WO2000015788A2 - Dna sequence encoding oncofetal ferritin protein - Google Patents
Dna sequence encoding oncofetal ferritin protein Download PDFInfo
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- WO2000015788A2 WO2000015788A2 PCT/IL1999/000485 IL9900485W WO0015788A2 WO 2000015788 A2 WO2000015788 A2 WO 2000015788A2 IL 9900485 W IL9900485 W IL 9900485W WO 0015788 A2 WO0015788 A2 WO 0015788A2
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- dna sequence
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P37/00—Drugs for immunological or allergic disorders
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- A61K38/00—Medicinal preparations containing peptides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- This invention relates to novel DNA sequences, amino acid sequences coded by them, detection method using said DNA sequences and pharmaceutical composition.
- Iron is known to be an essential element of the makeup of every living organism, but may also become toxic at physiological pH values by virtue of its tending to oxidize, hydrolyze and precipitate as insoluble ferric oxide polymers.
- the protein ferritin found in all living cells, is the body's means for ensuring that iron toxicity does not occur. Ferritin functions by storing iron in the cells in a soluble and readily available form. The iron stored in cells may then be mobilized whenever needed by the body, for example for erythropoiesis.
- the isoferritins extracted from different tissues or organs typically exhibit different isoelectric points, with the isoelectric focusing pattern of human tissues forming a continuous spectrum; those tissues associated with high iron storage have ferritins at the basic end of the spectrum (e.g. spleen and liver), while iron poor tissues, (e.g.
- a specific type of acidic isoferritin has been shown to be characteristic of neoplastic cells and placental cells (Drysdale and Singer, Cancer Res., 44:3352, 1974). This protein is also known as oncofetal ferritin or placcental isoferritin (PLF). Human placental ferritin has been shown to be composed predominantly of a single subunit type comigrating with a liver ferritin standard on SDS-PAGE (Brown et al, Biochem. J., 182:763, 1979). However, an immunoradiometric assay performed with anti-human spleen ferritin has shown tissue specific antigenicity for PLF (Brown et al, supra).
- the sequence of the gene coding for oncofetal ferritin could not be found in regular cDNA libraries, probably due to the fact that its expression in these libraries is extremely low.
- the protein is secreted only by the placenta during pregnancy or by cancer cells in malignant diseases such as lymphoproiiferative disorders, breast cancer and in HIV infection.
- Breast cancer is a malignant disease effecting different populations at a rate of one to every 9-13 of women. Early diagnosis of breast cancer is known to considerably improve the prognosis of the patient. Diagnosis of breast cancer is based today mainly on imagining techniques, such as mammographs verified at times by biopsies. Blood-based assays of breast cancer have been reported in the literature, for example, biomarkers such as CA 15.3. (Daly, L. et al, Comparison of a novel assay for breast cancer mucinto and CA 54 15.3 care ino embryonic antigen, J. Clin. Oncol, 10: 1057-65); the CA 549(2) marker (Dermers, I.M..
- U.S. 4,882.270 discloses an assay for the detection of breast cancer based on determination of oncofetal ferritin.
- the assay is based on binding of the oncofetal ferritin to specific monoclonal antibodies.
- Pathological pregnancy is a term commonly used to describe a multitude of symptoms which create difficulties in carrying a child to term and include spontaneous abortion and miscarriage, premature contractions, toxemia, premature delivery.
- U.S. 4,954.434 discloses the fact that low levels or absence of PLF in pregnant women can serve as a marker for potentially high risk pregnancy. Detection of this state is again achieved by monoclonal antibodies which has PLF specificity.
- This patent also concerns treatment and prevention of actual and potentially pathological pregnancy by the administration of this protein. However, since the sequence of the protein was not known at the date of the patents, the treatment suggested involved administration of partially purified protein and not of recombinant pure proteins.
- Patents 5,571,678, 5,120,640 and 5,283,177 are all directed to methods for assaying the presence and evaluating the prognosis of acquired immunodeficiency associated with HIV induction, by determining levels of placental isoferritin by monoclonal antibodies. All the above detection methods concern antibody-based assays. While such assays are known to be useful in conditions where the level of the protein to be detected is quite high, they are notorious for eliciting a false-negative answer where the protein level is low. against this, assays based on amplification of mRNA (RT-PCR) are much more sensitive and can detect even minute expression of mRNA. Thus there is need today for a RT-PCR method for detection of oncofetal ferritin for detection of breast cancer and for diagnosis of high risk pregnancies at its early stage.
- the present invention is based on the finding of the sequence of the oncofetal ferritin gene and of the oncofetal ferritin protein.
- the inventor was the first to discover the full sequence of the gene that codes for the protein termed hereinafter as "oncofetal ferritin 1 (OFFl) protein ".
- the present invention concerns a DNA sequence coding for the subunit of the oncofetal ferritin protein termed "oncofetal ferritin 1 " (OFFl) protein selected from the group consisting of:
- Fig. 1 was isolated from a cDNA library obtained from breast cancer patients while the DNA sequence of Fig. 4 was obtained when using PCR amplification where the sequence of Fig. 1 was used as a template.
