RU2834057C2 - Compositions and methods for isolating, detecting and analysing foetal cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method for detecting foetal cells in a blood sample obtained from a pregnant subject, involving: a) bringing the sample into contact with a first antibody conjugated with magnetic particles; b) recovering cells associated with a first antibody conjugated with magnetic particles to obtain an enriched sample; c) bringing said enriched sample into contact with a second antibody conjugated with a label against TREML2 and cytokeratin (CK); d) identifying a cell associated with a second antibody, conjugated with a label, as a foetal cell, as well as to a method for detecting foetal cells in a blood sample obtained from a pregnant subject, comprising: a) bringing the sample into contact with a first antibody or an antigen-binding fragment thereof, conjugated with magnetic particles against TREML2 and cytokeratin (CK); b) identifying the cell associated with the first antibody or its antigen-binding fragment conjugated with magnetic particles as foetal cells.

EFFECT: invention provides the possibility of expanding the methods of non-invasive prenatal testing.

24 cl, 18 dwg, 11 tbl

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 62/874,306, filed July 15, 2019, the disclosure of which is incorporated herein by reference in its entirety.

LEVEL OF TECHNOLOGY

[0002] Over the past four decades, researchers have been attempting to isolate fetal cells from pregnant women to develop prenatal diagnostic tools. In the early 1970s, the amniocentesis procedure was first developed, followed by the chorionic villus sampling (CVS) technique in the 1980s. Amniocentesis and chorionic villus sampling (CVS) are two invasive techniques used in routine clinical practice to diagnose chromosomal aberrations such as common fetal aneuploidies (an extra copy of a chromosome), such as trisomy 13, 18, and 21 (resulting in Down syndrome).

[0003] The ability to isolate fetal cells and fetal DNA from maternal blood during pregnancy has opened up opportunities for improved non-invasive prenatal testing. Recently, non-invasive prenatal testing (NIPT) has introduced cell-free DNA (cfDNA) screening, which is considered a good predictor of trisomy 21. However, the screening performance is inferior to invasive diagnostic tools, and confirmatory testing is still needed. In addition, according to professional societies, NIPT does not predict copy number variants (CNVs) or microdeletions/duplications (Practice bulletin nl63 Obstet Gynecol. 2016; 127(5) 979-981). Thus, current cell-free NIPT does not yet detect subchromosomal deletions and duplications with high sensitivity, specificity, and positive predictive value.

[0004] Direct analysis of fetal cells from maternal circulation remains challenging given the small number of fetal cells in maternal blood. A variety of enrichment methods have been tested, including filters, density gradients, fluorescence-activated cell sorting (FACS), microfluidic technologies, and immunomagnetic beads. Although fetal cells can be isolated, these methods lack stability and reproducibility. This is due to the extremely small number of circulating fetal cells (0.1-10 cells per ml of maternal blood, containing approximately 1-5 million cells), which currently hinders the development of reproducible protocols. The challenge is to remove all extraneous nucleated blood cells without losing the very small number of circulating fetal cells in the first trimester of pregnancy.

[0005] Given these limitations and the fact that amniocentesis and chorionic villus sampling (CVS) are procedures associated with a risk of pregnancy loss, there is a need to develop new cell-based NIPD (non-invasive prenatal diagnosis) procedures to collect fetal cells from the blood of pregnant women for screening for congenital defects and inherited diseases.

[0006] Fetal nucleated red blood cells (nRBCs) and trophoblast cells are known to be present in the maternal circulation, but developing a reliable cytogenetic cell-based form of NIPT has been challenging. Recently, the possibility of developing a cell-based form of NIPT with anomaly detection accuracy similar to that currently achieved using amniocentesis and CVS has been proposed (Amy M. Breman, et al., Prenatal Diagnosis, 2016, 36(11): 1009-1019).

[0007] The present document describes fetal cell markers and agents that bind thereto. In addition, the present document describes compositions, kits, and methods for isolating, detecting, and analyzing fetal cells based on the fetal cell markers.

ESSENCE OF THE INVENTION

[0008] Described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with a first antibody, wherein said sample comprises a plurality of cells; (b) isolating the cells bound to the first antibody to obtain an enriched sample; (c) contacting the enriched sample with a second antibody; and (d) identifying a cell bound to the second antibody as a fetal cell, wherein said first antibody or said second antibody: (i) is an antibody that binds to a trigger receptor analogue 2 protein expressed on myeloid cells (TREML2); or (ii) comprises an antigen-binding fragment that binds to a TREML2 protein.

