TITLE OF THE INVENTION:

AGENTS FOR THE PRE-SYMPTOMATIC DETECTION, PREVENTION AND TREATMENT OF BREAST CANCER IN HUMANS

Field of the Invention:

The invention relates to diagnostic reagents with therapeutic potential for the detection, prevention and treatment of breast and ovarian cancer, in humans. More specifically, the invention relates to ten genes, BC531L, BC532L, BC533 , BCAR2 , BCAR3L, BCRB1L, BCRB2L, BCFLT1L, BCFLT2L, and BRCE151 (BREX) , that are implicated in causing breast cancer, to their expressed proteins products and the use of these proteins as diagnostic probes for pre-symptomatic detection of breast and associated cancers. The invention additionally relates to nucleic acid molecules that influence the expression of these genes. The invention also relates to therapeutic methods that directed against the proteins and specific sequences in the nucleic acids.

Cross-reference to Related Applications:

This application is related to PCT/EP94/00651 , WIPO publication Number WO 94/21791.

Background of the Invention:

I. Breast Cancer

Breast cancer affects one in ten women and one in four thousand men. About five percent of these cases in women (.025% in men)

are associated with germ line heritable mutations. The rest, believed to be caused by somatic mutations of mostly unidentified genes, are termed "sporadic". As an inherited trait breast cancer is one of the most common genetic diseases in the industrial world; in fact, one out of 100 women alive today will develop breast cancer due to inheritance unless a cure for the disease is found. About 40% of cases are diagnosed before the patient has attained the age of 30.

At the moment there is neither a cure for breast cancer nor a non-invasive method for early detection of the disease. Breast cancer is presently treated using surgery, endocrine therapy and chemotherapy (Salmon, S.E., Semin. Oncol., 17:50-52 (1990) ; Hortobagyi, G.N. , Breast Cancer Res. Treat 21:3-13 (1992)) . Endocrine therapy results in complete or partial remissions in only 30% of patients (Jiang, S.-Y. et al . , J. Natl. Cane. Inst. 84:580-591 (1992) ; Muss, H.B., Breast Cancer Res. Treat 21:15-26 (1992)) . Chemotherapy, despite the development of new antineoplastic agents, has had only limited success in treating breast cancer (Hortobagyi, G.N., Breast Cancer Res. Treat 21:3-13 (1992) ) . Thus, surgery is the only presently proven treatment for breast cancer; its use has long been controversial (Albert, S. et al . , Cancer 41:2399-2408 (1978) . Surgery is often combined with endocrine therapy or chemotherapy regimes .

Biological agents, such as interferon and interleukin, have been found to be capable of producing definite anti-tumour responses. Unfortunately, such advances have not yet led to improved regimens for managing breast cancer (Hortobagyi, G.N., Breast Cancer Res. Treat 21:3-13 (1992)) . Monoclonal antibodies have also been employed to help the natural immune system. However, the antibodies available to date have not had the specificity or sensitivity to successfully target tumour cells.

The above approaches were based mainly on two overlapping hypotheses about the origin of breast cancer. One is that breast cancer originates in the breast and metastasizes serially through

lymph nodes before spreading to the rest of the body. This was the impetus for use of radical mastectomy combined with very early detection. The second hypothesis proposes that the disease invades the blood stream very early and spreads slowly (8 - 10 years) before it is detected in the breast by mammography or other specialized examination. This spurred the use of chemical adjuvant chemotherapy which did not improve the survival rate. Although these thoughts still pervade clinical approaches to breast cancer management, it is becoming increasingly obvious that killing tumour cells is not the answer but in fact the environment of the cell must be changed. This has to be done, essentially, by unravelling and manipulating the molecular events which control the expression of, until now, unknown genes which have disruptive effects on cell growth and development. One environmental mechanism through which cancer arises is cell exposure to a 'carcinogenic' chemical or to radiation. Such an exposure may damage the DNA leading to either an impairment of expression of a specific gene or the production of a mutant gene, or the expression of a gene which is normally repressed throughout life. The cell may then proceed to proliferate uncontrollably and alternately result in tumour formation.

Two classes of critical genes have been found experimentally to be influenced by the above mentioned conditions. One class of such genes has been referred to as "oncogenes". Oncogenes are genes which are naturally in an "inactivated" state, but which, through the effect of the DNA damage are converted to an "activated" state capable of inducing tumorigenesis (i.e. tumour formation) . Oncogenes have been identified in 15-20% of human tumours. The products of oncogenes ("oncoproteins") can be divided into two broad classes according to their location in the cell.

Oncogene products which act in the cytoplasm of cells have readily identifiable biochemical or biological activities (Green, M.R., Cell 56: -3 (1989)) . Those that act in the nucleus of a cell have been more difficult to characterize. Some nuclear

oncoproteins (such as E1A and myc) have transcriptional regulatory activity, and are believed to mediate their activities by the transcriptional activation of cellular genes (Kingston, R.E., Cell 41;3-5 (1985)) . Other nuclear oncoproteins appear to have a complex array of activities (such as DNA binding activity, ability to initiate viral DNA synthesis, ATPase activity, helicase activity, and transcriptional regulatory activity) (Green, M.R., Cell 56:1-3 (1989)) .

The second class of genes are the anti-oncogenes, or "tumour- suppressing genes". In their natural state these genes act to suppress factors which cause cell proliferation. Damage to such genes leads to a loss of this suppression, and thereby sometimes results in tumorigenesis. Thus, the deregulation of cell growth, which is a major characteristics of tumour cell proliferation may be mediated by either the activation of oncogenes or the inactivation of tumour-suppressing genes (Weinberg, R.A., Scientific Amer., Sept. 1988, pp 44-51) .

Oncogenes and tumour-suppressing genes have a basic distinguishing feature. The oncogenes identified thus far have arisen only in somatic cells, and thus have been incapable of transmitting their effects to the germ line of the host animal. In contrast, mutations in tumour-suppressing genes are identified in germ cell lines and are thus transmissible to the offspring.

Mutations in three tumour-suppressing genes and one putative tumour-suppressing gene have been shown to be involved in inherited breast cancer. To date, human familial breast cancer has been associated with point mutations and deletions in four genes located on three different chromosomes . Three of the four genes, BRCA1 on chromosome 17q21-23 (Miki, Y et al . , Science, 265:66-71 1994) , TP53 on chromosome 17pl3 (Malkin D., et al . , Science 250. 1233-1237 1990) and "BRCA2" on chromosome 13ql2-13 (Wooster A. et al . , Science 265:2088-2090 1994; Nature 579:789- 792(1995) ) , are involved in female breast and ovarian cancer. The fourth, the androgen receptor on chromosome Xqll-12 (Wooster A.,

et al . , Nature Genet. 2:132-134 1992) , and BRCA2 are involved in male breast cancer. The KFLT gene lies in a region close to BRCA2 on chromosome 13 and is a likely candidate for involvement in some aspects of BC. Germ line mutations in the retinoblastoma gene (RB gene) have not been implicated, so far, in breast or ovarian cancer,- however, somatic mutation in the RB gene are associated with sporadic breast cancers and somatic mutations in the TP53 gene are associated with sporadic breast and ovarian cancers (T'Ang.J.M., e . al . , Science 242:263-265 (1988)) . The exclusion of BRCAl from the nucleus (or the complete absence) has been shown to be associated specifically with all forms of breast cancer (Chen Y. et al . , Science 270:789-791 (1995)) . In spite of the fact that about 56 germ-line mutations have been discovered in the BRCAl gene (Shattuck-Eidens D. et al . , JAMA 273:535-541

(1995)) and about a dozen in BRCA2 (Wooster, R. et al . , Nature

579:789-792 (1995)) somatic mutation in these genes do not appear to be associated with breast and/or ovarian cancer (Futreal A. , et al . , Science, 266: 120-122 (1994) .

The odds of an individual developing breast cancer cannot be predicted from genetic analysis, because the absence of a germ cell mutation is no guarantee against becoming a victim of the disease. A woman with normal BRCAl, TP53 and (from what is known so far) BRCA2 genes, has the same risk as a woman with a hereditary mutation in one of these genes, of getting the disease even if in the latter case it occurs a little later in life. In fact there is a high probability that a person with a predisposing hereditary mutation can develop the disease from a factor (presently unknown) unrelated to the mutation.

As mentioned above only about 5% of all breast cancer victims have mutations in, at least, one of the predisposing genes. The other 95% of breast cancers are considered to be of sporadic origin. The high ratio of sporadic to inherited breast cancer (19 to 1) and the fact that there are no clinical or pathological differences in familial versus sporadic breast and ovarian cancer other than the age of onset (Lynch, H.T., et al . Gynecol . Oncol.

36, 48-53 (1990) , is a clear indication that mutations in the breast cancer related genes are not direct causes of the pathophysiological symptoms of the disease. Instead, common biochemical factors must be affected by mutations in all predisposing genes and by other "sporadic causes" of the disease. BRCAl appears to be a common factor in all forms of breast cancer, but neither the disruptions or mutations in BRCAl is an originating factor. The original factor must act at some point before the tumour suppressing activity of BRCA1/BRCA2 is disrupted. The latter activity may be related to transcriptional activation functions of these two genes ( Milner, J et. al. Nature 386,772-773 (1997)) .

SUMMARY OF THE INVENTION

The object of the present invention was to discover naturally occurring molecules which are expressed in a disease specific manner that were useful as specific, universal, diagnostic markers for breast and ovarian cancer (breast cancer) and which were also potential targets for therapeutics to treat breast cancer.

According to the invention the solution to the problem was in an approach to discovering disease specific genes which we have developed. The application of this procedure to the present problem led to the discovery of a number of pathogenic proteins (L-oncoproteins) which are expressed by L-oncogenes. These L- oncogene were located within the chromosomal loci occupied by the tumour suppressor genes described above. These molecules are expressed in humans with the disease but are not expressed in humans not afflicted with breast cancer. In contrast to oncogenes which must be activated by mutation, L-oncogenes are pre- activated oncogenes, i.e., the are expressed in the activated form in response to a number of environmental signals. The invention, therefore, concerns agents and use of these agents as markers for early diagnosis and targets for therapeutic approaches for all forms of human breast cancer. Such agents

include ten novel proteins implicated by unique association with breast cancer as well as analogues and derivatives of these molecules, and nucleic acid molecules encoding such molecules, or influencing their expression.

BRIEF DESCRIPTION OF THE FIGURES

FIG. la (SEQ ID NO:l) shows the cDNA sequence of BC531L.

FIG. lb (SEQ ID NO:2) shows the amino acid sequence of the deduced protein BC531 (in WO 94/21791 these sequence was designated "BC534")

FIG. Ic (SEQ ID NO:3) shows the cDNA sequence of BC532L

The sequence is identical to the sequences on the complementary strand of p53 exon 1 and exon 2.

FIG. Id (SEQ ID NO:4) shows the amino acid sequence of the deduced protein BC532 (in WO/94/21791 this sequence was designated "BC538") .