- the two sequences differ only in the 5' non-coding region which include 2 single nucleotide substitutions as well as a single base insertion and one deletion.
- the DNA sequences of the invention also include DNA sequences which code for the same amino acid sequences as those of Figs. 1 or 4. It is known that due to the degenerative nature of the genetic code, a large number of alternative DNA sequences, may code for the same proteins. Thus all sequences which code for the same amino acid sequences are encompassed by the scope of the invention.
- the present invention further concerns DNA sequences having at least 80% homology either to the DNA sequence of Fig. 1 or 4, or to the DNA sequences coding the same types of amino acid sequences, the homology determined by hybridization under stringent conditions. Examples of highly stringent conditions are given in (vi) above and of moderately stringent conditions are given in (vii) above.
- highly stringent conditions are given in (vi) above and of moderately stringent conditions are given in (vii) above.
- the artesian will appreciate the fact that there exists a large number of sequences capable of hybridization under such conditions, some of which code for more physiologically active OFFl proteins than others. Those sequences which fall under the scope of the invention, are those which code for a functionally equivalent gene product, as will be explained hereinbelow.
- the present invention also encompasses DNA sequences that hybridize to the sequences of Figs. 1 or 4, or to the cDNA inserts contained in the deposited cells, under highly stringent conditions, as specified above. Such DNAs can be used as probes to detect the OFF-1 gene or mRNAs (e.g. by hybridization or PCR amplification assays). Alternatively DNAs that hybridize under highly stringent or less stringent conditions (specified above), yet which encode a functionally equivalent gene product are also encompassed by the invention.
- the present invention further concerns fragments of all the above sequences, which code for an OFFl protein having functionally equivalent gene product will be explained hereinbelow.
- the term "functionally equivalent gene product” refers to an amino acid sequence, which is physiologically active in a manner similar to that of the native OFFl protein. Such an activity can be tested, for example, by determining the immunosuppressive activity in cell mediated immunity, as is well known in the an.
- the present invention further concerns expression vectors comprising said DNA sequences, as well as a host cells transfected with such expression vectors. Expression may be obtained in any suitable pro-or eucaryotic expression systems using known methods e.g. as described in Genentech EP 200341.
- Suitable expression vectors are DNA sequences encoding OFFl and operably linked to suitable control sequences capable of effecting the expression of OFFl in the host.
- control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation.
- the vector also should include DNA encoding a selection gene.
- Vectors include plasmids, viruses (including phages) and integratable DNA fragments, i.e. fragments that are integratable into the host genom by recombination.
- Preferred host cells are cells derived from multicellular organisms. In principle, any higher eucaryotic cell culture is workable whether from vertebrate or invertebrate culture. Examples useful in host cell lines are Chinese Hamster Ovary (CHO) cell lines and COS 7 cell lines.
- the cells and tissues may be engineered to express an endogenous gene under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.
- gene targeting can be used to replace a gene ' s existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.
- Such regulatory sequences may be comprised of promoters, enhancers, scaffoid-a ⁇ achment regions, negative regulator/ elements, iranscrintionai initiation sites, regulatory protein binding sites or combinations of said sequences.
- sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.
- the targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, __e_, inserting a new promoter or enhancer or both upstream of a gene.
- the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
- the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.
- the naturally occurring sequences are deleted and new sequences are added.
- the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA. allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.
- the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
- Markers useful for this purpose include the Herpes simplex virus thymidine kinase (TKJ gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
- TKJ gene Herpes simplex virus thymidine kinase
- gpt bacterial xanthine-guanine phosphoribosyl-transferase
- the present invention of course concerns also a recombinant protein coded by the above DNA sequence.
- Said recombinant protein is in fact the first recombinant OFF-1 protein produced, and enables production of large amounts of such protein in a pure form for therapeutic and detection purposes as will be explained hereinbelow.
- the present invention further concerns anti-sense RNA sequences, which are complementary to the mRNA sequences transcribed from the above DNA sequences, and thus are capable of neutralizing the expression of native OFF-1 gene in cells.
- the present invention further concerns DNA sequences coding for said anti-sense mRNA, as well as expression vectors comprising said DNA sequences.
- the present invention also concerns pharmaceutical compositions of two types.
- the pharmaceutical compositions of the invention comprise DNA sequences coding for the OFF 1 protein, expression vectors comprising said DNA sequences, for expression in specific target cells, or the recombinant OFFl protein itself.
- the above agents may be used in the immunization of a subject against diseases which are manifested by abnormally high expression of the OFFl. Examples of such diseases are: cancer, in general, and breast cancer and lymphomas in particular as well as HIV infections.
- the pharmaceutical composition according to the activating aspect of the invention may be used, as such, for increasing the level of the OFF 1 protein for the treatment of conditions manifested by lower than normal levels of the OFF 1 protein.
- the pathological conditions may be alleviated.