[0009] In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC). In some embodiments, the fetal cell is a trophoblast cell.

[0010] 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 attached to a first exogenous aggregation enhancing factor (EAEF), wherein the first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0011] In some embodiments, step (a) comprises adding a second EAEF to induce aggregation of magnetic particles, wherein said second EAEF comprises another member of the specific binding pair.

[0012] In some embodiments, step (b) includes applying a magnetic field to the sample.

[0013] In some embodiments, step (b) comprises adding a member of a specific binding pair to the enriched sample to eliminate aggregation of magnetic particles in the enriched sample.

[0014] In some embodiments, the method, prior to step (a), further comprises adding to the sample at least one aggregation inhibiting agent selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibiting agent is a chelating agent. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0015] In some embodiments, the second antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in any of SEQ ID Nos. 2-5.

[0016] In some embodiments, the method further comprises isolating single cells of the fetus prior to step (d).

[0017] In some embodiments, the isolation of single fetal cells is accomplished by isolating single fetal cells bound to a second antibody.

[0018] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0019] In some embodiments, the isolation of single fetal cells is based on immunofluorescence technology. In some embodiments, the isolation of single fetal cells is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the isolation of single fetal cells is performed using DEPArray.

[0020] In some embodiments, step (d) includes performing a sequencing-based analysis. In some embodiments, the sequencing-based analysis includes a short tandem repeat (STR) analysis.

[0021] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in a fetal cell.

[0022] In some embodiments, the first antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in any of SEQ ID Nos. 2-5.

[0023] In some embodiments, the antibody that binds to a TREML2 protein or antigen-binding fragment that binds to a TREML2 protein comprises one or more CDRs selected from: (i) a complementarity determining region (CDR) 1 of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 6; (ii) a CDR2 of an HCVR comprising the amino acid sequence of SEQ ID NO: 7; (iii) a CDR3 of an HCVR comprising the amino acid sequence of SEQ ID NO: 8); (iv) a CDR1 of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 9; (v) a CDR2 of an LCVR comprising the amino acid sequence of SEQ ID NO: 10; and (vi) CDR3 of LCVR comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, said antibody that binds to a TREML2 protein or antigen-binding fragment that binds to a TREML2 protein comprises 2, 3, 4, 5, or 6 CDRs selected from (i)-(vi).

[0024] In some embodiments, an antibody that binds to a TREML2 protein is an anti-TREML2 antibody. In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABPN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0025] Also described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with a magnetic reagent, wherein said sample comprises a plurality of cells, and the magnetic reagent comprises a magnetic particle conjugated to a first antibody, wherein said first antibody binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with an anti-TREML2 antibody or an antigen-binding fragment thereof; and (c) identifying a cell bound to the anti-TREML2 antibody as a fetal cell.

[0026] In some embodiments, the method further comprises, before step (c), isolating cells bound to the first antibody. In some embodiments, isolating cells comprises exposing the sample to a magnetic field to enrich the sample for cells bound to the first antibody.

[0027] In some embodiments, said magnetic particle is a colloidal magnetic particle. In some embodiments, said colloidal magnetic particle is a magnetic particle of a ferrofluid. In some embodiments, the size of the colloidal magnetic particle is less than 200 nm. In some embodiments, the size of the colloidal magnetic particle is approximately 80-200 nm. In some embodiments, the size of the colloidal magnetic particle is approximately 90-150 nm. In some embodiments, the magnetic mass of said colloidal magnetic particle is at least 50%. In some embodiments, the magnetic mass of said colloidal magnetic particle is at least 60%. In some embodiments, the magnetic mass of said colloidal magnetic particle is approximately 70-90%. In some embodiments, said colloidal magnetic particle comprises a crystalline core of a superparamagnetic material surrounded by coating molecules.

[0028] In some embodiments, the magnetic particle is attached to a first exogenous aggregation enhancing factor (EAEF), wherein said first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0029] In some embodiments, the method during step (a) comprises adding a second EAEF to enhance particle aggregation, wherein the second EAEF comprises another member of the specific binding pair.