FIG. le (SEQ ID:NO:5) , shows the cDNA sequence for BC533L

(formerly BC5381) which is encoded by sequences homologous to regions on the anti sense strand of p53 exon 2 and flanking sequences of intron 1;

FIG. If (SEQ ID NO:6) , shows the deduced protein sequence

(BC533) .

FIG. Ig (SEQ ID NO:7) , shows the transcription regulatory region of BC531L, BC532L and BC533L (BC53P) . The region is identical to sequences on the anti sense strand of p53 intron 5. The promoter system consists of correlated CAATT box, TATA box and cap site and two (very strong) tandem arranged TATA boxes.

FIG. 2a SEQ ID: O:8, shows the cDNA for BCAR2L which is encoded by sequences homologous to the antisense strand of exon 1 of the androgen receptor gene (AR gene) .

FIG. 2b (SEQ ID NO:9), shows the deduced protein sequence

(BCAR2) .

FIG. 2c (SEQ IDNO:10) , shows the cDNA for BCAR3L which is encoded by sequences homologous to the antisense strand of exon 1 of the

AR gene.

FIG. 2d (SEQ ID NO:11) , shows the deduced protein sequences

β

(BCAR3 ) .

FIG. 2e (SEQ ID NO:12) , shows the 5' regulatory region of the BCAR2L and BCAR3L genes (BCARP) . It contains a CAATT promoter element coordinated with a strong TATA box and a cap site. The region is identical to sequences on the anti sense strand of intron B of the AR gene .

FIG. 3a (SEQ ID N0:13) , shows the cDNA for BCRB1L which is encoded by sequences on the antisense strand of intron 2 in the RB gene

FIG. 3b (SEQ ID N0:14) , shows the deduced protein sequence (BCRB1) .

FIG. 3c (SEQ ID N0:15) and (SEQ ID N0:16) , shows the 5' regulatory region of the BCRB1L gene (BCRBP1 and BCRBP2) . It contains two TATA box promoter elements which are homologous to sequences in intron 2 of the RB gene.

FIG. 3d (SEQ ID NO:17) , shows the cDNA for BCRB2L which is encoded by sequences on the antisense strand of intron 2 of the RB gene.

FIG. 3e (SEQ ID NO:18) , shows the deduced protein sequence (BCRB2) .

FIG. 3f (SEQ ID:NO:19) , shows the 5' regulatory region of the BCRB2L gene, (BCRBP3) . It contains coordinated TATA box and cap site. It is homologous to regions on the antisense strand of intron 2 of the RB gene.

FIG. 4a (SEQ ID NO:20) , shows the cDNA for BCFLT1L which is encoded by sequences homologous to the antisense strand of the non-translated 3' end of the KFLT gene.

FIG. 4b (SEQ ID NO:21) , shows the deduced protein sequence (BCFLT1) .

FIG. 4c (SEQ ID NO:22) , shows the cDNA for BCFLT2L which is encoded by sequences homologous to the antisense strand of the non-translated 3' end of the KFLT gene in the same region as SEQ ID:NO:20, but in a different reading frame.

FIG. 4d (SEQ ID NO:23), shows the deduced protein sequence (BCFLT2) .

FIG. 4e (SEQ ID N0:24) , (SEQ ID NO:25) , (SEQ ID NO:26) and (SEQ ID NO:27) shows the 5' regulatory region of the BCFLT1L and

3

BCFLT2L genes. It contains five TATA boxes four of which are correlated with cap sites. The sequence is homologous to sequences in the 3' untranslated end of KFLT gene.

FIG. 5a (SEQ ID NO:28), shows the cDNA for BREX) which is encoded by sequences homologous to exon 16 of the antisense strand of the

BRCAl gene

FIG. 5b (SEQ ID NO:29) , shows the deduced protein (BREX) .

FIG. 5c (SEQ ID NO:30 (BRCE152) , shows the deduced protein

(BRCE152) . The proteins are translated from different reading frames on the same cDNA and not been associated with the disease so far.

FIG. 5d (SEQ ID NO:31) shows the 5' regulatory region of BREX.

It contains three TATA boxes correlated to cap sites and a GC box. The sequence is homologous to a region of the anti sense of

BRCAl intron 17.

FIG. 5e (SEQ ID NO:32) shows the deduced sequence of a protein encoded within the regulatory region of BREXLpro, in the same reading frame as BRCE152.

FIG. 6a Evolutionary relationship between the ten cDNAs described in figures 1-5

FIG. 6b Evolutionary relationship between the proteins deduced from the cDNAs .

FIG. 7a. Homology and similarity between BRCAl exon 4 BCRB2L.

FIG. 7b. Domain homology between BRCAl protein and BCRB2

FIG. 7c Domain homology between BRCAl protein and BRCA2

FIG. 7d Homology between potentially important functional domains in BRCA1/BRCA2 and BRCE151. The latter may be a so called

"antisense homology box" (Baranyi L. et al Nature Medicine 1:894-

901 (1995) ) to BRCAl" .

FIG. 7e Functional homology between the c terminal putative nuclear transit sequence of BC531 and the two putative nuclear transit sequences of BRCAl .

FIG. 7f Domain homology between BRCAl and BC532

FIG. 7g Domain homology between p53, BREX and FLT4

FIG. 7h Homology between BC532 and BREX

FIG. 8. An identical core promoter sequence "ATAAAA" in at least one promoter element in the regulatory region of each

L-oncogenes.

FIG. 9a & 9b Organisation of BC53L genes.

FIG. 9c Organization of BREXL genes

FIG. 10 Sequence of primers used in RT-PCR to detect L-oncogene mRNA in clinical specimens.

FIG. lla Sequence of PCR product amplified from a breast tumour obtained with BC531L pp set

FIG. 12a Sequence of epitopes against which mono-specific polyclonal rabbit antibody was raised. The antibodies were used to detect the expression of L-oncoproteins in clinical specimens.

FIG 12b Outline of ELISA antigen trap test .

FIG 12c Outline of ELISA anmtibody trap test.

FIG 12d & e Detection of BC531Lp & BC532 in proteins isolated from breast tumours and normal breast of unrelated individuals.

FIG. I2f & g Detection of BC531 & BC532 in proteins isolated from primary tumours of unrelated women.

FIG. 12h & i Detection of BC531 and BE532 in the nuclear fraction of proteins isolated from advanced breast tumours with lymph node involvement .

FIG. 12 j & k detection of BC531 and BC532 in blood obtained from 18 women with tumours less than 1 cm in diameter.

FIG. 12 1 detection of breast cancer within a mixed sample of 228 women with breast cancer and normal women.

FIG. 12 m PCR amplification of BREX cDNA from breast tumour tissue.

FIG. 12 n Detection of anti-BREX endogenous antibody in a mixed population BC and non BC samples; pre-symptomatic detection of

BC in clinically normal humans.

FIG. 12 o Immunodetection of BREX and BRCAl in breast tumour tissue from the same patient.

FIG. 12 p silver stain of BREX affinity purified from serum for a breast cancer patient on an anti-BC531/BC532/BREX mixed affinity column.

FIG. 12 q silver stain of BREX-IgG complex/IgG fragments complexes affinity purified on a anti BREX epitope affinity column.

FIG. 13 How oncogene interact to causing breast cancer.

//

DETAILS OF THE INVENTION.

In detail, the invention provides a nucleic acid molecule, substantially free of natural contaminants, that encodes a protein selected from the group consisting of BC531L, BC532L, BC533L, BCAR2L, BCAR3L, BCRB1L, BCRB2L, BCFLT1L, BCFLT2L and BREXL. In particular, the invention provides the above-described nucleic acid molecule wherein the sequence is SEQ ID N0:1, SEQ ID NO:3, SEQ ID NO: 5 , SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:28

SEQ ID NO:l

5' - CCC AGA ATG CAA GAA GCC CAG ACG GAA ACC GTA GCT GCC CTG GTA GGT TTT CTG GGA AGG GAC AGA AGA TGA CAG GGG -3'

SEQ ID NO:3

TCC ATG GCA GTG ACC CGG AAG GCA GTC TGG CTG CCA ATC CAG GGA AGC GTG TCA CCG TCG TGG AAA GCA CGC TCC CAG CCC GAA CGC AAA GTG TCC CCG GAG CCC AGC AGC TAC CTG CTC CCT GGA CGG TGG CTC TAG AC -3'

SEQ ID NO:5

TCC ATG GCA GTG ACC CGG AAG GCA GTC TGG CTG CTG CAA CAG GAA AAG TGG GGA TCC AGC ATG AGA CGC TTC CAA CCC TGG GTC ACC TGG GCC TGC AGA GGA GGA GGC CCC TCC GGG AAC ACC ATG CCA GTG TCT GAG ACA GTC CGG CTT CCT GTG GTG CAG GAA AAG AAT GGC TGC TTC ACA TTC TCT CTT CCA ATG TTT CAC CAC AAC CCA AGC ACT CCT GCC CCA CCC CAC ACC AGC CAT GCA CTT CTT TGA -3'

SEQ ID NO:8

ATG TCT TTA AGG TCA GCG GAG CAG CTG CTT AAG CCG GGG AAA GTG GGG CCC AGC AGG GAC AAC GTG GAT GGG GCA GCT GAG TCA TCC TCG TCC GGA GGT GCT GGC AGC TGC TGC GGC AGC

CCC TTG CTG GCG GCC ACG GCG GCT CCA GGC TCT GGG ACG CAA CCT CTC TCG GGG TGG CAC TCC AGG GCC GAC TGC GGC TGT GAA GGT TGC TGT TCC TCA TCC AGG ACC AGG TAG -3'

SEQ ID NO:10

5' - GG CCA ATG GGG CAC AAG GAG TGG GAC GCA GCA CGG GTG GAA CTC CCA AAA GTG GGG CGT ACA TGC AAT CCC CCC GAA GCT GTT CCC CTG GAC TCA GAT GCT CCA ACG CCT GGA CTC CCA GGC CCA TGG ACA CCG ACA CTG CCA TAC ACA ACT CCA TGG CGT TGT CAG AAA TGG TCG AAG TGC CCC CTA ACT AAT TGT CCT TGG AGG AAG TGG GAG CCC CCG AGG CCT CCC TCG CAC TCC CGC TGC TGC TGC CTT CGG ATA CTG CTT CTT GCT GCT GTT GCT GAA GAA GTT GCA TGG TGC TGC TGG CCA CGC TCA GGA TGT CCT TAA -3'

SEQ ID NO:13

CT CGC ATG ACG CAG GGC CAG GGT AAG ACA GAC GAG GTG AGC CAC CTG GAC GAG AAG GAG AGC TCC GAA GAC AAG AGC AGC TCC CTG GAC AGT GAC GAG GAC CTG GAC ACG GCC ATC AAG GAC TTA AGG TCC AAG CGA AAG CTC AAG AAG AGG TGC AGG GAA CCC AGG GCT GCG TGC AGG AAG GTC AGG TTC AGC ACT GCC CAG ATG CAC TTC CTG GAG CAG CTG GGC GGG CTG CCG AGA GAC TGG AAA GAC AGA AGC CCG CCG GTG CTT AAT AGC TGA