- Pathological conditions treated by these pharmaceutical compositions are pathological pregnancies manifested by spontaneous abortion and miscarriage, premature contractions, toxemia and premature delivery.
- the pharmaceutical compositions may be used to inhibit transplant rejection, for example, specific T-cell mediated immunity like that of a mother against her embryo.
- compositions of the invention may also be used for the treatment, alleviation, or prevention of autoimmune diseases, such as: Coeliac disease, Rheumatoid arthritis, and Multiple Sclerosis which are T-cell mediated autoimmune diseases.
- autoimmune diseases such as: Coeliac disease, Rheumatoid arthritis, and Multiple Sclerosis which are T-cell mediated autoimmune diseases.
- the pharmaceutical composition of the activation aspect of the invention may be used to support normal pregnancies, for example, for increasing the chances of success of in vitro fertilization (IVF), in both human and non human subjects.
- IVF in vitro fertilization
- the pharmaceutical composition of the invention may be used to enhance growth of bone marrow progenitor cells, for example, where due to some pathological condition their number has decreased. Such a condition occurs, for example, in patients treated by mega doses of cytotoxic drugs which kill bone marrow cells such as cancer patients. HIV infected patients and the like.
- the ⁇ harmac ⁇ uticai comnositions of the inventions could be used in patients who need bone marrow replacements such as those with genetic metabolic defects or autoimmune diseases.
- DNA coding for OFFl can be used in the development of a chimera which will enable further grafting of organ allografts or xenografts identical with the bone marrow donor.
- a preparation comprising OFF 1 can be used ex vivo as a growth factor for bone marrow progenitor cells, for example, from bone marrow obtained from a donor prior to implantation.
- bone marrow progenitor cells are granulocyte monocyte progenitor cells.
- the pharmaceutical compositions of the invention comprise anti-sense mRNA to the OFF-1, expression vectors comprising DNA sequences coding for said anti-sense mRNA, or antibodies against the OFF-1 protein.
- the neutralizing aspect of the invention is intended to lower the levels of OFF-1, where it is abnormally high as compared to normal tissue, notably for the treatment of cancer, especially breast cancer.
- the neutralizing aspect of the invention may be used in order to reduce the normal level of OFF 1 which is required to maintain a pregnancy to term and thus cause abortion (Moroz, C, 9 International Congress of Immunology, San Francisco, 1995).
- the present invention further concerns a method for the detection of cancer, or for the evaluation of the prognosis of a cancer patient, by determining the level of the OFFl gene expression in said patients.
- U.S. Patent 4,882,270 discloses a method for detecting breast cancer, by using antibodies against isoferritin placental protein.
- This method detects said protein in early stages of the cancer only on lymphocytes not in the serum.
- the protein can be detected in the serum only at a verv late stage of the disease when the rumor has a ⁇ rea ⁇ v meiastasized.
- the problem with lymphocyte-based detection is double fold: first, technical issues concerning detection are quite severe, since there is a requirement to isolate fresh lymphocytes from the blood and assay within hours without an opportunity to retest at a later date. This severe technical problem prohibits the use of this method of detection in widely used screening assays. Second, during progressive stages of cancer, the number of positive lymphocytes decreases dramatically, and thus it is not possible to detect the cancer, using lymphocytes directed anti-ferritin antibodies. Thus, according to said U.S.
- the detection methods of the present invention are based on RNA amplification, and are sensitive enough to detect even slightly elevated levels of the OFFl mRNA present in small amounts of the patient's blood in virtually all stages of cancer .
- the method of the invention detects the mRNA in circulating cancer cells, whereas the protein is shed from tumors and binds to lymphocytes.
- the level of OFFl protein is also a good indicator of the prognosis of cancer, for example the change in mRNA level after removal of the tumor by surgery or chemotherapy may indicate disease prognosis.
- cancers which can be detected by the methods of the invention are breast cancer, hepotomas, leukemias, lymphomas and embryonal tumors such as neuroblastoma and hepatoblastoma.
- Down Syndrome In addition, elevated levels of OFFl expression, are typical of Down Syndrome. In Down Syndrome there are elevations of embryonal proteins like ⁇ -fetoprotein (AFP). Also the Syndrome is associated with decreased immunoreactivity and high incidence of cancer. Thus by detecting high levels of this protein it is possible to determine also Down ' s disease.
- AFP ⁇ -fetoprotein
- the present invention concerns a method for the detection of diseases connected with pathological pregnancies, comprising detecting a lower level than normal of the OFF-1 expression.
- pathological pregnancies groups together a large number of disorders including spontaneous abortion and miscarriage, premature contractions, toxemia, premature delivery.
- the detection of the level of the OFF-1 expression can be carried out by utilizing reverse transcriptase polymerized chain reaction (RT-PCR).
- RT-PCR reverse transcriptase polymerized chain reaction
- This method considerably amplifies the OFF-1 mRNA present in the blood enabling its detection, even in minute levels.
- the present invention further concerns a method for the isolation of the cDNA of the invention as specified in Fig. 1 or 4 as will be appreciated in the "Detailed Description " section of the specification.
