KR-102964326-B1 - COMPOSITIONS AND METHODS FOR ISOLATING, DETECTING, AND ANALYZING FETAL CELLS

Abstract

The present invention provides compositions, kits, and methods for the isolation, detection, and analysis of fetal cells. The invention also provides methods for the preparation of fetal cell samples and for performing fetal genetic testing. The compositions, kits, and methods may include or use anti-TREML2 antibodies. Alternatively, or additionally, the compositions, kits, and methods include or use antibodies conjugated to colloidal magnetic particles and/or exogenous aggregation-enhancing factors.

Inventors

• 카스타그놀리 파올라
• 도피니 안나
• 차오 웬-샨

Assignees

• 에이. 메나리니 바이오마커스 싱가포르 피티이. 리미티드.

Dates

Publication Date

20260512

Application Date

20200714

Priority Date

20190715

Claims (20)

1. As a method for isolating fetal cells from a sample of a pregnant subject, (a) a step of contacting a sample containing a plurality of cells with a plurality of magnetic particles, wherein the magnetic particles are coupled to a first exogenous aggregation-enhancing factor (EAEF) and conjugated to a first antibody that binds to an endothelial marker selected from CD105 and CD71; (b) a step of isolating cells bound to the first antibody to produce a concentrated sample; (c) contacting the concentrated sample with a second antibody that binds to a fetal cell marker selected from Triggering Receptor Expressed on Myeloid Cells Like 2 (TREML2) and cytokeratin (CK) expressed on bone marrow cells; and (d) a step of isolating fetal cells bound to the second antibody. A method including
2. In paragraph 1, A method in which the fetal cells are fetal nucleated red blood cells (fnRBCs).
3. In paragraph 1, A method in which the fetal cell is a trophoblast.
4. In paragraph 1, A method in which the magnetic particles are colloidal magnetic particles.
5. In paragraph 4, A method in which colloidal magnetic particles are ferrofluid magnetic particles.
6. In paragraph 4, A method comprising step (b) applying a magnetic field to a sample.
7. In paragraph 6, A method in which the first EAEF comprises one member of a specific binding pair selected from the group comprising biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, avidin-biotin, biotin analog-avidin, destiobiotin-streptavidin, destiobiotin-avidin, iminoviotin-streptavidin, and iminoviotin-avidin.
8. In Paragraph 7, A method comprising step (a) inducing aggregation of magnetic particles by applying a second EAEF, wherein the second EAEF comprises another member of a specific bonding pair.
9. In paragraph 8, A method comprising step (b) applying a member of a specific bonding pair to the concentrated sample to reverse the aggregation of magnetic particles in the concentrated sample.
10. In paragraph 1, A method comprising a plurality of magnetic particles, additionally including magnetic particles conjugated to an antibody that binds to an epithelial marker.
11. In Paragraph 10, A method in which the epithelial marker is an epithelial cell adhesion molecule (EpCAM).
12. In paragraph 1, A method in which a second antibody is an antibody or antigen-binding fragment that binds to TREML2.
13. In paragraph 1, A method in which a second antibody is an antibody or antigen-binding fragment that binds to CK.
14. In paragraph 1, A method comprising the step of isolating a single fetal cell, wherein the single fetal cell bound to a second antibody is isolated.
15. In paragraph 1, A method comprising, in addition, step (c), contacting the concentrated sample with an anti-CD45 antibody.
16. In paragraph 1, A method comprising, in addition, step (c) contacting the concentrated sample with a nuclear stain.
17. In paragraph 1, A method in which the label is a fluorescent label.
18. In Paragraph 17, A method in which the step of isolating fetal cells is based on immunofluorescence technology.
19. In Paragraph 17, A method in which the step of isolating fetal cells includes fluorescence-activated cell sorting (FACS).
20. In Paragraph 17, A method in which the step of isolating fetal cells includes DEPArray.

