically detects the expression of only one MAGE gene.
The present invention accordingly provides a means for detecting a cancerous condition at a very early stage of the disease and thus allows the timely treatment of patients where disseminated tumor cells are detected. It was surprisingly found by the present invention that, in contrast to prior art reports, disseminated tumor cells of many different histological origins can be detected using the above-recited primers, even if the analyzed patient has no clinical evidence of disease. It has to be emphasized that the prior art methods and primers as well as the regions of the MAGE genes to which the primers described in the prior art hybridize are not suitable for detecting disseminated tumor cells in samples of patients with malignancies of many different histological origins since the sensitivity of said primers has, in accordance with the present invention, proven not to be high enough for such an analysis. The term “specifically hybridizing” as used in accordance with the present invention is intended to mean that these primers do not cross-amplify nucleic acids from the genes of other MAGE tumor specific antigens in a sensitive RT-PCR. Such RT-PCRs can be devised by the person skilled in the art according to conventional protocols. The specific hybridization occurs under stringent conditions. Such conditions can be devised by the person skilled in the art without undue burden according to conventional protocols; see, e.g., “Nucleic Acid Hybridization, A Practical Approach”; edited by Hames & Higgins, IRL Press, Oxford 1985.
The primers of the invention are advantageously used in PCR amplification of nucleic acid sequences derived from the desired MAGE genes or fragments thereof. The primers of the invention are devised to avoid the amplification of genomic DNA but to amplify a DNA sequence derived from the reverse transcription of messenger RNA. In this way, the PCR product can be unambiguously correlated with express

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