- the present invention further concerns primers for use in the above isolation method. These primers may also be used in RT-PCR. for the detection purposes of the invention.
- the primers are selected from the group consisting of:
- Fig. 1 shows the nucleic acid sequence of clone T16 isolated from T47D breast cancer cDNA library. Initiation and termination codons of the open reading frame are indicated by dark bars;
- Fig. 2A shows a comparison of the nucleic acid sequences (upper sequence) of clone 4.7 isolated from a placenta cDNA library exhibiting normal human FTH, and the sequences (lower sequence) of clone T16 isolated from human breast cancer T47D cDNA library. Initiation and termination codons of the open reading frame are marked by dark boxes;
- Fig. 2B shows in a schematic representation the comparison of the two sequences shown in Fig. 2A. Differences in nucleic acid sequences are represented by the shaded areas;
- Fig. 3 shows a comparison of sequence homology between cDNA clone T16 and human mitochondrial cytochrone oxidase I DNA
- Fig. 4 shows a comparison of nucleic acid sequences between placental cDNA obtained by PCR amplification using T16 specific primers
- T16 cDNA sequence obtained from the T16 cDNA clone (upper sequence) and T16 cDNA sequence obtained from the T16 cDNA clone (lower sequence). Identical nucleic acid sequences are indicated by a dotted line. Initiation and termination codons are indicated by a dark bar;
- Fig. 5 shows the nucleic acid sequence and deduced amino acid sequence of the cDNA of OFF 1 ;
- Fig. 6A shows the relative expression of FTH in mRNA and OFF 1 RNA among total mRNA isolated from different tissues and optimized with ⁇ -actin expression
- Fig. 6B shows the relative expression of FTH mRNA hybridized with 32 P Pstl 3' fragment of liver FTH cDNA (530 bp), from either normal HBL human lactating breast or from cancer cell lines (MCF-7 and T47D);
- Fig. 7 shows the sequence of clone T16. primers used for PCR are indicated in the above sequence;
- Fig. 8 shows the restriction enzyme map sequence of clone T16
- Fig. 9A shows SDS-PAGE (12%) of total cell lysates (lane 2) 10 ⁇ l from E.coli containing the vector pGEX alone or the pGEX constructed to contain C48 fragment (lane 4) or total constructs containing full length OFFl (lane 6).
- the recombinant proteins are marked by arrows;
- Fig. 9B shows the same as Fig. 9A but reactive with CMH-9 in antibodies (lanes 4 and 6 indicating presence of protein);
- Fig. 10 shows the effect of the protein of the invention on CFU-GM colony formation obtained from three healthy donors
- Fig. 11 shows the effect of the protein of the invention and its combination with GM-CSF or CMH9 Moab on CFU-GM colony formation
- Fig. 12 shows a scheme summarizing the effect of OFFl on immunoregulation.
- plaque hybridization was carried out at 42°C, 5xSSC and subsequent washing 3 times with 2xSSC 0.1% SDS at room temperature and twice with lxSSC, 0J% SDS at 68°C.
- Clones were isolated that gave hybridization signals after two rounds of screening. PCR amplification was performed on clones using one primer from the ⁇ gtl l vector ( ⁇ gtl l F-1060 or ⁇ gtl l R-1061. Table 1) and one FTH gene-specific primer (either 17R to amplify toward the 5' end of 17F to amplify toward the 3' end: as indicated in Table 1) as described above, PCR amplification was performed for 30 cycles under standard conditions. PCR products from the clones derived from both the 5' and the 3' end of the cDNA clone were selected according to their size, so that their sequence would produce a contig of maximum length.
- PCR products were purified by Qiagen PCR purification columns according to the manufacturer's instructions and were sequenced using standard protocols for the ABI373 or 377 primer mated sequencer with the 1060 and 1061 primers (Table 1) and specific primers until the full sequence was determined.
- the cDNA sequences and its deduced protein sequence were used to search the complete combined Gene Bank/EMBL database and the complete Swiss Prot database with BLAST and FASTA programs, respectively.
- the preparation was stored for 5 mins. at room temperature, and then 0.2 ml chloroform per 1 ml of Tri Reagent were added and vortexed for 5 sees.
- the resulting preparation was stored for 2-15 mins. at room temperature and centrifuged 12,000 g 15 mins. at 4°C.
- RNA pellet was washed with 75% ethanol by vortexing and centrifugation.
- RNA or 14 ⁇ l whole blood RNA of Tube A was placed in total volume of up to 15 ⁇ l in DEPC water, heated at 70°C for 10 mins. and cooled immediately on ice. Then it was spun briefly and 10 ⁇ l of mix was added a follows: 5 ⁇ l M-MLV RT 5xReaction buffer;
- PCR For PCR the following reagents were used: 1 ⁇ l cDNA (or 0.5 ⁇ l cDNA for PCR of normal ferritin (FTH) H (Chain); 5 ⁇ l lOx reaction buffer for DNA polimerase; 1 ⁇ l forward primer ( ⁇ 10 pmole); 1 ⁇ l reverse primer ( ⁇ 10 pmole); 1 ⁇ l dNTPs (12.5 mM), 0.5 u Taq polymerase from Appligen (0.1 ⁇ l); Takara 0.2 gel DDW water up to 50 ⁇ l.