[0030] In some embodiments, the method further comprises adding a member of the specific binding pair (a third EAEF) to the enriched sample to eliminate aggregation of the magnetic reagent in the sample, thereby facilitating cell identification.

[0031] In some embodiments, the method further comprises adding to the sample, before step (a), at least one aggregation inhibiting agent selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibiting agent is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0032] In some embodiments, the method further comprises isolating cells using an anti-TREML2 antibody or a second antibody before step (c). In some embodiments, the second antibody is selected from an anti-cytokeratin antibody and an anti-HLAG antibody.

[0033] In some embodiments, the anti-TREML2 antibody or second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0034] In some embodiments, the cell isolation is based on immunofluorescence technology. In some embodiments, the cell isolation is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the cell isolation is performed using a DEP Array.

[0035] In some embodiments, identifying a cell comprises performing a sequencing-based analysis.

[0036] In some embodiments, the sequencing-based analysis comprises short tandem repeat (STR) analysis.

[0037] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in a fetal cell.

[0038] In some embodiments, the fetal cell is a fetal erythroblast or a fetal trophoblast cell.

[0039] In some embodiments, an anti-TREML2 antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) selected from: (i) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 1; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8); (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0040] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0041] Also described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with a first antibody, wherein said sample comprises a plurality of cells, and wherein the first antibody binds to a trigger receptor analogue 2 protein expressed on myeloid cells (TREML2) (anti-TREML2 antibody), or an antigen-binding fragment thereof; and (b) identifying a cell bound by the first antibody as a fetal cell.

[0042] In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC).

[0043] 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 colloidal magnetic particle has a size of less than 200 nm. In some embodiments, the colloidal magnetic particle has a size of about 80-200 nm. In some embodiments, the colloidal magnetic particle has a size of about 90-150 nm. In some embodiments, the magnetic mass of the colloidal magnetic particle is at least 50%. In some embodiments, the magnetic mass of the colloidal magnetic particle is at least 60%. In some embodiments, the magnetic mass of the colloidal magnetic particle is about 70-90%. In some embodiments, the colloidal magnetic particle comprises a crystalline core of a superparamagnetic material surrounded by coating molecules.

[0044] In some embodiments, the method further comprises applying a magnetic field to the sample.

[0045] In some embodiments, the magnetic particles are attached to a first exogenous aggregation enhancing factor (EAEF), wherein said first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0046] In some embodiments, the method further comprises, prior to step (b), adding a second EAEF to enhance aggregation of magnetic particles, wherein the second EAEF comprises another member of the specific binding pair.

[0047] In some embodiments, the method further comprises isolating cells bound to the first antibody to obtain an enriched sample.

[0048] In some embodiments, the method further comprises adding a third EAEF to the enriched sample to eliminate aggregation of magnetic particles in the enriched sample, wherein the third EAEF is capable of binding to the first EAEF or the second EAEF. In some embodiments, the third EAEF is a member of a specific binding pair.

[0049] In some embodiments, the method, prior to step (a), further comprises adding to the sample at least one aggregation inhibiting agent selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibiting agent is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0050] In some embodiments, the first antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0051] In some embodiments, the method further comprises, before step (b), isolating cells bound to the first antibody, wherein said isolating cells is based on fluorescence-based technology. In some embodiments, isolating cells bound to the first antibody is performed by fluorescence-activated cell sorting (FACS). In some embodiments, isolating cells bound to the first antibody is performed using a DEP Array.

[0052] In some embodiments, step (b) includes performing a sequencing-based analysis. In some embodiments, the sequencing-based analysis includes short tandem repeat (STR) analysis. In some embodiments, the method further includes analyzing fetal cells. In some embodiments, analyzing fetal cells includes performing genomic or genetic analysis. In some embodiments, performing genetic analysis includes detecting the presence or absence of one or more genetic abnormalities in a fetal cell.

[0053] In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from: (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 2, 3, 4, 5, or 6 CDRs selected from (a)-(f).

[0054] In some embodiments, the first antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0055] Also described herein is a cell-based method for genetic testing of a fetus, comprising: (a) contacting a sample obtained from a pregnant subject with an anti-TREML2 antibody or antigen-binding fragment thereof, wherein said sample comprises a plurality of cells; (b) isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of said one or more nucleic acid molecules, wherein said report provides a probability of the presence of one or more genetic abnormalities in the fetus.