Description

Compositions and methods for isolating, detecting, and analyzing fetal cells Cross-reference regarding related applications The present application claims priority to U.S. provisional application No. 62/874,306 filed July 15, 2019, the disclosures of which are incorporated by reference in their entirety. Over the past 40 years, researchers have attempted to isolate fetal cells from pregnant women to develop prenatal diagnostic tools. Amniocentesis was first developed in the early 1970s, and chorionic villus sampling (CVS) was developed in the 1980s. Amniocentesis and chorionic villus sampling (CVS) are two invasive methods used in routine clinical practice to diagnose common fetal aneuploidy (excess copies of chromosomes), such as trisomy of chromosomes 13, 18, and 21 (which causes Down syndrome). The ability to isolate fetal cells and fetal DNA from maternal blood during pregnancy has opened up interesting opportunities for improved non-invasive prenatal testing. Recently, cell-free DNA-based testing (cfDNA), known as a non-invasive prenatal test (NIPT), has been introduced for prenatal screening and has been recognized for its high predictive accuracy regarding trisomy 21. Nevertheless, screening performance is lower than that of invasive diagnostic tools, so confirmatory testing is still required. Furthermore, according to a professional society (Practice Bulletin n163 Obstet Gynecol. 2016; 127(5) 979-981), NIPT does not predict copy number variations (CNV) or microdeletions/duplications. Therefore, current cell-free NIPT is not yet suitable for detecting subchromosomal deletions and duplications with high specificity, sensitivity, and positive predictive values. Direct analysis of fetal cells in the maternal circulatory system has been difficult until now due to the scarcity of fetal cells in maternal blood. Numerous different enrichment methods have been tested, including filters, density gradients, fluorescence-activated cell sorting (FACS), microfluidics, and immuno-magnetic beads. While these methods can recover circulating fetal cells, they lack consistency and reproducibility. This is because the number of circulating fetal cells (0.1 to 10 cells per 1 ml of maternal blood containing approximately 1 to 5 million cells) is extremely small, which has hindered the establishment of reproducible protocols to date. The challenge is to remove all contaminating nucleated blood cells while minimizing the loss of circulating fetal cells during the first three months of pregnancy. Given these limitations and the fact that amniocentesis and chorionic villus sampling (CVS) are procedures associated with the risk of pregnancy loss, there is a need to develop a new cell-based NIPD (non-invasive prenatal diagnosis) procedure that selects fetal cells from the pregnant woman's maternal blood to screen for congenital defects and genetic diseases. Although fetal nucleated red blood cells (nRBCs) and trophoblastic cells are known to exist in the maternal circulation, developing a reliable cytogenetic cell-based form of NIPT has been challenging. Recently, the potential for developing a cell-based form of NIPT capable of detecting abnormalities with accuracy comparable to that obtained from amniocentesis and CVS has been proposed (Amy M. Breman, et al., Prenatal Diagnosis, 2016, 36(11):1009-1019). The present invention discloses a fetal cell marker and an agent that binds thereto. The present invention further discloses a composition, kit, and method for isolating, detecting, and analyzing fetal cells based on a fetal cell marker. The present invention discloses a method for detecting fetal cells in a sample of a pregnant subject, the method comprising: (a) contacting the sample with a first antibody, wherein the sample comprises a plurality of cells; (b) isolating the cells bound to the first antibody to produce a concentrated sample; (c) contacting the concentrated sample with a second antibody; and (d) identifying the cells bound to the second antibody as fetal cells, wherein the first antibody or the second antibody comprises (i) an antibody that binds to a trigger receptor-like 2 (TREML2) protein expressed on bone marrow cells; or (ii) an antigen-binding fragment that binds to the TREML2 protein. In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC). In some embodiments, the fetal cell is a cytotrophoblast. In some embodiments, the first antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the magnetic particles are coupled to a first exogenous aggregation-enhancing factor (EAEF), the first EAEF comprises a member of a specific binding pair selected from the group comprising biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-a