SUBSTANCES FOR DETECTING BLOODCLOTS

Abstract: A DNA sequence encoding the functional A chain of human t-PA, the DNA sequence being attached at its 3' end through an inert DNA sequence to the 5' end of another DNA sequence capable of being transcribed into a termination codon, was expressed in a mammalian host. The A chain of the invention can be used to detect blood clots in vivo, for example by radiolabelling the polypeptide.

Description: SUBSTANCES FOR DETECTING BOODCLOTS Background of the Invention The invention relates to detecting bloodclots and the production of substances used for same. Tissue plasminogen activator (t-PA) is a serine protease that is a thrombolytic agent. It is normally produced as a single chain of 527 amino acids and converted into a biologically active two-chain form in the presence of plasminogen. The two-chain form has an A chain and a B chain, covalently linked by a single disulfide bridge. The A chain binds fibrin, and the B chain has sjrine protease activity. DNA encoding t-PA includes, starting at the 5' end, a sequence encoding a leader sequence; a sequence encoding A chain; and a sequence encoding B chain. Attached to the 3' end of the DNA encoding t-PA is DNA that is transcribed into a messenger RNA termination codon. Fibrin is formed by the proteolytic action of thrombin on fibrinogen during blood clotting. The proteolytic action causes the formation of fibrin monomers which aggregate to form a blood clot. Summary of the Invention One aspect of the invention features a DNA sequence encoding the functional A chain of human t-PA, the DNA sequence being attached at its 3' end through an inert DNA sequence to the 5' end of another DNA sequence capable of being transcribed into an mRNA termination codon. The term functional A chain of t-PA, as used herein, means a peptide sequence substantially identical to some portion of the peptide sequence of naturally occurring human A chain, the peptide sequence retaining some or all of the fibrin binding activity of human t-PA A chain. A termination codon is responsible for the termination of translation; the common termination codons are UAA, UAG, and UGA. A DNA sequence encoding functional A chain, as used herein, means a DNA sequence corresponding to the mRNA sequence that is translated into functional A chain. The DNA sequence that is transcribed into a termination codon is attached to the DNA sequence encoding functional A chain so that translation of the RNA transcribed from the sequences ceases after the functional A chain has been produced. The two sequences may be connected through an inert DNA sequence. Thus the resultant DNA sequence encodes a peptide, or portion thereof, that has essentially no biological activity, other than fibrin binding activity. The peptide, or portion thereof, encoded by the inert DNA sequence is attached at the C-terminal end of the functional A chain. In preferred embodiments, the DNA sequence is substantially identical to (i.e., has at least 90% homology with) the region of the complete structural gene (i.e., the full DNA sequence) encoding the A chain, and the inert DNA sequence has 100 bases or less (more preferably 50 bases or less). In other preferred embodiments the DNA sequence includes less than the region of the complete structural gene encoding the A chain. In other preferred embodiments, the 3' end of the DNA sequence is attached directly to the 5' end of the DNA sequence capable of being transcribed into a termination codon, i.e., there is' no inert DNA sequence. The invention features, in another aspect, a method of detecting bloodclots in a mammal, the method including the steps of (1) providing labelled A chain of t-PA; (2) introducing the labelled A chain into the bloodstream of the mammal, the labelled A chain circulating in the bloodstream, the circulating labelled A chain contacting and binding to the fibrin of a bloodclot; and (3) detecting the labelled A chain bound to the bloodclot. In preferred embodiments the A chain is radiolabelled with 125I and the detecting step involves radioi aging. The invention provides an inexpensive, readily available source for pure A chain, which can be used to detect_ and monitor bloodclots. Other features and advantages of the invention will be apparent from the following description of the preferred embodiment, and from the claims. Description of the Preferred Embodiment The structure and use of the preferred embodiment are now described, after first briefly describing the drawings. Brief Description of the Drawings Fig. 1 is the DNA sequence encoding the amino acid sequence of human t-PA A chain. Fig. 2 is a diagrammatic representation of the construction of a vector of the invention. Fig. 3 is a diagrammatic representation of the construction of an expression vector of the invention. Structure Referring to the Fig. 1, there is shown the amino acid sequence of human t-PA A chain, along wi h ■ the corresponding DNA sequence. According to the preferred embodiment, the DNA sequence encoding the A chain of human t-PA lias at its 3 ' end DNA that can be transcribed into a termination codon. The DNA is cloned into an expression vector that contains a promoter and polyadenylation signal, the vector is transfected into mammalian cells, and the cells are grown by standard procedures to produce A chain, which is then isolated for use. Construction of cDNA Encoding the A Chain of Human t-PA cDNA encoding human uterine t-PA is described in Wei et al., U.S.S.N. 782,686, which is assigned to the same assignee as the present application and is hereby incorporated by reference. . The Sail fragment (2100 bp) , which includes the cDNA encoding t-PA, is isolated from the yeast expression vector pYBDT-10, described in the above application. Referring to Fig. 2, the Sail fragment (2100 bp) is cleaved with EcoRl, and the fragments are separated on acrylamide gels. The internal EcoRl fragment (472 bp) is cleaved with Ddel, and electrophoresced on acrylamide gel to isolate the 184 bp fragment. The 472 bp and 184 bp fragments include the DNA encoding functional A chain. The Sail - EcoRl fragment (730 bp) that encodes the H, terminal portion of t-PA is ligated with the 184 bp EcoRl - Ddel fragment and the synthetic Ddel - Sail oligomer shown below. The sequence that is transcribed into a termination codon is circled. 5' TGAGA CAG TAC AGC CAG CCT CAG TTT CGC (TAA)GTC GACC 3' 3' CT GTC ATG TCG GTC GGA GTC AAA GCG AΪT CAG CTGG 5' The ligation reaction mixture is subjected to Sail cleavage and the 950 bp fragment can'be cloned into the Sail site of pBR322 for storage. Construction of an Expression Vector for A Chain The 950 bp Sail fragment can be inserted into any suitable mammalian expression vector, most preferably those which are tranεfected into rodent epitheliod cells. Preferred expression vectors include the BPV vectors described in Wei et all., supra, Wydro et al., U.S.S.N. 890,401, which is assigned to the same assignee as the present application and is hereby incorporated by reference, and Hsiung et a_l. , 1984, J. Molec. and App. Genet. 2:497. The vectors include a mouse metallothionein promoter (MT) from which inserted genes can be transcribed, and bovine papilloma virus DNA (BPV) to effect transfection of mammalian cells. CLH3axBPV (Fig. 3) also includes poly-adenylation sequences derived from SV40, which can affect expression from a gene inserted into the vector. The illustrated expression -plasmid also includes a portion of the E. coli plasmid pML, which permits shuttling between procaryotic and eucaryrtic systems. No selection is required for the maintenance of this plasmid in rodent host cells, and it is maintained in high (approximately 100 copies/cell) copy number. The 950 bp Sail fragment is cloned into the Xhol of the BPV vector (Fig. 3). Transfection of C127 Cells BPV vectors are transfected into suitable mammalian cells, e.g., C127 (mouse) cells by the standard calcium-phosphate precipitation method. Foci (transformed cells) appearing after two weeks are separated by cloning rings and grown in tissue.culture flasks. The media are assayed for A chain production by analysis of fibrin-binding activity. A chain is isolated by standard methods, for example, ion exchange or affinity chromatography. Deposit The following deposit has been made on with the Agricultural Research Culture Collection (NRRL). where the deposit was given the following accession number: Deposit Accession No. pYBDT-10 B-15884 Applicant's assignee, Integrated Genetics, represents that the NRRL is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited microorganism, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant's assignee acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit. Use Becuase it binds to fibrin, the A chain of the invention can be used to detect blood clots in vivo, for example, by radiolabelling the A chain, injecting the labelled A chain into the body of a human patient, and then radioimaging the body, or part thereof. The binding affinity of the A chain provides good selectivity and sensitivity. The A chain can be labelled using any conventional label such as a radiolabel (where radioimaging is involved) or fluorophore. Most preferably the A chain is radiolabelled with 125I, by conventional methods, e.g., those described by Wahl et al., 66. Hybridorna 111 (1987) and Sharkey et al., 8J. Proc. Natl. Acad. Sci. USA 2843 (1984). To carry out in vivo imaging in the detection, and localization, of bloodclots a patient can be given an intravenous injection of approximately 50 uCi of sterile I-A chain in physiological saline. Whole body scan scinitigrams can then be taken using a gamma camera interfaced with a computer and fitted with a medium energy, parallel hole collimator, and I images can-be obtained about the 125I photopeaks. Indium, can also be used as the radiolabel. The A chain of the invention can also be labelled with a paramagnetic ion, e.g. Gd or Mn + , to provide a targeted NMR contrast agent. " The paramagnetic ion can be complexed with the A chain via a chelating agent such as DTPA using conventional techniques, e.g., the method described in Khaw et al., 23 J. Nucl. Med. 1011 (1982). The contrast agent can be administered to a patient and NMR imaging carried out; the agents will provide NMR contrast between bloodclots, to which the targeted agents are bound, and other areas of the circulatory system. In addition to being useful for detecting and localizing bloodclots, in vivo imaging using the labelled A chain of the invention can provide a sensitive means for evaluating and monitoring the efficacy of thrombolytic agents, e.g., urokinase, streptokinase, and t-PA, in dissolving previously detected bloodclots. Other Embodiments Other embodiments are within the following claims. For example, any portion of A chain that binds to fibrin, e.g., the kringle 2 portion described in Dodd, EPO publication number 0196920, and Ichinoεe et al., 7_8 J. Clin. Inv. 163 (1986), can be used in accordance with the invention. The relevant portion (e.g., kringle 2) can be produced from appropriate vector transformed cells containing the DNA encoding the A chain portion; the DNA is attached at its 3' end to a DNA sequence capable of being transcribed into a termination codon.