[0056] In some embodiments, the cells bound to the TREML2 antibody or antigen-binding fragment thereof are fetal cells.

[0057] In some embodiments, the fetal cells are fetal erythroblasts. In some embodiments, the fetal cells are fetal trophoblast cells.

[0058] In some embodiments, analyzing said one or more nucleic acid molecules comprises performing a karyotype analysis.

[0059] In some embodiments, analyzing the one or more nucleic acid molecules comprises performing a sequencing-based analysis. In some embodiments, the sequencing-based analysis comprises a short tandem repeat (STR) analysis.

[0060] In some embodiments, the one or more genetic abnormalities are selected from a trisomy, a sex chromosome abnormality, and a structural abnormality. In some embodiments, the trisomy is selected from trisomy 3, trisomy 4, trisomy 6, trisomy 7, trisomy 8, trisomy 9, trisomy 10, trisomy 11, trisomy 12, trisomy 13, trisomy 16, trisomy 17, trisomy 18, trisomy 20, trisomy 21, and trisomy 22. In some embodiments, the sex chromosome abnormality is selected from monosomy X, trisomy X, and Klinefelter syndrome. In some embodiments, the structural abnormality is a copy number variation (CNV). In some embodiments, the structural abnormality is a CNV deletion or a CNV duplication.

[0061] In some embodiments, the anti-TREML2 antibody is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a colloidal magnetic particle. In some embodiments, the colloidal magnetic particle is a ferrofluid magnetic particle.

[0062] In some embodiments, step (b) includes applying a magnetic field to the sample.

[0063] In some embodiments, the method further comprises, prior to step (a), contacting the sample with a first antibody, wherein the first antibody binds a protein selected from EpCAM, CD105, and CD71.

[0064] In some embodiments, the method further comprises, prior to step (a), isolating cells bound to the first antibody.

[0065] In some embodiments, the first antibody is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a colloidal magnetic particle. In some embodiments, the colloidal magnetic particle is a ferrofluid magnetic particle. In some embodiments, the colloidal magnetic particle has a size of less than 200 nm. In some embodiments, the colloidal magnetic particle has a size of about 80-200 nm. In some embodiments, the colloidal magnetic particle has a size of about 90-150 nm. In some embodiments, the magnetic mass of the colloidal magnetic particle is at least 50%. In some embodiments, the magnetic mass of the colloidal magnetic particle is at least 60%. In some embodiments, the magnetic mass of the colloidal magnetic particle is about 70-90%. In some embodiments, the colloidal magnetic particle comprises a crystalline core of a superparamagnetic material surrounded by coating molecules.

[0066] In some embodiments, isolating cells bound to the first antibody is accomplished by exposing the sample to a magnetic field. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0067] In some embodiments, the isolation of cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is based on immunofluorescence technology. In some embodiments, the isolation of cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the isolation of cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is performed using DEPArray.

[0068] In some embodiments, the method further comprises contacting the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof with a second antibody or antigen-binding fragment thereof.

[0069] In some embodiments, the second antibody is an anti-TREML2 antibody or an antigen-binding fragment thereof. In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0070] In some embodiments, the method further comprises isolating cells bound to the second antibody or antigen-binding fragment thereof. In some embodiments, isolating cells bound to the second antibody or antigen-binding fragment thereof is based on immunofluorescence technology. In some embodiments, isolating cells bound to the second antibody or antigen-binding fragment thereof is performed by fluorescence-activated cell sorting (FACS). In some embodiments, isolating cells bound to the second antibody or antigen-binding fragment thereof is performed using DEPArray.

[0071] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0072] In some embodiments, an anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from: (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 2, 3, 4, 5, or 6 CDRs selected from (i)-(vi).

[0073] Also described herein is a method for obtaining a fetal cell sample from a sample obtained from a pregnant subject, comprising: (a) contacting a sample obtained from the mother's body and comprising fetal cells and maternal cells with a first antibody conjugate, wherein said first antibody conjugate comprises (i) a first antibody; and (ii) a colloidal magnetic particle, wherein said first antibody is conjugated to said colloidal magnetic particle, and (b) isolating cells associated with said first antibody conjugate by exposing the sample obtained from the mother's body to a magnetic field, thereby obtaining a fetal cell sample.

