Compositions and methods for isolation of circulating tumor cells (CTC)
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C12N-015/79
G01N-033/53
A61K-039/23
C07H-021/04
C12Q-001/6897
C12Q-001/6886
C12N-015/86
출원번호
15517143
(2015-10-05)
등록번호
10941452
(2021-03-09)
국제출원번호
PCT/US2015/053982
(2015-10-05)
국제공개번호
WO2016/057387
(2016-04-14)
발명자
/ 주소
Dorsey, Jay F.
Kao, Gary D.
Hahn, Stephen M.
출원인 / 주소
The Trustees of the University of Pennsylvania
대리인 / 주소
Howson & Howson LLP
인용정보
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0인용 특허 :
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초록▼
A method useful for identifying and isolating live circulating tumor cells is described. The method utilizes an adenoviral vector comprising a replication-competent adenovirus in which the E1 gene region is expressed under the control of a telomerase-specific promoter and further comprises a second
A method useful for identifying and isolating live circulating tumor cells is described. The method utilizes an adenoviral vector comprising a replication-competent adenovirus in which the E1 gene region is expressed under the control of a telomerase-specific promoter and further comprises a second expression cassette containing a marker protein, optionally fused to a detectable cell surface marker to permit detection of circulating tumor cells lacking cell surface markers. The method involves combining ex vivo a test sample from a patient suspected of having circulating tumor cells, an adenoviral probe system, and culture media for the cells. The test sample is incubated with the adenoviral system for a sufficient time to permit expression of the reporter protein. The marker gene expression can thereafter be quantitated and the marker-expressing cells may optionally be collected for further analysis.
대표청구항▼
1. A method useful for isolating live circulating melanoma tumor cells, said method comprising (a) combining ex vivo a test sample from blood of a melanoma patient suspected of having circulating melanoma tumor cells, an adenoviral probe system, and culture media for the cells, said adenoviral probe
1. A method useful for isolating live circulating melanoma tumor cells, said method comprising (a) combining ex vivo a test sample from blood of a melanoma patient suspected of having circulating melanoma tumor cells, an adenoviral probe system, and culture media for the cells, said adenoviral probe system comprising: a first replication-competent recombinant adenovirus comprising a nucleic acid sequence which comprises at least a first telomerase specific promoter which directs expression of an adenovirus E1a gene and/or E1b gene following infection of circulating tumor cells (CTC) with the first recombinant adenovirus, and a coding sequence for a first fluorescent reporter protein which is expressed in the adenovirus-infected CTC following expression of the adenovirus E1a gene and/or E1b gene;(b) incubating the test sample and the adenoviral probe system for a sufficient time to permit expression of the first fluorescent reporter protein, optionally replenishing the media; and(c) collecting cells expressing the first fluorescent reporter protein, whereby expression of the first fluorescent reporter protein indicates the presence of live CTC in the sample. 2. The method according to claim 1, wherein the telomerase specific promoter is selected from the group consisting of a human telomerase reverse transcriptase (hTERT) promoter, a human telomerase RNA (hTR) promoter, and a human telomerase RNA gene (hTERC) promoter. 3. The method according to claim 1, wherein the first replication-competent recombinant adenovirus further comprises a second fluorescent protein gene sequence under the control of regulatory sequences which direct expression thereof in the live CTC following expression of the E1a gene. 4. The method according to claim 1, wherein the first replication-competent recombinant adenovirus comprises a nucleic acid sequence encoding a fusion protein, said fusion-protein comprising a tumor associated cell surface marker protein and the first fluorescent reporter protein. 5. The method according to claim 4, further comprising capturing the infected CTC by a ligand specific for the tumor associated cell surface marker protein or a ligand specific for the first fluorescent reporter protein. 6. The method according to claim 5, wherein said ligand is an antibody. 7. The method according to claim 6, wherein said antibody is conjugated or bound to a magnetic bead. 8. The method of claim 4, further comprising identifying CTC using a probe for the tumor associated cell surface marker and a probe for the first fluorescent protein. 