Claims: Claims 1. A DNA sequence encoding the functional A chain of human t-PA, said DNA sequence being attached at its 3 ' end through an inert DNA sequence to the 5' end of another DNA sequence capable of being transcribed into a termination codon. 2. The DNA sequence of claim 1 wherein said DNA sequence is substantially identical to the complete structural-gene encoding said A chain. 3. The DNA sequence of claim 1 wherein said iner- DNA sequence comprises 100 bases or less. 4. The DNA sequence of claim 1 wherein said DNA sequence includes less than the complete structural gene encoding said A chain. 5. A vector comprising the DNA sequence of claim 1. 6. The DNA sequence of claim 1 wherein said 3' end of said DNA sequence is attached directly to said 5' end of said other DNA sequence. 7. The use of an indicating agent for identifying a suitable medicament for treating a bloodclot characterized in that said indicating agent comprises a functional A chain of t-PA capable of binding to fibrin and having a detectable label associated therewith. 8. The use of claim 7 wherein said A chain is radio! belled. 9. The use of claim 8 wherein said radiolabel is 125 I.

Inventors: REDDY VEMURI BHASKARA, REDDY, VEMURI, BHASKARA
Applicants: INTEGRATED GENETICS INC, INTEGRATED GENETICS, INC.
Application year: 1988
Main classification: C12N15
IPC codes: C12N9