[0074] In some embodiments, said magnetic particle is a colloidal magnetic particle. In some embodiments, said colloidal magnetic particle is a magnetic particle of a ferrofluid. In some embodiments, the size of the colloidal magnetic particle is less than 200 nm. In some embodiments, the size of the colloidal magnetic particle is approximately 80-200 nm. In some embodiments, the size of the colloidal magnetic particle is approximately 90-150 nm. In some embodiments, the magnetic mass of said colloidal magnetic particle is at least 50%. In some embodiments, the magnetic mass of said colloidal magnetic particle is at least 60%. In some embodiments, the magnetic mass of said colloidal magnetic particle is approximately 70-90%. In some embodiments, said colloidal magnetic particle comprises a crystalline core of a superparamagnetic material surrounded by coating molecules.

[0075] In some embodiments, the magnetic particle is attached to a first exogenous aggregation enhancing factor (EAEF), wherein said first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0076] In some embodiments, the method further comprises adding a second EAEF to the sample obtained from the mother, wherein the second EAEF comprises another member of the specific binding pair.

[0077] In some embodiments, the first antibody is an anti-TREML2 antibody.

[0078] In some embodiments, the first antibody is an anti-CD71 antibody.

[0079] In some embodiments, the first antibody binds to a protein selected from EpCAM and CD105.

[0080] In some embodiments, obtaining a sample of fetal cells further comprises contacting cells isolated from the sample obtained from the mother's body with a second antibody.

[0081] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0082] In some embodiments, obtaining a sample of fetal cells further comprises isolating cells bound to the second antibody.

[0083] In some embodiments, the isolation of cells bound to the second antibody is based on immunofluorescence technology. In some embodiments, the isolation of cells bound to the second antibody is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the isolation of cells bound to the second antibody is performed using a DEP Array.

[0084] Also described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with a first antibody conjugate, wherein said sample comprises a plurality of cells, and said first antibody comprises a first antibody conjugated to a colloidal magnetic particle; (b) isolating cells bound to the first antibody by exposing the sample to a magnetic field, thereby producing an enriched sample; (c) contacting the enriched sample with a second antibody, wherein said second antibody binds to a marker on the surface of a fetal cell; and (d) identifying a cell bound to the second antibody as a fetal cell.

[0085] In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC). In some embodiments, the fetal cell is a fetal trophoblast cell.

[0086] In some embodiments, said colloidal magnetic particles are magnetic particles of a ferrofluid. In some embodiments, the size of the colloidal magnetic particle is less than 200 nm. In some embodiments, the size of the colloidal magnetic particle is approximately 80-200 nm. In some embodiments, the size of the colloidal magnetic particle is approximately 90-150 nm. In some embodiments, the magnetic mass of said colloidal magnetic particle is at least 50%. In some embodiments, the magnetic mass of said colloidal magnetic particle is at least 60%. In some embodiments, the magnetic mass of said colloidal magnetic particle is approximately 70-90%. In some embodiments, said colloidal magnetic particle comprises a crystalline core of a superparamagnetic material surrounded by coating molecules.

[0087] In some embodiments, the magnetic particles are attached to a first exogenous aggregation enhancing factor (EAEF), wherein said first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0088] In some embodiments, step (a) comprises adding a second EAEF to enhance aggregation of magnetic particles, wherein said second EAEF comprises another member of the specific binding pair.

[0089] In some embodiments, step (b) comprises adding a member of a specific binding pair to the enriched sample to eliminate aggregation of magnetic particles in the enriched sample.

[0090] In some embodiments, the method, prior to step (a), further comprises adding to the sample at least one aggregation inhibiting agent selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibiting agent is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0091] In some embodiments, said second antibody is an antibody that binds to the TREML2 protein or comprises an antigen-binding fragment that binds to the TREML2 protein.

[0092] In some embodiments, the method further comprises isolating single fetal cells before step (d). In some embodiments, isolating single fetal cells is performed by isolating single fetal cells bound to the second antibody.

[0093] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0094] In some embodiments, the isolation of single fetal cells is based on immunofluorescence technology. In some embodiments, the isolation of single fetal cells is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the isolation of single fetal cells is performed using a DEP Array.

[0095] In some embodiments, step (d) includes performing a sequencing-based analysis. In some embodiments, the sequencing-based analysis includes a short tandem repeat (STR) analysis.

[0096] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in a fetal cell.

[0097] In some embodiments, said first antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein.