- FTH ferritin
- 5 ⁇ l lOx reaction buffer for DNA polimerase 1 ⁇ l forward primer ( ⁇ 10 pmole); 1 ⁇ l reverse primer ( ⁇ 10 pmole); 1 ⁇ l dNTPs (12.5 mM), 0.5 u Taq polymerase from Appligen (0.1 ⁇ l); Takara 0.2 gel DDW water up to 50 ⁇ l.
- T16 transcripts do not yield bands after the first PCR, in order to amplify the results the initial PCR is followed by nesting PCR, using 1 ⁇ l of PCR 1 diluted 1 : 100 in the above PCR program.
- PCR1 XTF ⁇ 16R nesting PCR 2: 17F ⁇ SPR
- T T47D breast cancer cells cDNA library
- P placenta cDNA library
- the sequence of the full length cDNA (0.9 KB) from clone T16 revealed a sequence of 890 bp; 109 bp in the 5' UTR 495 bp (165 aa) in the coding region and 286 bp in the 3' UTR. Full sequences are shown in Fig. 1.
- the nucleic acid sequence of cDNA from clone p4, 7 revealed a sequence of 890 bp which is completely homologous to the known FTH sequence (as compared in Fig. 2) and represents a normal ferritin heavy chain (Cohen et al, 1996, Drysdale 1988). Partial homology was found between clone p4. 7 and clone T16 (Fig. 2A, 2B).
- the homologous sequences were clone 4. 7 139-511 and clone T16 87-460. The later included 22 bp in the 5' UTR followed by 351 bp (117 aa) in the above coding region. No further homology was found between the above two clones. As can be seen in Fig. 2A and 2B, homology is indicated by a broken line.
- Example 7 PCR amplification of a T16 compatible placental cDNA
- T16 specific primers i.e. ⁇ gtl l F (1060) or ⁇ gtl l R (1061) and primer 16R for the 5' end and primer SPF for the 3' end (Table 1; Fig. 7) schematically as shown below:
- RNA by reverse transcriptase and PCR amplification was isolated from human T47D breast cancer and HBL 100 breast epithelial cell lines, and from human peripheral blood lyphocytes ((PBL), non activated and from concanavalin activated PBL.
- PBL peripheral blood lyphocytes
- RNA (5 ⁇ g) from the different cells was used to prepare cDNA by reverse transcriptase using random primers.
- T16 cDNA as amplified by PCR using the following primers:
- T16 gene in a variety of human tissues, including breast epithelial and breast cancer cell lines were analyzed by northern blotting, according to state of the art procedure and the results are shown in Fig. 6A and 6B.
- the northern blot revealed a 0.9 kb transcript in all the cells tested.
- each tissue mRNA indicates that all the peaks on the blot for a single probe FTH (normal H chain) or T16 (OFFl) amount to 100%) and therefore each mRNA bar represents a ratio relative to the other tissues.
- FTH normal H chain
- T16 OFFl
- the expression vector (pGEX-5X-l) used for gene fusion construction was the GST Gene Fusion System (Pharmacia).
- the OFFl coding region (designated as "FL ", full-length) of about 0.5 kb was prepared by PCR with the following 5' end primer: 5' GTGGGATCCCCATGACGACCGCGTCCA, in order to add a Bam HI
- a deletion construct for encoding the unique C-terminal 48 aa, designated as "C48 ", of the OFFl was also prepared from “FL " OFFl PCR product, by cleavage with restriction enzymes 5' ECORI and 3' Xhol .
- the PCR program was as follows (cycles):
- the PCR products were gel-purified and then ligated into the pGEX 5X-1 plasmid at 5' BamHI and 3' Xhol sites.
- the resultant recombinant plasmids canproduce fusi ⁇ on proteins in which the N-terminus is the GST and the C-terminus is the OFFl, or c48 of OFFl.
- E.coli strain BL-21 was transformed with the vector alone or the two recombinant plasmid DNA to produce pGST cells, pGST-FL cells, pGST-C48 cells following the standard protocol (Maniatis Sambrook J. Fritch EF & Maniatis T., Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Harbor Lab. Press Plainview, N.Y. Snd Ed.)).
- LB LB broth containing 100 ⁇ g/ml of ampicillin at 37°C overnight.
- the overnight cultures were diluted 1000-fold using fresh LB broth plus ampicillin, and incubation continued at 37°C.
- samples were taken every 30 mins. to measure the optical density at 600 nm.
- Isopropyl ⁇ -D-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM, and incubation was continued at 37°C for 4 hours. After IPTG induction, the cultures were harvested and cell pellets were obtained by centrifugation. Pellets were resuspended in 100 ⁇ l of Laemmli sample buffer. Thirty micrograms of protein samples, determined by the Bradford assay, was subjected to SDS/PAGE.