SEQ ID NO:17

GT GGC ATG ATC ACG GCT CAC TGC AAA CAC TGC CAC CCA CCT TCA AGC AAT TCT CCT GCC TCA CCC ACC CAA GTA GTC GAG ATT ACA GGC ATC TGC TAC CAT GCC TGG CTC ATT TTT GTA TTT TTA GTA GAG ATG GGG TTT CAC CAT GTT GGC CAG GCT GGT CTA GAA CTC CTG ACC TCA ATG ATC ACC TGC CTG GGC CAC CCA AAG TGC TGG GAT TAC AGG TTT GAG CCA CCA CGC CCA GCC TTA TTT TTA TTT ATT TTT TTT TGC AAC AGG GTC TTC CTG TGT TGC CCA GGC TGG AGT GCA GTG GTG CGA TCT TGG CTT ACT GTA ACC TCC ACC TCC CAG GTT CAA GAG ATC GTC CCA CCT CAG CCT CCC AAG TAG -3'

SEQ ID NO:20

ATG ACT AGA AAT ATA GGA CCA AAC CAT GTC TGT CTT ATA TCT GTA GCA TAT ATT CTT GGT TTG TAT AAA AGT AAC TTT AAA ATT CCA GTT TCC TTA AAT AGT TAT GCA CAA AAC ACA CAT ACA CCC ACA CCC ACA CAC ACA CAC ACA CAC ACA CAC ACA CAC ACA CAC ACA CAG TTA CAC CAC TGT CGG CCA AAG ATG CAC ACC TCC TTT AAT CAA TTT AAA TGA -3'

SEQ ID NO:22

ATG CAC AAA ACA CAC ATA CAC CCA CAC CCA CAC ACA CAC ACA CAC ACA CAC ACA CAC ACA CAC ACA CAC AGT TAC ACC ACT GTC GGC CAA AGA TGC ACA CCT CCT TTA ATC AAT TTA AAT GAG GCT AGC GAG TAT CTG TTT GAT GTT TGC ATT CTT GTG GGC TAG -3'

SEQ ID NO:28

5' - ATGGT ATG TTG CCA ACA CGA GCT CAC TCT GGG GCT CTG TCT TAC GAA GGA TCA GAT TCA GGG TCA GAG AAG AGG CTG ATT CCA GAT TCC AGG TAA -3'

The invention also provides a protein, substantially free of natural contaminants, selected from the group consisting of BC531, BC532, BC533, BCAR2 , BCAR3, BCRB1, BCRB2, BCFLT1, BCFLT2 and BREX. In particular, the invention provides the above-described protein having a sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: , SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:29. The invention also provides two hypothetical proteins (expression of these two proteins protein has not been detected so far in clinical specimens) having a sequence of SEQ ID NO:30 and SEQ ID NO:32.

SEQ ID NO:2

M Q E A Q T E T V A A L V G F L G R D R R

SEQ ID NO:4

M A V T R K A V L P I Q G S V S P S W K A R S Q P E R K V S P E P S S Y L L P G R L

IH

SEQ ID NO: 6

M A V T R K A V W L L Q E E K W G S S M R R F Q P W V T W A C R G G G P S G N T M P V S E T A R L P V V Q E K N G C F T F S L P M F H H N P S T P A P P H T S H A L L

SEQ ID NO:

M S L R S A E Q L L K P G K V G P S R D N V D G A A E S S S S G G A G S C C G S P L L A A T A V P G S G T Q P L S G W H S R A D C G C E G C C S S S R T A

SEQ ID NO: 11

M G H K E W D A A R V E L P K V G R T C N P P E A V P L D S D A P T P G L P G P W T P T L P Y T T P W Q C Q K W S K C P L T N C P W R K W E P P R P P S H S R C C C L R I L L P A A V A E E V A W C W P R S G C P

SEQ ID NO:14

M T Q G Q G K T D E V R H L D E K E s S E D

K S S S L D S D E D L D T A I K D L R S K R

K L K K R C R E P R A A c R K V R F s T A Q

M H F L E Q L G G L P R D W K D R S P P V L

N S

SEQ ID NO:18

M I T A H C K H C H P P s S N S P A S P T Q

V V E I T G I C Y H A W L I F V F L V E M G

F H H V G Q A G L E L L T s M I T c L G H P

K C W D Y R F E P P R P A L F L F I F F c N

R V F L C C P G W S A V V R S W L T V T S T

S Q V Q E I V P P Q P P K

SEQ ID NO:21

M T R N I G P N H V C L I S V A Y I L G L Y K S N F K I P V S L N S Y A Q N T H T P T P T H T H T H T H T H T H T Q L H H C R P K M H T F S N Q F K

SEQ ID NO:23

M H K T H I H P H P H T H T H T H T H T H T H S Y T T V G Q R C T P P L I N L N E A S E Y L F D V C I L V G

SEQ ID NO:29

M L P T R A H S G A L S Y E G S D S G S S E K R L I P D S R

SEQ ID NO: 0

M V C C Q H E L T L G L C L Q K D Q I Q G H Q R R G

IS

SEQ ID NO:32

M D I L L L T L S V E A V N S G F S L L T L S S I A L Y P A V S V V

The invention also provides a reagent capable of diagnosing the presence of a molecule selected from the group consisting of BC531, a BC531 encoding nucleic acid molecule, BC532, a BC532 encoding molecule, BC533, a BC533 encoding molecule, BCAR2, a BCAR2 encoding molecule, BCAR3, a BCAR3 encoding molecule, BCRB1, a BCRB1 encoding molecule, BCRB2, a BCRB2 encoding molecule, BCFLT1, a BCFLT1 encoding molecule, BCFLT2 , a BCFLT2 encoding molecule and BREX, a BREX encoding molecule.

The invention particularly concerns the embodiments wherein the reagent is a nucleic acid molecule produced from nucleic acid molecules having a sequence complementary to SEQ ID NO: 7

(nucleotides 12-23 "BC53P1"), (nucleotides 90-106, "BC53P2") or

(nucleotides 160-171, "BC53P3") , or produced from nucleic acid molecules having a sequence complementary to SEQ ID NO:12

(nucleotides 46-90, "BCARP") , or having a sequence complementary to SEQ ID NO: 15,"BCRBP1", SEQ ID NO: 16, "BCRBP2" SEQ ID NO:19,

"BCRBP3", SEQ ID NO:24, "BCFLTP1", SEQ ID NO:25, "BCFLTP2", SEQ

ID NO:26, "BCFLTP3" SEQ ID NO:27 , "BCFLTP4" or SEQ ID NO:32,

BREXP.

SEQ ID NO:7

5' - GAGCTTTTTTTAAAAGCCAAGGAATACACGTGGATGAAG AAAAAGAAAAGTTCTGCATCCCCAGGAGAGATGCTGAGGGTGT GATGGGATGGATAAAAGCCCAAATTCAAGGGGGGAATATTCAA CTTTGGGACAGGAGTCAGAGATCACACATTAAGTGGGTAAACT ATAAAAAAACACTGAGGAAGCCTAGGGGTTG -3'

SEQ ID NO:12

5' - AAAGTGGACAGAGTATGGCACCAAACCCTAAGTTATTTGATAGG GCCTTGCCAATGACTCTATTTCTGAGATGATAAAATCCTGGGCC

ll

CTGAAAGGTTAGTGTCTCTCTCTGGAAGGTAAAGGAGAAAGGGA AAGAGAAGTGCATGTGCAAGACC

SEQ ID NO: 15

5' - AAAGCACCACAGAATAAAAGCGGGGTCAAGA

SEQ ID NO: 16

5'- TGCCGGCCCCCATGTAAGGCAGCAACAGGTCCC -3'

SEQ ID NO:19

5' - GATTTTACACATAAAAAAAGATTTTATATTTCATAT CTCAATGATTTTTAAA-3'

SEQ ID NO:24

5' - GGGTTCAAAGGAGAATACAAAGAGCAGAGAATATATA GGCAGACATTCAGGCTGAAATGAATTTGCTTAATAAA GGCAAAACT-3'

SEQ ID NO:25

5'- TCAAATATATCTACACTGGATAATTTGCATTTTCATGG- 3'

SEQ ID NO:26

5' - AAACAAGGATAAATTTGAGTTTATTTTAGGAAAAAAAA -3 '

SEQ ID NO:27

5' - GAACATAAAAGCAGTTGCCAAGTTTATCAATTAAGTTGGT-3 '

SEQ ID NO:32

5' - CTGTGATTGNTTTCTAGATTTCTTCCTCTAGGTTATTAAT TGACAATACCTACATAAAACTCTTTCCAGAATGTTGTTAAGTCT TAGTCATTAGGGAGATACATATGGATACACTCACAAATTCTTCT GGGGTCAGGCCAGACACCACCATGGACATTCTTTTGTTGACCCT TTCAGTTGAAGCTGTCAATTCTGGCTTCTCCCTGCTCACACTTT CTTCCATTGCATTATACCCAGCAGTATCAGTAGTCTGAGCAGCA GCTGGACTCTGGGCAGATTCTGCAACTTTCAATTGGGGAACTTT CAATGAGAGGTTGAAGATGGT -3'

n-

The invention also concerns a nucleic acid molecule obtainable by mutating a nucleic acid molecule having a sequence of SEQ ID NO:l SEQ ID NO:3 , SEQ ID NO:5, SEQ ID NO: 8 ; SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:22 or, SEQ ID NO:2. A protein molecule (especially an antibody, or a fragment of an antibody) , which is capable of binding to a molecule having a sequence of SEQ ID NO:2, SEQ ID NO: , SEQ ID NO:6, SEQ ID NO:) , SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23 Or SEQ ID NO: 29.

The invention also provides a method of treating breast cancer by providing an individual, in need of such treatment, an effective amount of an antibody or anti peptide substance against BC531, BC532, BC533, BCAR2, BCAR3 , BCRB1, BCRB2, BCFLT1, BCFLT2 or BREX. The antibody or antipeptide may be either directed against one of the proteins, all of them or a combination of some them. The invention also provides a method for treating breast cancer by use of a reagent or a combination of reagents to prevent the activation of the promoters (BC531P/2P/3P, BCARP, BCRBP1/P2/P3, BCFLTPI/P2/P3/P4 and BREXP) .

DESCRIPTION OF THE PREFERRED EMBODIMENTS :

The success of the present invention came mainly from our realization that there must be other genes within the loci occupied bs Tp53 and BRCAl which were associated with the biochemistry of breast and ovarian cancer, and that at least one of these genes must also have a related component on other genes known to be associated with the disease. Therefore, we used a procedure which we had successfully used to find alternative genes which were putative causative factors of other "genetic

l6 diseases", to search for such genes which might segregate with breast cancer within the loci encoding Tp53 gene on chromosome 17. We then used the potential genes detected in this regions as probes to scan sequences on the BRCAl gene, the RB gene, the AR gene, and sequenced regions in the proposed "BRCA2" locus on chromosome 13, which were available to us, to look for similar sequences .