[0098] In some embodiments, the TREML2 protein-binding antibody or TREML2 protein-binding antigen-binding fragment comprises, consists of, or consists essentially of one or more CDRs selected from: (i) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8); (iv) a light chain variable region (LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) CDR3 of LCVR comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, said antibody that binds to a TREML2 protein or antigen-binding fragment that binds to a TREML2 protein comprises 2, 3, 4, 5, or 6 CDRs selected from (i)-(vi). In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0099] In some embodiments, the antibody that binds to the TREML2 protein is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul HBD563661.

[0100] Also described herein are anti-TREML2 antibodies. In some embodiments, an anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from: (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises two or more of the CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises three or more of the CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises four or more of the CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises five or more of the CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises all of the CDRs of (a)-(f).

[0101] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0102] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a colloidal magnetic particle. In some embodiments, the colloidal magnetic particle is a ferrofluid magnetic particle. In some embodiments, the colloidal magnetic particle has a size of less than 200 nm. In some embodiments, the colloidal magnetic particle has a size of about 80-200 nm. In some embodiments, the colloidal magnetic particle has a size of about 90-150 nm. In some embodiments, the magnetic mass of the colloidal magnetic particle is at least 50%. In some embodiments, the magnetic mass of the colloidal magnetic particle is at least 60%. In some embodiments, the magnetic mass of the colloidal magnetic particle is about 70-90%. In some embodiments, said colloidal magnetic particle comprises a crystalline core of superparamagnetic material surrounded by coating molecules.

[0103] In some embodiments, the magnetic particle is attached to a first exogenous aggregation enhancing factor (EAEF), wherein said first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0104] The present document describes an anti-TREML2 antibody conjugate comprising (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) a magnetic particle, wherein said magnetic particle is conjugated to the anti-TREML2 antibody.

[0105] In some embodiments, said magnetic particle is a colloidal magnetic particle. In some embodiments, said colloidal magnetic particle is a magnetic particle of a ferrofluid. In some embodiments, the size of the colloidal magnetic particle is less than 200 nm. In some embodiments, the size of the colloidal magnetic particle is approximately 80-200 nm. In some embodiments, the size of the colloidal magnetic particle is approximately 90-150 nm. In some embodiments, the magnetic mass of said colloidal magnetic particle is at least 50%. In some embodiments, the magnetic mass of said colloidal magnetic particle is at least 60%. In some embodiments, the magnetic mass of said colloidal magnetic particle is approximately 70-90%. In some embodiments, said colloidal magnetic particle comprises a crystalline core of a superparamagnetic material surrounded by coating molecules.

[0106] In some embodiments, the magnetic particle is attached to a first exogenous aggregation enhancing factor (EAEF), wherein said first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0107] In some embodiments, an anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from: (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of two or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of three or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of four or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of five or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of all of the CDRs of (a)-(f).

[0108] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0109] Additionally described herein are kits for isolating, detecting, and analyzing fetal cells. In some embodiments, the kit comprises, consists of, or consists essentially of (a) an antibody that binds to the trigger receptor analogue 2 protein expressed on myeloid cells (TREML2) (anti-TREML2 antibodies), or an antigen-binding fragment thereof; and (b) a magnetic reagent comprising colloidal magnetic particles.

[IT] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0111] In some embodiments, an anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more complementarity determining regions (CDRs) selected from: (a) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) CDR3 of LCVR comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0112] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label to form a conjugated antibody. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0113] In some embodiments, the size of the colloidal magnetic particles is less than 200 nm. In some embodiments, the colloidal magnetic particles are ferrofluid particles.

[0114] In some embodiments, said colloidal magnetic particles are conjugated to an antibody or antigen-binding fragment thereof.

[0115] In some embodiments, the antibody is an anti-TREML2 antibody. In some embodiments, the anti-TREML2 antibody or antigen binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from: (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 2, 3, 4, 5, or 6 CDRs selected from (i)-(vi).

[0116] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0117] In some embodiments, the kit further comprises, consists of, or consists essentially of an inhibitory agent selected from the group consisting of a reducing agent, an immune complex, a chelating agent, diaminobutane. In some embodiments, the kit further comprises, consists of, or consists essentially of a chelating agent. In some embodiments, the chelating agent is EDTA.