- One-dimensioned SDS/PAGE was performed according to Laemmli using 12.5% (wt/vol) polyacrylamide gels.
- proteins were transferred from polyacrylamide gels to immobilon polyvinylidene difiuoride membranes (Millipore) with Tris/glycine electroblotting buffer according to Towbin et al. .
- Protein bands cross-reacting with CM-H-9 monoclonal antibody (MoAb) were identified by reaction with horseradish peroxidase conjugated with goat anti-mouse IgG (Bio-Rad). The conditions of immunoreactions were according to the manufacturer's specification (DAKO).
- Triton-XlOO • Sonicate 90/7 for 10 sec, three times on ice (in 50 ml tube)
- T16 transcript in breast carcinoma is consistent with the differential cDNA cloning strategy which suggest its utility as a biomarker in breast cancer detection. 25 patients suspected of having cancer were tested for their T16 transcript by using RT-PCR in their peripheral blood as described in the experiment.
- CM-H-9 was produced against human placental ferritin as previously described in the above two U.S. patents.
- the MoAb was obtained from ascites fluid following precipitation with 50% saturated ammonium sulphate solution, and purification on sephadex G-200 colomn.
- Bone marrow samples from 11 healthy volunteer donors were processed by density gradient separation using Histopaque-107 (Sigma diagnostics, St. Louis, MO, USA) to obtain a purified population of mononuclear cells. Colony assays were performed in a plating medium containing final concentrations of 0.92% methyl cellulose (M-281 powder, 4,000 centipoise, Fisher Scientific Co., Fair Lawn, NJ, USA), rehydrated in Iscove's modified Dulbecco's medium containing 36 mM sodium bicarbonate (Gibco, Grand Island, NY, USA), 30% fetal bovine serum (FBS) (HyClone, Logan, UT, USA)0.292 mg/ml glutamine, 100 U/ml penicillin and 0.01 mg/ml streptomycin (Biological Industries, Beit Haemek, Israel).
- FBS fetal bovine serum
- p43 (PLF) was added at concentration of 1 ⁇ g/mL and in neutralization experiments, p43 (PLF) was preincubated with lOx excess of CM-H-9 MoAb at 37°C for 30 min and the complex added to the assay as above.
- the colony assay medium contained 10 mononuclear cells/ml and each 1 ml was plated into triplicate wells (333 ⁇ l/well) of a 24 well tissue culture plate (Greiner, Germany). Water was added to spaces between cells to maximize humidity during incubation of the cultures. The cultures were incubated at 37°C in 5% C0 2 and 55% relative humidity. Plates were scored after 14 days for colonies containing more than 50 cells.
- p43 (PLF) was tested for its capacity to influence colony formation of human bone marrow progenitor CFU-GM.
- p43 alone exhibited a concentration dependent stimulatory effect on bone marrow progenitor cells obtained from three donors. The highest number of colonies was obtained with l ⁇ g/lmL of p43 (Fig. 10). At higher concentrations the number of cells was lower. All subsequent experiments were further carried out at concentration of 1 ⁇ g/mL of p43 (PLF).
- p43 known to act as an immunosuppressive cytokine is active as a growth factor on human bone marrow progenitor cells.
- Thl autoreactive T helper 1
- induced regulatory responses such as Th2 cells
- a polypeptide encoded by the DNA molecule of the invention may be used for this purpose.
- In-vitro activation of human lymphocytes without and with C48/OFF1 treatment was carried out in a mixed lymphocyte culture (MLC).
- Human peripheral blood lymphocytes (2x16 /ml) (responder cells) were incubated with non-related Mitomycin C (40 ⁇ gr/ml) treated lymphocytes (2x10 /ml) in RPMI-1640 medium containing 10% Fetal Calf serum and antibiotics.
- the cultures were incubated for 24h in a humidified incubator at 37°C with 5%> C0 2 .
- Supematants were collected and the concentration of the secreted cytokines were measured using an ELISA assay kit.
- the level of IL-2 R, and interferon (Thl cytokines) and IL-6 and LL-10 (TH2 cytokines) were determined.
- sIL-2R Soluble interleukin 2 receptor
- C48/OFF 1 induces preferentially a TH2 type immunoresponse associated with diminished cellular immunity.
- mice Female ICR mice were obtained from our institution's breeding facilities and were fed a standard diet and tap water ad libidum.
- zymosan-induced arthritis A homogeneous suspension of 25 mg zymosan A (Saccharomyces cerevisiae), dissolves in 1 ml endotoxin-free saline, was obtained by boiling twice followed by sonic emulsification. Arthritis was induced by intrarticular injection of 0.5 mgr/20 1 zymosan into each right and left knees.