9. The method of claim 4, wherein the tumor associated cell surface marker protein is epithelial cell adhesion molecule (EpCam) and the first fluorescent reporter protein is green fluorescent protein (GFP). 10. The method according to claim 1, wherein the coding sequence for the first fluorescent reporter protein is operably linked to an expression control sequence and is in the site of a deletion in the adenovirus immediate early E3 gene region. 11. The method according to claim 1, wherein the adenoviral probe system further comprises at least a second, different recombinant adenovirus. 12. The method according to claim 1, wherein the first fluorescent reporter protein is cherry picker, and wherein the method further comprises capturing the infected CTC by a ligand which is an anti-cherry picker antibody. 13. The method of claim 12, wherein said antibody is conjugated or bound to a magnetic bead. 14. The method according to claim 1, further comprising counting the collected CTC expressing the first fluorescent reporter protein. 15. The method according to claim 1, wherein the adenoviral probe system further comprises a coding sequence for a second fluorescent reporter protein which is distinguishable from the first fluorescent reporter protein, wherein the second fluorescent reporter protein is specifically expressed in CTC, and wherein the method further comprises counting the collected CTC expressing the second fluorescent reporter protein. 16. The method of claim 1, wherein the first fluorescent reporter protein is expressed under the control of a constitutive promoter. 17. The method of claim 1, further comprising detecting cell size of the CTC expressing the first fluorescent reporter protein in step (c). 18. The method of claim 1, further comprising measuring fluorescence intensity of the expressed first fluorescent reporter protein in step (c). 19. The method of claim 1, further comprising (d) combining ex vivo a control sample from blood of a healthy subject, the adenoviral probe system, and the culture media for the cells;(e) incubating the control sample and the adenoviral probe system for the time of step (b), optionally replenishing the media as in step (b);(f) detecting cell size of cells from the blood of the healthy subject expressing the first fluorescent reporter protein after step (e) and fluorescence intensity of the expressed fluorescent reporter protein to establish background levels of cell size and fluorescence intensity;(g) detecting cell size of the cells from the melanoma patient collected in step (c) and fluorescence intensity of the first fluorescent reporter protein expressed in the collected cells; and(h) counting the cells from the melanoma patient collected in step (c) when the cells do not have a cell size or a fluorescence intensity above the background levels determined in step (f), whereby the cell count indicates the live CTC level in the sample. 20. The method according to claim 1, further comprising (d) analyzing the live CTC from step (c) for a selected tumor associated mutation. 21. A method for tracking resistance of a targeted therapy comprising: (a) combining ex vivo a test sample from blood of a melanoma patient suspected of having circulating tumor cells (CTC) and who has been treated with a targeted therapy, an adenoviral probe system, and culture media for the cells, said adenoviral probe system comprising: a first replication-competent recombinant adenovirus comprising a nucleic acid sequence which comprises at least a first telomerase specific promoter which directs expression of an adenovirus E1a gene and/or E1b gene following infection of CTC with the first recombinant adenovirus, and a coding sequence for a first fluorescent reporter protein which is expressed in the infected CTC following expression of the adenovirus E1a gene and/or E1b gene;(b) incubating the test sample and the adenoviral probe system for a sufficient time to permit infection of CTC with the adenoviral probe system and expression of the first fluorescent reporter protein in the CTC, optionally replenishing the media;(c) collecting the CTC expressing the first reporter protein, whereby expression of the first reporter protein indicates the presence of live CTC in the sample; and(d) counting the CTC expressing the first fluorescent protein, whereby the presence of a level of live CTC above a control CTC count threshold indicates resistance to the targeted therapy. 22. The method of claim 21, wherein the control CTC count threshold is determined using a blood sample from a healthy subject. 23. The method of claim 21, wherein the control CTC count threshold is 1.1 CTCs/mL.
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