[0118] In some embodiments, the kit further comprises, consists of, or consists essentially of an exogenous aggregation enhancing factor (EAEF). In some embodiments, said EAEF comprises, consists of, or consists essentially of one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0119] Additionally, the present document describes a kit comprising (a) a first antibody capable of binding to a protein expressed on the surface of a cell of a fetus, wherein said first antibody is linked to colloidal magnetic particles; and (b) an anti-TREML2 antibody or antigen-binding fragment thereof.

[0120] In some embodiments, the first antibody binds to a protein selected from EpCAM, CD105, and CD71.

[0121] In some embodiments, said colloidal magnetic particles are ferromagnetic fluid particles.

[0122] In some embodiments, an anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from (a) HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0123] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0124] In some embodiments, the kit further comprises, consists of, or consists essentially of an inhibitory agent selected from the group consisting of a reducing agent, an immune complex, a chelating agent, diaminobutane. In some embodiments, the chelating agent is EDTA.

[0125] In some embodiments, said kit further comprises, consists of, or consists essentially of an exogenous aggregation enhancing factor (EAEF), wherein said first EAEF comprises, consists of, or consists essentially of one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

BRIEF DESCRIPTION OF DRAWINGS

[0126] Fig. 1 shows a typical method for isolating, detecting and analyzing rare cells.

[0127] Fig. 2 shows a typical structure of a ferromagnetic fluid.

[0128] Fig. 3 shows a typical method for detecting rare cells.

[0129] Fig. 4 shows a typical method for isolating and detecting rare cells.

[0130] Figures 5A-E show gating of cells using a FACS instrument.

[0131] Fig. 6 shows a typical method for isolating, detecting and analyzing rare cells.

[0132] Fig. 7 shows a diagram of the aggregation of a ferromagnetic fluid due to controlled aggregation.

[0133] Fig. 8: Schematic of the fetal cell enrichment process: Fetal cells from whole blood of pregnant women are enriched and stained. Pure single cells are isolated using DEPArray™ for whole genome amplification and genomic analysis.

[0134] Fig. 9 Gating strategy for erythroblasts isolated from a fetal blood sample: (1) FSC-A/SSC-A to gate the bulk cell population (2) gating live cells negative for Sytox Green (3) FSC-H/W to exclude double cells (4) gating cells double positive for GPA/Hoechst (5) gating CD71+/CD45- cells (6) gating for TLS1/TREML2 and overlaying with isotype control to determine % TLEML2-positive cells.

[0135] Fig. 10 Gating strategy for erythroblasts isolated from a bone marrow sample: (1) FSC-A/SSC-A to gate the bulk cell population (2) gating live cells negative for Sytox Green (3) FSC-H/W to exclude double cells (4) gating cells double positive for GPA/Hoechst (5) gating CD71+/CD45- cells (6) gating for TLS1/TREML2 and overlaying isotype control to determine % TLEML2-positive cells.

[0136]

[0137] Figures 11A-11J show TLS1/TREML2 expression on fetal erythroblasts isolated from various fetal blood (FB) samples of different clones.

[0138] Fig. 12A-12L show TLS1/TREML2 expression on adult erythroblasts isolated from various bone marrow (BM) samples of different clones.

[0139] Figure 13 shows a scatter plot analysis of TLS1/TREML-2-positive trophoblast cells identified using DEPArrayl™ after addition and enrichment with CD105-FF and EpCAM-FF.

[0140] Figure 14 shows a CellBrowser® image gallery: Trophoblast cells show positive staining with TREML-2-PE antibody, CK-APC, and nuclear staining.

[0141] Figure 15A shows a scatter plot analysis of Draq5/Hoechst-positive erythroblasts spiked into healthy donor blood and enriched with CD71-FF. Figure 15B shows a gallery of CellBrowser® images: erythroblasts show positive staining with CD71-PE antibody, with Draq5 and Hoechst nuclear staining, and negative staining with CD45-FITC antibody.

[0142] Figure 16A shows a scatter plot analysis of Draq5/Hoechst-positive erythroblasts spiked into healthy donor blood and enriched with TLS1/TREML-2-FF. Figure 16B shows a gallery of CellBrowser® images: erythroblasts show positive staining with CD71-PE antibody, with Draq5 and Hoechst nuclei stained, and negative staining with CD45-FITC antibody.

[0143] Figure 17 shows the analysis of CTP in single fetal cells isolated from maternal blood.