- Knee joints were dissected, fixed, decalcified, dehydrated, and embedded in paraffin. Standard frontal section of 7 ⁇ m were prepared and stained with eosin-hematoxilin. Disease severity was assessed by calculation of arthritic index as follows:
- grade 0 normal synovia
- grade 1 1-5 cellular infiltrates in the microscopic field
- grade 2 6-20 cellular infiltrates
- grade 3 21-50 infiltrates
- Lymphocytic nodules grade 1: 1-3 nodules per sectio; grade 2:
- Histiocytes grading grade 1: 1-10 cell; grade 2: 11-20 cells; grade
- Synovial thickness grade 0: presence of 1 layer of synovial cells; grade 2: 2-3 layers; grade 3: 4-6 layers; grade 4: more than 6 layers.
- Treatment Patients may be treated with c48 or OFFl at a dose of e.g. 0.5-5mgr per kilogram weight injected subcutaneously twice weekly for three months. Other treatment protocols may be determined by those skilled in the art. Treatment may be repeated with disease recurrence.
- T cells in the donor marrow are the cause of graft- versus-host disease (GVHD).
- GVHD graft- versus-host disease
- the present treatment protocol was undertaken to develop ex-vivo treatment with OFFl to induce a state of alloantigen-specific tolerization resulting in the lack of GVHD generation in vivo and development of bone marrow chimerism.
- mice C57BL/6 (H2 ) and BALB/c (H2 ) mice were purchased from
- mice Harlane. Donors and recipients were 8-10 wk of age at the time of BMT. All mice were housed in a specific pathogen- free facility in microisolator cages.
- C57B1 donor bone marrow (responder) was harvested 2 days before transplantation.
- Balb/c recipient (stimulator) spleen cells were irradiated at a midplane dose of
- the cells were resuspended in RPMI-1640 medium with 5% mouse serum to 4x10 /ml and incubated with 1 ⁇ g/ml of OFFl for 30 min.
- the erythrocyte depleted mononuclear cell fraction of the marrow was resuspended at a concentration of 4x10 /ml in the above medium in a Donor: Recipient ratio of 1 :1.
- the cells were cultured in tissue culture flasks for 36 hours at 37°C in 5% C02, washed and resuspended to 2x10 cells/ lml in PBS containing OFFl (1 ⁇ g/ml).
- mice were treated for 14 days by i.p. injection of 10 ⁇ g OFFl. Control mice were injected with PBS (BMT+PBS)
- Bone marrow (B.M.) and splenocytes (spl.) were removed from transplanted Balb/c mice 6 weeks post transplantation and tested with anti-K monoclonal antibody for the presence of C57B1 Donor lymphocytes.
- Recipients Patients with haploidentical bone marrow to the donor (i.e., marrow from a donor who shared only one or two major histocompatibility-complex haplotypes with the recipient).
- the patients undergo leukopheresis to collect 200 million to 600 million mononuclear cells per kilogram of body weight for use as the recipient's alloantigen-presenting cells. These cells are cryopreserved with the use of a standard method. Subsequently, the patient receive 1400 cGy of total-body irradiation followed by cyclophosphamide, and methylprednisolone.
- Donor marrow is harvested two days before transplantation.
- Prophylaxis against GVHD consists of a short-course of cyclosporine starting on the day before transplantation, either by continuous infusion at 0J mg per kilogram per hour or by a bolus of 1.5 mg per kilogram over a period of 2 to 3 hours every 12 hours.
- Cryopreserved mononuclear cells derived from recipient's blood are thawed, washed, and irradiated at a midplane dose of 3300 cGy.
- the cells are resuspended at a concentration of 5x10 /ml in RPMI 1640 medium with 5% human AB serum.
- c48 or OFFl is added at a concentration of 0.5-5 ⁇ g per milliliter for 30 minutes before the addition of the donor marrow cells.
- the erythrocyte-depleted mononuclear-cell fraction of the marrow is resuspended at a concentration of 5x10 cells per milliliter in RPMI 1640 medium with 5 percent human AB serum and added to the mixture of recipients cells and c48 or OFFl, in a dono ⁇ recipient ratio of 1 : 1.
- the cocullture is incubated in tissue-culture flasks for 36 hours at 37 ° C in 5 percent CO2. washed, and then infused into recipient.
- Engraftment of Anergic Haploidentical Bone Marrow A median of 3 million CD34+ donor cells per kilogram of the recipient's weight are treated in the co-culture system, and a median of 2.2 million CD34+ cells per kilogram are infused into the recipient.
- the transfused donor bone marrow may contain mature T cells, with medians of 28 million CD3+ cells per kilogram, including 14 million CD3+CD4+ cells per kilogram and 9 million CD3+CD8+ cells per kilogram, after the ex vivo treatment.
- control and with BMT+OFFl were used as responder cells (R) and were reacted with C57B1 (K ) splenocytes as stimulator cells (S) in MLR.
- control mice BMT+PBS
- splenocytes obtained from the BMT+OFFl chimeric mice were completely anergic and did not react at any S/R ratios.
- splenocytes from chimeric BMT+OFFl Balb/c mice responded in MLR against splenocytes from a non related alloantigen from ICR splenocytes, similarly to the response of control BMT+PBS (Table 7).
- the latter results indicate a specific anergy of BMT+OFFl to the donor alloantigens but not to other non-related alloantigens.