We call the above mentioned (alternative genes) "piggy-back genes" (pb genes) , and we refer to this technology as "disease gene discovery by positional searching" (DGDPS) . As a working definition, pb genes are transcribed in any orientation within the chromosomal locus occupied by another characterised functional gene.

Following is a description of DGDPS procedure.

This procedure has an advantage over gene isolation by cloning from a genomic or cDNA library, because it overcomes three important drawbacks, (1) the possibility that some DNA sequences cannot be cloned by the conventional methods, (2) that some mRNA sequences are of such low abundance that they are not represented in the cDNA library, and (3) the products of some cloned sequences are highly toxic to bacterial or other hosts.

In general, first we identified a gene closely related to a gene already genetically linked to a certain disease, then isolated the mRNA transcribed from the gene from disease tisεue or patient's blood, then synthesized cDNA from the isolated mRNA with reverse transcriptase then amplified the novel cDNA with specific primers which flanked the entire coding region of the cDNA, then we identified the cDNA from the size following electrophoresis on agarose gel, and finally isolated the unique cDNA from the agarose gel. This allowed us to select out the desired molecule, if it was expressed, without having to probe

several hundred thousand cDNA clones .

The present invention derives in part from the discovery and cloning of 10 (ten) novel genes, BC531L, BC532L, BC533L, BCRB1L, BCRB2L, BCAR2, BCAR , BCFLTIL, BCFLT2L, and BREXL, using the above approach. Following are examples of positional searching as it was applied in the present invention:

(1) We examined the sequenced regions within the p53 and BRCAl loci on chromosome 17 and selected potential complete "orfs", i.e. with orfs acceptable translation initiation sequences (see Kozak, M. Nucleic Acid Res. 12:857-872 1984) and translation termination stop codons (TAA, TAG or TGA) in place,

(2) next we used the method of Bucher et al . , J. Mol. Biol. 212; 563 - 578 (1990) to identify putative promoter regions associated with the orf within 100-1000 bp 5' upstream of the translation initiation sequence and we identified potential poly-A addition signals (the consensus poly-A addition sequence is "AATAAA") within a region of -1000 bp 3' downstream from the stop translation codon of the potential orf,

(3) then, orfs fulfilling the above two characteristics were translated into putative protein sequences using the universal code,

(4) then we analyzed the putative protein with our proprietary computer assisted protein finger printing technology and obtained information about the potential biochemical characteristics of the deduced proteins,

(5) next the biochemical characteristics of the deduced proteins were correlated with the known biochemical characteristics of breast cancer,

(6) next RNA encoding proteins with properties correlating with the disease characteristics were selected as potential disease related candidates,

(7) next transcription of the selected sequences was investigated in clinical material, [detection of the presence of transcribed

2 θ mRNA sequences encoding the protein in a cell can be determined by any means capable of detecting mRNA.

In this invention reverse transcriptase-PCR (RT-PCR) was done using the Stratagene RAP-PCR RT-PCR kit according to the manufacturer's instructions, with unlabelled primers to detect cDNAs encoding the deduced proteins in RNA isolated from frozen human breast tumours, normal breast and lymphocytes. RNA from frozen tissue was extracted by grinding about 20 mg frozen breast tissue in a tissue homogenizer in the presence of diethylpyrocarbonate (DEPC) . Total RNA was isolated using the Stratagene micro RNA isolating Kit and poly (A) + was isolated using Stratagene Poly(A) + Quick mRNA isolation kit following the manufacturer's instruction. Isolated RNA was treated with 10 units of RNAse free DNAse at 37oC for 15 minutes. DNAse was inactivated by treating for 2 mins at 100 oC. cDNA synthesis using mRNA as template was carried out with the first strand protocol supplied with the and RT-PCR was done using the cycling conditions recommended by the manufacturer. Forward and reverse PCR primers were prepared to regions flanking the entire protein coding region of the orf of the selected protein (see table 1 for sequence of the primers and for the size of the expected amplified product) . The amplified cDNA was electrophoresed on agarose gels and the size was determined by comparison with DNA size markers which were electrophoresed along side. To verify the sequence of the cDNA, the region of agarose containing the desired size cDNA was extracted into H20, precipitated with ethanol and a portion was cycle sequenced using the primers in "12" and Perkin Elmer ampli-Taq on the Perkin Elmer 376 A DNA sequencer using a non radioactive method described by Liu, C. et al.Nucl. Acid. Res 21:333-334 (1993) .

(8) next it was determined that the proteins were actually expressed: in order to do this epitopes were identified within the amino acid sequence of the deduced protein using the method of Hopp, T.P. and Woods, K.R. Proc. Natl Acad Sci. USA 78:3824- 3828 (1981) and their sequences compared to sequences in

databases; epitopes (see table #2) having no homologue with sequences in public databases were selected. Mono-specific polyclonal rabbit antibodies were prepared against these and purified by immunoaffinity chromatography on Pharmacia LKB, CNBr- activated sepharose 4B according to the recommendation of the manufacturer (see section #11 on Immumology, in Current Protocols in Molecular Biology (Vol 1) Ausubel, F.M. et al (ed) John Wiley & Sons NY. NY. 1991) . Enzyme-based immunoassay formats (ELISAs) were done with a colorimetric assay using horse radish peroxidase conjugated IgG as the second antibody and orthophosphate diamine

(OPD) as substrate (see "ELISA and other Solid Phase Immunoassays" (Kemeny, D.M. et al . (eds) John Wiley & sons, N.Y, N.Y (1988) . Two versions of ELISA were used to determine the presence of the specific breast cancer related proteins in body fluids (blood, serum, saliva) obtained from breast cancer patients and the presence of an endogenous IgG produced by all breast cancer patients against these proteins.

To detect the expression of the deduced proteins in human material, proteins were isolated from breast tumours and normal tissue by precipitating the homogenized tissue with >80% ammonium sulphate and dialysis against SDSPAGE buffer (the buffer conditions are described in Laemmeli, U.K. Nature 227:680-685

(1970) ) . Dialysed proteins were boiled in SDSPAGE buffer and either electrophoresed in 17.5% acrylamide gel, following which the proteins were Western blotted onto nylon membrane and treated with the affinity purified antibody or spotted onto positively charged membranes and treated as above. Interaction of the antibody with the protein bound to the membrane was visualized with a chemiluminescent kit purchased from BioRad Inc. , according to the manufacturer's instructions (also see Blake M.S. et al.Anal. biochem. 136:175-179 (1984) . Interaction of the of the antibody with the protein localized within cells in breast and ovarian tissue and other tissue obtained from patients was visualized by immunohistochemical methods.

THE MOLECULES OF THE INVENTION

One embodiment of the invention relates to the discovery of ten pb-genes (L-oncogenes) the sequence and SEQ ID of which are given above, and the protein products of these genes (L-oncoproteins) , the sequence and SEQ ID also given above, using the DGDPS procedure. The transcription of the L-oncogenes is programmed by promoter elements located within the sequences shown in the embodiment of figures IG (BC531L/532L/533L) , 2E (BCAS2L/BCAR3L) , 3C (BCRB1L) , 3F (BCRB2L) , 4E (BCFLT1L/BCFLT2L) and 5D (BREXL) respectively. The chromosomal location and selected potential biological features of features of the L-oncoproteins which may be relevant to a role in breast and ovarian cancer, are set forth in the examples provided below and in Table 1. The organization of three of the genes designated BC531L, BC532L, BC533L deduced from our search procedure, is shown in the embodiment of figure 9a and 9b and the organisation of BREXL is shown in the embodiment of figure 9c.

Example 1

BC531 (SEQ ID NO:2, FIG IB) is a putative DNA binding protein, by similarity to human SNUB2 , yeast GCN1 and herpes virus major DNA binding protein. The protein can also affect the activity of a variety of intracellular proteins, including a human tyrosine kinase. As shown in the embodiment of figure 7e, the sequence domain in the c- terminal has the basic feature of the two putative nuclear transit sequences identified in BRCAl (Boulikas, T. J.Cell Biochem. 55:32-34 (1994)) and therefore might be able to compete with or mimic the function of BRCAl nuclear transport sequences . A situation which might lead to the exclusion of BRCAl from nuclear sites in cells where BC531 is expressed. BC531 has been identified in the nuclear and cytoplasmic protein fractions of breast tumours cells, but not in normal cells (figure I2h & 12i) .

Example 2

BC532 (SEQ ID NO:4, FIG ID) is potentially a highly phosphorylated protein which iε related to transcriptional factors e.g., ATP-4 and the transactivating transacting factor. It may be involved in intracellular phosphorylation reactions. It can mimic or modulate a variety of proteins, especially growth related factors and receptor, including two tyrosine kinase receptor precursors. As seen in the embodiment of figure 71, BC532 has a strong relationship to BREX the L-oncoprotein which is expressed within the region of the BRCAl gene exon 16- intron 16-intron 15, and by virtue of a unique structural relationship to BRCAl may play a role in the regulation of the latter protein. BC532 also have regions of domain homology with BRCAl (figure 7G) .

Example 3

BC533 (SEQ ID NO:6, FIG IF) has a GXGxxG protein kinase signal and similarities to transcription factors. It can mimic or influence the activity of a variety of proto oncogenes and growth factors, including a tyrosine kinase receptor precursor. BC533 contains the first 10 amino acids of BC532.

Example 4

BCAR2 (SEQ ID NO:9, FIG 2B) is a putative transcription activator which can mimic or influence transducer-type molecules. It can secrete two smaller biologically active polypeptides. It can mimic or influence the activity of a number of hormone related factors and other proteins including a tyrosine kinase receptor precursor. As shown in the embodiment of figure 7D the n-terminal region of the protein has significant homology to a domain in the c-terminal region of BRCAl.

Example 5

BCAR3 (SEQ ID NO:11, FIG 2D) is a putative transcription activator. It can effect or mimic the activities of a wide variety of proteins, including a tyrosine kinase receptor precursor. BBCAR2 and BCAR3 are transcribed from the same promoters; their expressions may be primarily related to male breast cancer.

Example 6

BCRBl (SEQ ID NO: 14, FIG 3B) can influence the expression and activity of several proteins including DNA polymerase and a tyrosine kinase. It has a bipartite nuclear localization sequence and can be highly phosphorylated.

Example 7

BCRB2 (SEQ ID NO:18, FIG 3E) contains two transmembrane helices and an Fe* ⁺ binding site. It is expressed from a mRNA which appears to be an allele of BRCAl exon 4, as shown in the embodiment of figure 7A; it contains sequences which are identical to BRCAl exon 4 splice sequences and hence can conceivably compete with exon 4 for inclusion in BRCAl mRNA. IfF the latter occurs it can lead to the expression of a hybrid protein which contains the first three exons of BRCAl and BCRB2. As shown in figure 7B, the protein has significant homology to small domains in BRCAl. BCRB2 can secrete three different active polypeptides by post-translation modification and can influence a variety of growth factors and other proteins including aromatases. Expression of the latter are usually changed in breast and ovarian cancer patients and are used as diagnostic parameters for the disease. The protein can also influence a tyrosine kinase receptor precursor and a tyrosine kinase.

Example 8

BCFLT1 (SEQ ID NO:21, FIG 4B) is expressed from an mRNA containing a 21 unit "CA" dinucleotide repeat. It is a putative leukotrine-type protein with some structural characteristics of homeobox proteins. It can modulate or mimic several important proteins, including a tyrosine kinase and a tyrosine kinase transforming protein.

Example 9

BCFLT2 (SEQ ID NO:23, FIG 4C) can be expressed alternatively to BCFLTl. It contains the identical 21 unit "CA" repeat present in BCFLT1. It is a homeobox type transcriptional activator which can influence or mimic a number of proteins especially Hox-related proteins; it can also modulate a tyrosine kinase and a DNA repair protein.

Hox genes are potentially powerful oncogenic factors (Maulbecker & C.C., and Gruss, P., Cell Growth & Differ., 4:431-441 1993) which are known to be involved in leukaemia (Celetti et al . , Int. J. Cancer, 53:237-244 1993) and other forms of cancers (De Vita et al . , Eur. J Cancer. 6:8887-8893 1993) ; recently a definite involvement of Hox genes was demonstrated in mouse mammary gland tumours (Freidmann, Y. el al . , 54:5981-5985 1994) , and it was suggested that Hox proteins play a role in the establishment of dispersed metastatic colonies during progression of breast cancer.

Example 10

BREX (SEQ ID NO:29) is a tachykinin-like polypeptide hormone which can effect many intracellular systems including activation of expression of the other L-oncogenes described herein, and FLT4 tyrosine kinase. As shown in figure 7H, it has significant homology to domains in p53 and FLT4. It contains a domain which

is identical to a εimilar domain in BRCAl which iε part of one of two BRCAl εulphation sites: The same domain is also 100% homologous about (70 % similar) to a domain in BRCA2 which is adjacent to a major glycosylation site. Tyrosine sulphation is important for proteins that enter the golgi vesicles to be glycosylated. BRCA1/BRCA2 are glycosylated granin type proteins which are secreted; glycosylation occurs mainly in the golgi vesicles and is necessary for the functional integrity of these proteins. Hence BREX may be able to obstruct maturation and transport activity of BRCA1/BRCA2. This and other data driven deductions led to the conclusion that BREX was involved in the earliest stages of BC whereas the other L-oncoproteins were likely involved at later stages of the disease.

A sequence region containing a potential L-oncogene was selected using BC532 as an "evolutionary relationship" probe. The evolutionary relationship between the L-oncogenes and between the L-oncoproteins was done according to the method of Higgins D.G and Sharp P.M., CABIOS =5:151-153 (1989) . The relatednesε between L-oncogenes, and between L-oncoproteins are shown in figure 6a & 6b.

In another embodiment of the invention it was discovered that all L- oncogenes contained at least one promoter element in the 5' regulatory region which harboured a conserved 6 bp core promoter sequence as shown in the embodiment of figure 8. This indicated that at least one promoter for each L-oncogene might be influenced by the same transcription factor/ mechanism or same variety of factors. Blocking such a factor may be sufficient to stop transcription of all the L-oncogenes.

Furthermore, according to this invention, the combination of non expression in normal cells and the complexity of the promoter systems located in the 5' regulatory region of SEQ ID N0:1, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:24,

SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 31, shown in figures IG, 2E, 3C, 3F, 4E and 5D, is an indication that the promoters can be regulated by a number of factors probably in a tissue specific manner. Preventing activation of selective L- oncogene promoters is, therefore, an ideal method to prevent expression of transcription of L-oncogenes, and hence expression of L-oncoproteins.

Example 11

Detection of L-oncogene transcription in breast tumour cells. RNA from breast tumour tissue and normal breast tissue was prepared and cDNA synthesis of RNA were done as described above. PCR primer set ("pp" sets) (table 2a) were used to amplify specific cDNA prepared with RNA isolation kit and PCR kit as described above. The expected size of the amplified cDNA was ascertained ("+") (which usually included the entire protein coding region for the respective protein) . No amplification was obtained when PCR was done on aliquots of the cDNA prepared from normal breast tissue ("-"). The amplified cDNA was eluted from agarose gels and cycle sequenced as described above. In every case the nucleotide sequence was identical to the predicted sequence. An example of the results obtained from cycle sequencing the PCR product from amplifying cDNA in breast tumours with pp-BC531 are shown in the embodiments of figures 11. The sequence is identical with the SEQ ID N0:1.

Example 12

Detection of L-oncoproteins in tissue from advanced breast tumours.

Detection of BC531/2/3 using monospecific antiserum Bc531abl and BC532abl (figure 12d-m) .

2 Θ

Protein was prepared from the supernatants of homogenized breast tumour tissue from normal persons and from frozen breast tumours described above. Protein concentration was determined using the BioRad micro protein assay kit, and each sample was diluted to give approx the same concentration of protein in 2μl . These were spotted on immunelite membrane, blocked with non-fat dry milk (NFDM) and treated with 1:2000 dilution of BC531abl or BC532abl overnight, at room temperature. BC531abl and Bc532abl complexed to BC531 and BC532 on the membranes was detected by use of a chemiluminescent labelled substrate linked to anti rabbit IGG by reaction with alkaline phosphate and visualized following exposure to x ray film from 2 mins to 10 mins . As shown in the embodiment of figure 12 d & e BC531 and BC532 were detected in fourteen advanced breast tumours but not in 2 normal breast tested.

Example 13

Detecting L-oncoproteins in tissue from primary breast tumours. Using the same procedure described above 18 primary tumours, obtained from unrelated women were tested for the presence of BC531 and BC532. As shown in the embodiment of figure 12e & 12f BC531abl and BC532abl detected BC531 and BC532 in the 15 confirmed primary breast tumours, but not in two normal controls or in three unconfirmed primary breast tumours.

Example 14

Discriminating between breast tumour affected tissue and normal breast tissue with BC531abl and BC532abl.

Proteins isolated from unmarked clinical specimens obtained from unrelated humans, containing a mixture of normal and cancerous breast tissue were tested for the presence of BC531 and BC532 with BC531abl and BC532abl, as described above. As shown in

Z3 figure 12H & 12i, the antibodies correctly identified the breast tumour tissue.

Example 15

Localization of BC531 and BC532 in crude nuclear fraction from tumour cells obtained from advanced breast tumours. The nuclear fraction isolated by differential centrifugation from four breast tumours with lymph node involvement and from four normal breast was treated with 1% triton x 100 to disrupt the nuclei. Proteins present in the cytoplasmic and nuclear fraction were tested with BC53labl and BC532abl as described above. Aε shown in figure 12j & 12k, BC531 and BC532 was detected in both the nuclear fraction and cytoplasm fraction of the tumour cells but not in any of the normal cells.

Example 16

Detection of BC531 and BC532 in normal and breast cancer patient blood.

Two (2) μl of blood serum from 14 normal and from five breast cancer patients were dotted onto immunolite membrane and probed for BC531 and BC532 with BC531abl and BC532abl. As shown in figure 121 & 12m, neither BC531 or BC532 was detect in any of the fourteen normal human plasma whereas both proteins were present in the five specimens from breast cancer patients .

Example 17

Detection of BREX mRNA in breast cancer and normal patients.

mRNA was isolated and converted to cDNA as described. PCR was done with the appropriate primer pair as shown in figure 12 m the correct size fragment was amplified in all five BC patients but not in three normal people tested.

3 Ό

Example 18

Detection of BREX protein and BREX protein complexed to human IgG in serum from an advanced breast cancer patient using the ELISA test described in figure 12 b/c . Serum from all breast and ovarian cancer positive patients was positive for BREX , ^• serum from four of five normal patients was negative for BREX and serum from 11 of 14 patients with other epithelial cell cancers and inflammatory diseases were negative for BREX serum one patient with pancreatic, one patient with stomach cancer and two benign BC patients were positive for BREX.

Example 19

Detection of BRCAl and BREX from tumour extract of the same patient .

Serum from a BC patient was analyzed by western blotting following non SDS cationic poly acrylamide gel electrophorosis and immunoreacting with anti-brex affinity purified epitope specific poly clonal IgG to detect BREX (figure 12 p and anti BRCAl monoclonal antibody to detect BRCAl using (figure 12 q) . The immunoreactive bands were determined with a species specific c emiluminescent labelled monoclonal IgG.

Therefore, according to this invention it is possible to use PCR primers and mono specific antibodies directed against L- oncoprotein epitopes to detect, with very high level of confidence, breast cancer and ovarian cancer in humans. Since the L-oncoproteins have the combined and singular biological properties to cause many biochemical characteristics of epithelial cell derived cancers, eliminating these molecules can lead to cessation of the symptoms; therefore, L-oncoproteins are suitable targets against which therapeutic reagents to stop breast cancer in humans can be directed.

Biochemical activity of the L-oncogenes relating to a role in breast and ovarian tumour formation: How L-oncogenes might initiate breast and ovarian cancer.

Aε can be seen from the expression profile of the L-oncogenes in tumour cells, shown in table 2a, BREXL was also present in all the specimens tested; anti-BREX antibody also detected BREX in all breast tumours tested (data not shown) . In immunohistochemical experiments (data not shown here) , done with anti BRCAl antibodies provided by others, anti-BRCAl and anti- BREXabl antibody co-reacted with what appeared to be the same structures in the cytoplasm of breast tumour cells, whereas anti- BC531 appeared mainly in the nucleus and scattered throughout the cytoplasm. While anti-BRCAl was located mainly in the nucleus in normal breast tissue, neither anti-BC531 nor anti-BREX reacted with cells from normal breast tissue. These results suggested to us that BRCAl and BREX were closely associated in the cytoplasm of breast tumour cells (Chen, Y. et al, Science 270:789-791 (1995) , have demonstrated conclusively that while BRCAl is found in the nucleus in all normal cells, it is almost exclusively found in the cytoplasm in breast and ovarian cancer cells) , whereas BC531 is associated with the nucleus in breast tumour cells. The latter supports the results shown in the embodiment of figure 12H and 12, which show that BC531 was detected in proteins isolated from a tumour cell nuclear pellet after treating the pellet with triton x-100. Similar experiments have not been done with BRCA2, because a specific BRCA2 antibody was not available to us .