- Donor marrow treated ex vivo with OFFl to induce anergy can reconstitute bone marrow with donor chimerism.
- Chimeric BMT are anergic to the donor alloantigen but not to other non-related alloantigens, indicating that the chimeric host is not completely immunosuppressed and tolerance is specific. Hyporesponsiveness against additional alloantigens can be further achieved by renewed ex vivo treatment with OFF 1 against an additional alloantigen in MLR.
Abstract
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AU56457/99A AU5645799A (en) | 1998-09-11 | 1999-09-08 | Dna sequence encoding oncofetal ferritin protein |
US09/786,867 US7217686B1 (en) | 1999-09-08 | 1999-09-08 | DNA sequence encoding oncofetal ferritin protein |
EP99943188A EP1112358A2 (en) | 1998-09-11 | 1999-09-08 | Dna sequence encoding oncofetal ferritin protein |
JP2000570315A JP2003512811A (en) | 1998-09-11 | 1999-09-08 | DNA sequence encoding oncofetal ferritin protein |
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IL12618198A IL126181A0 (en) | 1998-09-11 | 1998-09-11 | Dna sequences proteins coded by them medicaments and detection methods using same |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1989009936A1 (en) * | 1988-04-08 | 1989-10-19 | Chaya Moroz | Isoferritin as a marker for pathological pregnancy |
US4882270A (en) * | 1981-05-15 | 1989-11-21 | Chaya Moroz | Monoclonal antibodies to placental isoferritin for use in detecting oncofetal ferritin associated with breast cancer and Hodgkins disease |
EP0426458A2 (en) * | 1989-10-31 | 1991-05-08 | Japan Antibiotics Research Association | Human monocyte growth factor |
US5571678A (en) * | 1981-05-15 | 1996-11-05 | Moroz; Chaya | Placental isoferritins for the prognosis and diagnosis of immunosuppression |
Family Cites Families (1)
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JPH0731482A (en) * | 1993-07-21 | 1995-02-03 | Life Technol Kenkyusho | Recombined growth factor for human monocyte, and dna sequence coding the same |
-
1998
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-
1999
- 1999-09-08 EP EP99943188A patent/EP1112358A2/en not_active Withdrawn
- 1999-09-08 AU AU56457/99A patent/AU5645799A/en not_active Abandoned
- 1999-09-08 WO PCT/IL1999/000485 patent/WO2000015788A2/en active Application Filing
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US4882270A (en) * | 1981-05-15 | 1989-11-21 | Chaya Moroz | Monoclonal antibodies to placental isoferritin for use in detecting oncofetal ferritin associated with breast cancer and Hodgkins disease |
US5571678A (en) * | 1981-05-15 | 1996-11-05 | Moroz; Chaya | Placental isoferritins for the prognosis and diagnosis of immunosuppression |
WO1989009936A1 (en) * | 1988-04-08 | 1989-10-19 | Chaya Moroz | Isoferritin as a marker for pathological pregnancy |
EP0426458A2 (en) * | 1989-10-31 | 1991-05-08 | Japan Antibiotics Research Association | Human monocyte growth factor |
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BOYD D ET AL.: "Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, no. 21, 25 September 1985 (1985-09-25), pages 11755-11761, XP000876843 * |
DATABASE EMORG [Online] EMBL ID MIHSXX, AC V00662, 9 June 1982 (1982-06-09) ANDERSON S ET AL.: "H. sapiens mitochondrial genome" XP002131126 * |
DATABASE GCG_GENESEQ_P [Online] Geneseq ID R71567, AC R71567, 1 November 1995 (1995-11-01) ZH LIFE TECHNOLOGY KENKYUSHO: "Human monocyte growth factor" XP002131125 * |
FISCH B ET AL.: "Placental isoferritin as a marker of early abortion in pregnancies induced by in vitro fertilization" PLACENTA, vol. 17, no. 4, May 1996 (1996-05), pages 247-251, XP000877132 * |
GARTY B Z ET AL.: "High serum levels of placental isoferritin p43 component in Down syndrome." CHILDREN'S HOSPITAL QUARTERLY, vol. 10, no. 1, 1998, pages 39-41, XP000876978 * |
HIGGY N A ET AL: "Differential expression of human ferritin H chain gene in immortal human breast epithelial MCF-10F cells" MOLECULAR CARCINOGENESIS , vol. 20, no. 4, December 1997 (1997-12), pages 332-339, XP000876881 * |
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MOROZ C ET AL: "T-cell mitogenesis stimulates the synthesis of a mRNA species coding for a 43-kDa peptide reactive with CM-H-9, a monoclonal antibody specific for placental isoferritin." PROC. NATL. ACAD. SCI. USA , vol. 86, no. 9, May 1989 (1989-05), pages 3282-3285, XP002131123 * |
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WO2000015788A3 (en) | 2000-05-25 |
AU5645799A (en) | 2000-04-03 |
EP1112358A2 (en) | 2001-07-04 |
JP2003512811A (en) | 2003-04-08 |
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