Our invention has led to the development of a concept shown graphically in the embodiment of figure 13, about how the expression of L-oncoproteins is coordinated in breast cancer. It assumes: (1) that BRCA1/BRCA2 is the master suppressor of L- oncogenes transcribed from the complementary region of the tumour suppressor genes involved in familial breast and ovarian cancer

3* and of the homeobox type proteins within the KFLT2 gene; (2) that BREX, expressed from the antisense strand of BRCAl gene is a BRCAl antisense homology box (the antisense homology box has been described recently by Baranyi, L. et al . Nature Medicine 1:894- 901 (1995) and iε incorporated herein by reference) ; that the transcription of BREX can be activated by a number of various environmental and intracellular factors,- (3) that the anti sense homology box blocks the transport or maturation of BRCAl and BRCA2, rendering the protein incapable of entering or leaving the golgi system. This results in a loss of tumour suppressive activity (i.e transcriptional control) of these proteins and allows expreεεion of the L-oncogenes and L-oncoproteins. It is possible that BC531, which has a sequence strongly resembling BRCAl nuclear transport sequences, when expressed enters the nucleus and influences the regulatory regions of the chromosome that control critical growth related genes. The other L-oncogenes working in concert influence other critical genes. This cascade of expressed previously suppressed genes results in the devastating symptoms of breast and ovarian cancer and possibly some other epithelial cell-derived cancers.

One intriguing factor, which can be seen from the putative biochemical activities of the L-oncogenes, is the potential of each oncogene to influence activity/expression of a tyrosine kinase protein. The latter is consistent with many recent reports on tyrosine kinase modulation in breast and ovarian cancer (Negata, K., et al. , EMBO J.13: 3517-3523 1994; Pechoux C, et al., Anti Cancer Res. 14:1343-1360 1994; Gia M., et al. , Anti Cancer Res. 14:1441-1450 1994; Hubbard A.L., et al. , Br. J. Cancer 70:434-4391994; Rajkumer T. and Gullik W.J., Br J. Cancer 70:459:463 1994) . In addition the L-oncogenes can mimic or influence activity of a variety of proto oncogenes, growth factors and other proteins previously implicated (see Lemoine N.R., Ann, Oncol. 5 [Suppl.4] : 31-37 1994) in the etiology of breast and ovarian cancers. According to this invention breast

and ovarian tumour formation may be initiated by a widespread interference of tyrosine kinase activity in breast and ovarian tissue, which effects a variety of growth control factors; however, other mechanisms (Foidart, J.M., et al . , Bull. Acad. Natl. Med., 178:533-544 (1994), may be simultaneously activated by all or a combination of the antisense oncogenes .

Also, according to this invention the speed of the tumour advancement to metastasis may be dependent on the number and/or type of the L-oncogenes involved. However, tumour formation, in the very early stages following initiation should not be irreversible if expression of the L-oncogene/protein is stopped. This appears to be consistent with the finding of Dr. William Black (reported in the New York times, November 9, 1994 "Doubts on early cancer detection") , that 39% of women, between 40-50 on which autopsies are done, have tiny, quiescent, breast and or ovarian tumours which, most probably, would not have progressed any further.

In summary the invention involves the discovery of a gene (BREX) expressed from within the region encoding the BRCAl gene on chromosome 17. BREX encodes a protein which can be classified an endogenous pathogen as the human immune system produces a endogenous anti BREX antibody against it. The pathogenic activity of BREX stems from out competing BRCAl and BRCA2 for sulphation and glycosylation sites respectively on the golgi membranes and the potential to mimic tachykinin type physiologically active peptides. The loss of BRCA1/BRCA2 leads to an activation cascade of a number of genes that are normally repressed in humans . The protein expressed by some of these genes may bind to sites on chromosomal DNA and influence the activation of critical developmentally and growth related genes and other pathogenic genes normally repressed in the human genome; at least one of these pathogenic genes might be involved in disrupting tyrosine kinase phosphorylation reactions.

3H

The Uses of the Molecules of the Present Invention

A. Diagnostic Uses

Since none of the L-oncogenes are expressed at a detectable level by normal cells, the detection of these molecules in a tissue or f uid sample (such as a biopsy sample, or a blood or urine or saliva) is indicative of the presence of breast or ovarian cancer in a human.

In this invention we have used two types immunological approaches for detecting L-oncoproteins in human material; however, the detection of these molecules may be done by any of a variety of immunological methods; a large number of suitable immunoassay formats have been described (Yolken, R.H., Rev. Infect. Dis . 4:35 (1982) ; Collins, W.P., In: Alternative Immunoassayε, John Wiley & Sonε, NY (1985) ; Ngo, T.T. et al . , In: Enzyme Mediated Immunoassay, Plenum Press, NY (1985) ; incorporated by reference herein. Using epitope specific, double affinity purified, monospecific antibodies as shown in the examples given above and in figures 12A and 12B, the presence of the target molecule is detected by an immunopositive reaction; the intensity of which is proportional to the concentration of the antibody bound to the target molecule.

The other approach which is applicable to diagnosis of breast and ovarian cancer is detecting the expression of L-oncogene mRNAε (BC531L, BC532L, BC533L, BCAR2L, BCAR3L, BCRB1L, BCRB2L, BCFLTIL, BCFLT2L and BREXL in a breast cancer patient or in a randomly selected population as described in the examples given above. Diagnosis is based on any method capable of detecting mRNA encoding these proteins in tissue and biological fluids described herein. Thus, molecules nucleic acid probes (DNA or RNA) capable of hybridizing to such molecules may be used in the diagnosis of breast and ovarian cancer.

In one embodiment, the assays may be performed on RNA extracted from blood, cells or tissue as described in the specifications herein.

Where the concentration of such mRNA in a sample is too low to be detected, such mRNA may be specifically amplified through the use of any of a variety of amplification protocols, such as PCR (Mullis, K.B., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Mullis K. et al., U.S. Patent 4, 683, 202; Erlich, H. , U.S. Patent 4,582,788; Saiki, R. et al . , US 4,683,194 and Mullis, K.B., et al . , Met. Enzymol. 155:335-350 (1987), all incorporated by (reference) or transcription-based amplification systems (Kwoh D et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173 (1989); incorporated by reference Gingeras TR et al. , PCT appl . WO 88/10315; Davey, C. et al. European Patent Application Publication no. 329,822), etc. (all incorporated by reference)

B. Prognostic Uses

The present invention additionally provides a sensitive and specific of monitoring of possible recurrence of the disease in an individual believed to be cured of breast or ovarian cancer. Thus, any of the above-described assays may be performed on an asymptomatic individual in order to assess if there is a re¬ initiation of these diseases.

C. Therapeutic Uses

Significantly, the present invention provides a means for treating Breast cancer. Such treatment may be either "prophylactic", one that is provided in advance of any clinical symptom BC in order to prevent or attenuate any subsequent onset of the disease or A therapeutic treatment one that is provided in response to the onset of a symptom of breast cancer, and

31, serves to attenuate an actual symptom of the disease.

In one embodiment, treatment is provided by administering to a patient an effective amount of an antibody, or an antibody fragment (F(ab') , F(ab')2, or single chain antibodies, or pseudo- homologous antibodies including "humanized" antibodies or a combination of the above that is capable of neutralizing BC531, BC532, BC533, BCAR2, BCAR3, BCRBl, BCRB", BCFLT1, BCFLT2 and/or BREX. As used herein, an effective amount is an amount sufficient to mediate a clinically significant change in the severity of a symptom, or a clinically significant delay in the onset of a symptom.

These may include monospecific polyclonal or monoclonal antibodies (or fragments of either) may be administered. More preferably, such molecules will be of non-immunogenic. The latter can be pseudo-homologous (i.e. humanised molecules prepared by recombinant or other technology. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies, but are less immunogenic, and are better tolerated by the patient.

Humanized anti BC531, BC532, BC533, BCAR2, BCAR3, BCRBl, BCRB2, BCFLT1, BCFLT2, and BREX can be produced, for example by replacing an immunogenic portion of each antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) . The production of humanized antibodies have been described in details in the following references (Robinson, R.R. et al. , International Patent Publication PCT/US8 6/02269; Akira, K. et al. , European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S.L. et al. , European Patent Application 173,494; Neuberger, M.S. et al. , PCT Application WO 86/01533; Cabilly, S. et al. , European Patent Application 125,023; Better, M. et al. , Science 240:1041-1043 (1988); Liu, A.Y. et al. , Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A.Y. et al., J. Immunol. 139:3521-3526 (1987); Sun,

3?

L.K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987) ; Nishimura, Y. et al . , Cane.Res. 47:999-1005 (1987) ; Wood, CR. et al . , Nature 314:446-449 (1985) ; Shaw et al . , J. Natl.Cancer Inst. 80:1553-1559 (1988) ; Morrison, S.L. (Science, 229:1202-1207 (1985) ; Oi, V.T. et al . , BioTechniques 4:214 (1986) ; Jones, P.T. et al . , Nature 321:552-525 (1986) ; Verhoeyan et al . , Science 239:1534 (1988) ; Beidler, CB. et al . , J. Immunol. 141:4053-4060 (1988)) which are incorporated herein by reference.

In another embodiment the desired therapy may be obtained by targeting the nucleic acid molecules, specifically the promoter sequences in BC53P, BCARP, BCRBP1, BCRBP2 BCRBP3, BCFLTP1, BCFLTP2, BCFLTP3, BCFLTP4 and BREXP4 of the present invention with "antisense" nucleic acid molecules (anti sense oligonucleotides) . To act as an antisense oligonucleotide, the nucleic acid molecule must be capable of binding to or hybridizing with that portion of the molecule which mediates the translation of the target mRNA. Antisense oligonucleotides are disclosed in European Patent Application Publication Nos. 263,740; 335,451; and 329,882, and in PCT Publication No. WO90/00624, all of which references are incorporated herein by reference.

As used herein, an "antisense oligonucleotide" is a nucleic acid (either DNA or RNA) whose sequence is complementary to the sequence BC53P to BCARP, to BCRBP1 to BCRBP2, BCFLTP1, BCFLTP2 BCFLTP3, BCFLTP4 and BREXLP described herein, such that it is capable of binding to, or hybridizing with, an endogenous promoter or heat shock sequence in a manner sufficient to impair its transcription, and significantly inactivate it in a cell, and thereby impair (i.e. attenuate or prevent) its the translation into protein.

The anti sense oligonucleotides may be transported into the cell using the Protein-Polycation Conjugates system (Beug, H. et al

3ff

United States patent 5,354, 844 11/10 1994) in an appropriate pharmaceutical compound.

In yet another embodiment of the present invention, anti sense nucleic acid molecule (s) may be administered using viral or retroviral vectors in accordance with the methods of "gene therapy". The topic is extensively covered in: Biotechnology, A Comprehensive Treatise, volume 7B, Gene Technology, VCH Publishers, Inc. NY, pp 399-458 (1989)) . The latter is incorporated herein by reference.

Although, as indicated above, such gene therapy can be provided to a recipient in order to treat (i.e. suppress, or attenuate) an existing condition, the principles of the present invention can be used to provide a prophylactic gene therapy to individuals who, are deemed at risk for the disease.

Administration of the Molecules of the Present Invention

Aε disclosed in Remington's Pharmaceutical Sciences (1980) , additional nucleic acid molecules may be employed to control the duration of action of any therapeutic subεtance adminiεtered to a patient .

Having now generally described the invention, through references and examples that makes it more readily understood by any one sufficiently skilled in the art, it must be pointed out that these are not intended to be limiting of the present invention, unless specified.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it iε capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and

35 including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential

features herein before set forth and as follows in the scope of the claims .

Note:

SEQ ID NO:30 and SEQ ID NO: 32 are not described in detail in the description of this invention. Although the association of these proteins with BRCAl and BREXL makes them suspect for involvement in breast cancer, we have not, so far, detected transcripts of these molecules in breast cancer tumour cells or in normal tissue.

o

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Bergmann, Johanna and Preddie, E. Rick

(ii) TITLE OF INVENTION : Agents for pre-symptomatic detection, prevention and treatment of breast cancer.

(iii) NUMBER OF SEQUENCES: 32

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DR. J. BERGMANN

(B) STREET: MORIKESTR. 22

(C) CITY: HAMBURG

(D) STATE:

(E) COUNTRY: GERMANY

(F) ZIP: 22587

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT

(B) FILING DATE: 30 November 1995

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: NONE

(B) REGISTRATION NUMBER:

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (4940) 862-576

(B) TELEFAX: (4940) 862-596

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

Hi

( iv ) ANTI - SENSE : NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BC531

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

CCCAGAATGC AAGAAGCCCA GACGGAAACC GTAGCTGCCC TGGTAGGTTT 50 TCTGGGAAGG GACAGAAGAT GACAGGGG 78

(2) INFORMATION FOR SEQ ID NO: 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BC531L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

Met Gin Glu Ala Gin Thr Glu Thr Val Ala Ala Leu Val Gly Phe

5 10 15

Leu Glu Arg Asp Arg Arg

20

INFORMATION FOR SEQ ID NO: 3 :

!i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 140 base pairs

(B) TYPE: nucleic acid

(C) STRANDNESS single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BC532L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3.

TCCATGGCAG TGACCCGGAA GGCAGTCTGG CTGCCAATCC AGGGAAGCGT 50

GTCACCGTCG TGGAAAGCAC GCTCCCAGCC CGAACGCAAA GTGTCCCCGG 100

AGCCCAGCAG CTACCTGCTC CCTGGACGGT GGCTCTAGAC 140

2 ) INFORMATION FOR SEQ ID NO :4 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(n) MOLECULE TYPE* cDNA

(ill) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v ) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE:BC532

(xi) SEQUENCE DESCRIPTION SEQ ID NO : 4

Met Ala Val Thr Arg Lys Ala Val Trp Leu Pro He Gin Gly Ser

5 10 15

Val Ser Pro Ser Trp Lys Ala Arg Ser Gin Pro Glu Arg Lys Val

20 25 35

Ser Pro Glu Pro Ser Ser Tyr Leu Leu Pro Gin Arg Try Leu

40 45

(2) INFORMATION FOR SEQ ID NO: 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 254 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BC533L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 :

TCCATGGCAG TGACCCGGAA GGCAGTCTGG CTGCTGCAAC AGGAAAAGTG 50

GGGATCCAGC ATGAGACGCT TCCAACCCTG GGTCACCTGG GCCTGCAGAG 100

GAGGAGGCCC CTCCGGGAAC ACCATGCCAG TGTCTGAGAC AGTCCGGCTT 150

CCTGTGGTGC AGGAAAAGAA TGGCTGCTTC ACATTCTCTC TTCCAATGTT 200

TCACCACAAC CCAAGCACTC CTGCCCCACC CCACACCAGC CATGCACTTCT 250

TTGA 254

(2) INFORMATION FOR SEQ ID NO: 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 873 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

HH

( iv) ANT I - SENSE : NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE:BC533

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Met Ala Val Thr Arg Lys Ala Val Trp Leu Leu Gin Glu Glu Lys

5 10 15

Trp Gly Ser Ser Met Arg Arg Phe Gin Pro Trp Val Thr Trp Ala

20 25 30

Cys Arg Gly Gly Gly Pro Ser Gly Asn Thr Met Pro Val Ser Glu

35 40 45

Thr Ala Arg Leu Pro Val Val Gin Glu Lys Asn Gly Cys Phe Thr

50 55 60

Phe Ser Leu Pro Met Phe His His Asn Pro Ser Thr Pro Ala Pro

65 70 75

Pro His Thr Ser His Ala Leu Leu

80

(2) INFORMATION FOR SEQ ID NO:7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 199 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BC53LP

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :

HS

GAGCTTTTTT TAAAAGCCAA GGAATACACG TGGATGAAGA AAAAGAAAAG 50 TTCTGCATCC CCAGGAGAGA TGCTGAGGGT GTGATGGGAT GGATAAAAGC 100 CCAAATTCAA GGGGGGAATA TTCAACTTTG GGACAGGAGT CAGAGATCAC 150 ACATTAAGTG GGTAAACTAT AAAAAAACAC TGAGGAAGCC TAGGGGTTG 199

(2) INFORMATION FOR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 234 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCAR2L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : ATGTCTTTAA GGTCAGCGGA GCAGCTGCTT AAGCCGGGGA AAGTGGGGCC 50 CAGCAGGGAC AACGTGGATG GGGCAGCTGA GTCATCCTCG TCCGGAGGTG 100 CTGGCAGCTG CTGCGGCAGC CCCTTGCTGG CGGCCACGGC GGCTCCAGGC 150 TCTGGGACGC AACCTCTCTC GGGGTGGCAC TCCAGGGCCG ACTGCGGCTG 200 TGAAGGTTGC TGTTCCTCAT CCAGGACCAG GTAG 234

INFORMATION FOR SEQ ID NO: 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

H.

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE :BCAR2L

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 :

Met Ser Leu Arg Ser Ala Glu Gin Leu Leu Lys Pro Gly Lys Val 1 5 10 15

Gly Pro Ser Arg Asp Asn Val Alp Gly Ala Ala Glu Ser Ser Ser

20 25 30

Ser Gly Gly Ala Gly Ser Cys Cys Gly Ser Pro Leu Leu Ala Ala

35 40 45

Thr Ala Val Pro Gly Ser Gly Thr Gin Pro Leu Ser Gly Trp His

50 55 60

Ser Arg Ala Asp Cys Gly Cys Glu Gly Cys Cys Ser Ser Ser Arg

65 70 75

Thr Ala

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 326 base pairs

(B) TYPE: nucleotides

(C) STRANDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vn) IMMEDIATE SOURCE:

(B) CLONE: BCAR3L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

HI

GGCCAATGGG GCACAAGGAG TGGGACGCAG CACGGGTGGA ACTCCCAAAA 50

GTGGGGCGTA CATGCAATCC CCCCGAAGCT GTTCCCCTGG ACTCAGATGC 100 TCCAACGCCT GGACTCCCAG GCCCATGGAC ACCGACACTG CCATACACAA 150 CTCCATGGCG TTGTCAGAAA TGGTCGAAGT GCCCCCTAAC TAATTGTCCT 200 TGGAGGAAGT GGGAGCCCCC GAGGCCTCCC TCGCACTCCC GCTGCTGCTG 250 CCTTCGGATA CTGCTTCTTG CTGCTGTTGC TGAAGAAGTT GCATGGTGCT 300 GCTGGCCACG CTCAGGATGT CCTTAA 326

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 105 amino acids

(B) TYPE: amino acids (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCRB3L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Met Gly His Lys Glu Trp Asp Ala Ala Arg Val Glu Leu Pro Lys

1 5 10 15

Val Gly Arg Thr Cys Asn Pro Pro Glu Ala Val Pro Leu Asp Ser

20 25 30

Asp Ala Pro Thr Pro Gly Leu Pro Gly Pro Trp Thr Pro Trp Leu

35 40 45

Pro Tyr Thr Thr Pro Trp Gin Cys Gin Lys Trp Ser Lys Cys Pro

50 55 60

Leu Thr Asn Cys Pro Thr Arg Lys Thr Glu Pro Pro Arg Pro Pro

65 70 75

Ser His Ser Arg Cys Cys Cys Leu Arg lie Leu Leu Pro Ala Ala

H$

80 85 90

Val Ala Glu Glu Val Ala Trp Cys Trp Pro Arg Ser Gly Cys Pro

95 100 105

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 155 base pairs

(B) TYPE: nucleic acid

(C) STRANDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: genomic DNA (in) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCARL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12

AAAGTGGACA GAGTATGGCA CCAAACCCTA AGTTATTTGA TAGGGCCTTG 50

CCAATGACTC TATTTCTGAG ATGATAAAAT CCTGGGCCCT GAAAGGTTAG 100

TGTCTCTCTC TGGAAGGTAA AGGAGAAAGG GAAAGAGAAG TGCATGTGCA 150

AGACC 155

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 278 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (m) HYPOTHETICAL, no

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE.

(B) CLONE: BCRB1L

Hi.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CTCGCATGAC GCAGGGCCAG GGTAAGACAG ACGAGGTGAG CCACCTGGAC 50 GAGAAGGAGA GCTCCGAAGA CAAGAGCAGC TCCCTGGACA GTGACGAGGA 100 CCTGGACACG GCCATCAAGG ACTTAAGGTC CAAGCGAAAG CTCAAGAAGA 150 GGTGCAGGGA ACCCAGGGCT GCGTGCAGGA AGGTCAGGTT CAGCACTGCC 200 CAGATGCACT TCCTGGAGCA GCTGGGCGGG CTGCCGAGAG ACTGGAAAGA 250 CAGAAGCCCG CCGGTGCTTA ATAGCTGA 278

(2) INFORMATION FOR SEQ ID NO: 14:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 90 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: no

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCRBl

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Thr Gin Gly Gin Gly Lys Thr Asp Glu Val Arg His Leu Asp 1 5 10 15

Glu Lys Glu Ser Ser Glu Asp Lys Ser Ser Ser Leu Asp Ser Asp

20 25 30

Glu Asp Leu Asp Thr Ala lie Lys Asp Leu Arg Ser Lys Arg Lys

35 40 45

Leu Lys Lys Arg Cys Arg Glu Pro Arg Ala Ala Cys Arg Lys Val

50 55 60

Arg Phe Ser Thr Ala Gin Met His Phe Leu Glu Gin Leu Gly Gly

65 70 75

Leu Pro Arg Asp Trp Lys Asp Arg Ser Pro Pro Val Leu Asn Ser

80 85 90

5Ό

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid (D) TOPOLOGY: linear

(C) STRANDNESS: single

(ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCRBL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: AAAGCACCAC AGAATAAAAG CGGGGTCCAG A 31

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleotide

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCRBL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TGCCGGCCCC CATGTAAGGC AGCAACAGGT CCC 33

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:

Si

(A) LENGTH: 377 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCRB2L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17

GTGGCATGAT CACGGCTCAC TGCAAACACT GCCACCCACC TTCAAGCAAT 50

TCTCCTGCCT CACCCACCCA AGTAGTCGAG ATTACAGGCA TCTGCTACCA 100

TGCCTGGCTC ATTTTTGTAT TTTTAGTAGA GATGGGGTTT CACCATGTTG 150

GCCAGGCTGG TCTAGAACTC CTGACCTCAA TGATCACCTG CCTGGGCCAC 200

CCAAAGTGCT GGGATTACAG GTTTGAGCCA CCACGCCCAG CCTTATTTTT 250

ATTTATTTTT TTTTGCAACA GGGTCTTCCT GTGTTGCCCA GGCTGGAGTG 300

CAGTGGTGCG ATCTTGGCTT ACTGTAACCT CCACCTCCCA GGTTCAAGAG 350

ATCGTCCCAC CTCAGCCTCC CAAGTAG 377

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 123 ammo acids (D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(m) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

5 ^* 2

(vn) IMMEDIATE SOURCE. (B) CLONE- BCRB2

(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 18-

Met He Thr Ala His Cys Lys His Cys His Pro Pro Ser Ser Asn

1 5 10 15

Ser Pro Ala Ser Pro Thr Gin Val Val Glu H e Thr Gly He Cys

20 25 30

Tyr His Ala Trp Leu He Phe Val Phe Leu Val Glu Met Gly Phe

35 40 45

His His Val Gly Gin Ala Gly Leu Glu Leu Leu Thr Ser Met He

50 55 60

Thr Cys Leu Gly His Pro Lys Cys Trp Asp Try Arg Phe Glu Pro

65 70 75

Pro Arg Pro Ala Leu Phe Leu Phe He Phe Phe Cys Asn Arg Val

80 85 90

Phe Leu Cys Cys Pro Gly Trp Ser Ala Val Val Arg Ser Trp Leu

95 100 105

Thr Val Thr Ser Thr Ser Gin Val Gin Glu He Val Pro Pro Gin

110 115 120

Pro Pro Lys

[2) INFORMATION FOR SEQ ID NO: 19

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 52 base pairs

(B) TYPE nucleic acid

(C) STRANDNESS. single

(D) TOPOLOGY linear

(n) MOLECULE TYPE genomic DNA (m) HYPOTHETICAL NO (iv) ANTI-SENSE. NO

(vi) ORIGINAL SOURCE-

(A) ORGANISM Homo sapiens

(vii) IMMEDIATE SOURCE

(B) CLONE BCRB2L

S3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GATTTTACAC ATAAAAAAAG ATTTTATATT TCATATCTCA ATGATTTTTA AA 52

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 227 base pairs

(B) TYPE: nucleic acid

(C) STRANDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCFLT1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

ATGACTAGAA ATATAGGACC AAACCATGTC TGTCTTATAT CTGTAGCATA 50

TATTCTTGGT TTGTATAAAA GTAACTTTAA AATTCCAGTT TCCTTAAATA 100

GTTATGCACA AAACACACAT ACACCCACAC CCACACACAC ACACACACAC 150

ACACACACAC ACACACACAC ACAGTTACAC CACTGTCGGC CAAAGATGCA 200

CACCTCCTTT AATCAATTTA AATGA 225

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

SH

(A) ORGANISM: Homo sapiens

(vn) IMMEDIATE SOURCE:

(B) CLONE: BCFLT1

(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 21:

Met Thr Arg Asn He Gly Pro Asn His Val Cys Leu He Ser Val 1 5 10 15

Ala Tyr He Leu Gly Leu Tyr Lys Ser Asn Phe Lys He Pro Val

20 25 30

Ser Leu Asn Ser Tyr Ala Gin Asn Thr His Thr Pro Thr Pro Thr

35 40 45

His Thr His Thr His Thr His Thr His Thr His Thr Gin Leu His

50 55 60

His Cys Arg Pro Lys Met His Thr Phe Ser Asn Gin Phe Lys

65 70

(2) INFORMATION FOR SEQ ID NO: 22:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 165 base pairs

(B) TYPE: nucleic acid

(C) STRANDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vn) IMMEDIATE SOURCE:

(B) CLONE: BCFLT2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.22. ATGCACAAAA CACACATACA CCCACACCCA CACACACACA CACACACACA 50 CACACACACA CACACACACA GTTACACCAC TGTCGGCCAA AGATGCACAC 100 CTCCTTTAAT CAATTTAAAT GAGGCTAGCG AGTATCTGTT TGATGTTTGC 150 ATTCTTGTGG GCTAG 165

SS

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 amino acids

(B) TYPE: amno acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCFLT2

( i) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Met His Lys Thr His He His Pro His Pro His Thr His Thr His 1 5 10 15

Thr His Thr His Thr His Thr His Ser Tyr Thr Thr Val Gly Gin

20 25 30

Arg Cys Thr Pro Pro Leu He Asn Leu Asn Glu Ala Ser Glu Tyr

35 40 45

Leu Phe Asp Val Cys He Leu Val Gly

50

2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 83 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

εl

(vii) IMMEDIATE SOURCE: (B) CLONE: BCFLTP

(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 24.

GGGTTCAAAG GAGAATACAA AGAGCAGAGA ATATATAGGC AGACATTCAG 50

GCTGAAATGA ATTTGCTTAA TAAAGGCAAA ACT 83

(2) INFORMATION FOR SEQ ID NO:25

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY linear

(n) MOLECULE TYPE: genomic DNA (m) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCFLT2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: TCAAATATAT CTACACTGGA TAATTTGCAT TTTCATGG 38

(2) INFORMATION FOR SEQ ID NO: 26-

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE- genomic DNA (ill) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE

(A) ORGANISM Homo sapiens

(vn) IMMEDIATE SOURCE:

(B) CLONE: BCFLT2

(xi) SEQUENCE DESCRIPTION SEQ ID NO 26

S?

AAACAAGGAT AAATTTGAGT TTATTTTAGG AAAAAAAA 38

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: BCFLT2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GAACATAAAA GCAGTTGCCA AGTTTATCAA TTAAGTTGGT 40

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 95 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE: CLONE: BRCE151L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

ATGGTATGTT GCCAACACGA GCTCACTCTG GGGCTCTGTC TTACGAAGGA 50

ε$

TCAGATTCAG GGTCAGAGAA GAGGCTGATT CCAGATTCCA GGTAA 95

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: proten

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS

(vii) IMMEDIATE SOURCE:

(B) CLONE: BRCE151L

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

Met Leu Pro Thr Arg Ala His Ser Gly Ala Leu Ser Tyr Glu Gly 1 5 10 15

Ser Asp Ser Gly Ser Ser Glu Lys Arg Leu He Pro Asp Ser Arg

20 25 30

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS

(vii) IMMEDIATE SOURCE:

(B) CLONE: BRCE152

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 0:

S3

Met Val Cys Cys Gin His Glu Leu Thr Leu Gly Leu Cys Leu Gin 1 5 10 15

Lys Asp Gin He Gin Gly His Gin Arg Arg Gly

20 25

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 235 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS

(vii) IMMEDIATE SOURCE:

(B) CLONE: BRCE151

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: TGACAATACC TACATAAAAC TCTTTCCAGA ATGTTGTTAA GTCTTAGTCA 50 TTAGGGAGAT ACATATGGAT ACACTCACAA ATTCTTCTGG GGTCAGGCCA 100 GACACCACCA TGGACATTCT TTTGTTGACC CTTTCAGTTG AAGCTGTCAA 150 TTCTGGCTTC TCCCTGCTCA CACTTTCTTC CATTGCATTA TACCCAGCAG 200 TATCAGTAGT CTGGAGCAGC AGCTGGACTC TGGGC 235

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: amino acids

(B) TYPE: amno acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS

(vii) IMMEDIATE SOURCE:

(B) CLONE: BRCE153

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Met Asp He Leu Leu Leu Thr Leu Ser Val Glu Ala Val Asn Ser 1 5 10 15

Gly Phe Ser Leu Leu Thr Leu Ser Ser He Ala Leu Tyr Pro Ala

20 25 30

Val Ser Val Val

Li

Legend to figures 1-5

over & under lined = CAATT promoter element underlined = TATA promoter element over lined = Cap site underline x 2 = bipartite nuclear transport sequence boxed = transmembrane helix x Fe ^* •x = heme iron binding site

Legend to Figure 12 &2

A. Sequence of epitopes used to raise monospecific polyclonal antibodies against L-oncoproteins.

B. ELISA test for detecting L-oncoproteins in human body fluids .

C. ELISA test for detecting endogenous anti L-oncoprotein antibody in human body fluids.

D. Detection of BC531 with BC531abl in metastatic breast tumours and normal breast, Al = normal breast.

E. Detection of BC532 using BC532abl, same as D.

F. Detection of BC531 with BC531abl in proteins isolated from 15 confirmed and 3 suspected primary breast tumours. Bl and B4 are from normal breast, A2, B2 and C2 were from suspected tumours.

G. Detection of BC532 using BC532abl. Al and Bl = normal breast, A2, B2 and C2 = suspected tumours.

H. Identification of tumour tissue from a mixture of normal and breast cancer tissue, using BC53labl. Al, Bl, Cl, A2 and B2 were correctly identified aε cancerous breast tissue; C2, DI and D2 were normal .

I. Detection of BC532 using BC532abl, same identification as H.

J. Detection of BC531 in nuclear and cytoplasmic fractions of breast tumours using BC531abl. A = BC cytoplasmic fraction, B = BC nuclear fraction, C = normal nuclear fraction and D = normal cytoplasmic fraction.

K. Detection of BC532 using BC532abl, same as J.

L. Detection of BC531 and BREX in serum of a mixed population of 228 women with and without BC. The samples were independently blinded.

M. Detection of BREX mRNA in breast with tumours (4) and normal breast (4) : RNA purified from tissue was converted to cDNA and cDNA was PCR amplified using "pp" BREXL (see figure 9. The presence of the predicted fragment was visualised by electrophoresis on 2% agarose. lanes 1-3 & 5 = tumours; lanes 1,4, 7&8 = normals.

N. Detection/pre-symptomatic detection of anti BREX endogenous IgG in a serum from a population of humans with breast and ovarian cancer and a population of humans without clinical symptoms of breast cancer using the ELISA procedure in "C" .

0. Detection of BREX in tumour tissue from a metastatic BC patient: the protein extract was subjected to cationic non- SDS PAGE; BREX was detected by western blotting followed by reaction with anti-BREXlab and a chemiluminescence mouse anti-rabbit IgG mab.

Detection of BRCAl in the same tumour extract as in "Q" . BRCAl protein was detected by anti-BRCAl mouse maB an a chemiluminescence labelled anti rabbit IgG.

P. Detection of BC531, BC532 and BREX by silver staining following tricine SDSPAGE of serum from a breast cancer patient was affinity chromatographed on a column prepared

from a mixture of sepharose-bound antBC531/BC532/BREX antibody. Detection of BREX-IgG complex/IgG fragments complex by silver staining following affinity chromatography as described in "P".