[0144] Figure 18 shows the results of CNV analysis in single cells of the fetus.

[0145] Figure 19 shows the results of CNV analysis in single cells of a healthy donor.

[0146] Fig. 20 shows a typical method for isolating and detecting rare cells.

DETAILED DESCRIPTION OF THE INVENTION

[0147] Disclosed herein are compositions, kits, and methods for isolating, detecting, and analyzing rare cells in a sample. In general, the compositions, kits, and methods described herein include agents that bind to the triggering receptor analog 2 protein expressed on myeloid cells (TREML2) (this protein is also referred to herein as TLS1). Alternatively or additionally, the compositions, kits, and methods described herein include antibody conjugates. The antibody conjugates comprise an antibody conjugated to a colloidal magnetic particle. The rare cell may be a fetal cell. The sample may be a sample isolated from a pregnant subject.

[0148] Methods for isolating, detecting and/or characterizing rare cells

[0149] Described herein are methods for isolating, detecting, and/or characterizing rare cells. In some embodiments, the rare cells are fetal cells. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the fetal cells are trophoblast cells. In general, the methods include using an anti-TREML2 antibody or antigen-binding fragment thereof to identify the cell as a fetal cell. Alternatively or additionally, the methods include using an antibody conjugated to a colloidal magnetic particle to isolate the fetal cell.

[0150] The present document describes a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with an anti-TREML2 antibody or antigen-binding fragment thereof, wherein said sample comprises a plurality of cells; and (b) identifying cells bound to the anti-TREML2 antibody as fetal cells.

[0151] Also described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with a first antibody or antigen-binding fragment thereof, wherein said sample comprises a plurality of cells; (b) isolating the cells bound to the first antibody or antigen-binding fragment thereof to obtain an enriched sample; (c) contacting the enriched sample with a second antibody or antigen-binding fragment thereof; and (d) identifying a cell bound to the second antibody as a fetal cell, wherein said first antibody or said second antibody is an antibody that binds to trigger receptor analogue 2 expressed on myeloid cells (TREML2) protein.

[0152] Also described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with an antibody or antigen-binding fragment thereof and a magnetic reagent, wherein said sample comprises a plurality of cells, and the magnetic reagent comprises a magnetic particle conjugated to a first antibody or antigen-binding fragment thereof, wherein said first antibody or antigen-binding fragment thereof binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with an anti-TREML2 antibody or antigen-binding fragment thereof; and (c) identifying a cell bound to the anti-TREML2 antibody as a fetal cell.

[0153] Also described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with a magnetic reagent, wherein said sample comprises a plurality of cells, and the magnetic reagent comprises a colloidal magnetic particle conjugated to a first antibody or antigen-binding fragment thereof, wherein said first antibody or antigen-binding fragment thereof binds to a protein selected from EpCAM, CD 105, and CD71; (b) contacting the sample with a second antibody or antigen-binding fragment thereof; and (c) identifying a cell bound to the second antibody as a fetal cell.

[0154] Also described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with a magnetic reagent and a second exogenous aggregation-enhancing factor (EAEF), wherein said sample comprises a plurality of cells, and wherein the magnetic reagent comprises a colloidal magnetic particle conjugated to a first antibody or antigen-binding fragment thereof, wherein said colloidal magnetic particle is conjugated to EAEF, and the first antibody or antigen-binding fragment thereof binds to a protein selected from EpCAM, CD105, and CD71; (b) contacting the sample with a second antibody or antigen-binding fragment thereof; and (c) identifying a cell bound to the second antibody as a fetal cell. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of a specific binding pair, wherein the specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-Fc antibody and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin and iminobiotin-avidin.

[0155] Also described herein is a method for detecting fetal cells in a sample obtained from a pregnant subject, comprising: (a) contacting the sample with a first antibody conjugate, wherein said sample comprises a plurality of cells, and said first antibody comprises a first antibody or antigen-binding fragment thereof conjugated to a colloidal magnetic particle; (b) isolating cells bound to the first antibody by exposing the sample to a magnetic field, thereby producing an enriched sample; (c) contacting the enriched sample with a second antibody or antigen-binding fragment thereof, wherein said second antibody binds to a marker on the surface of a fetal cell; and (d) identifying a cell bound to the second antibody as a fetal cell.

[0156] Also described herein is a method for obtaining a fetal cell sample from a sample obtained from a pregnant subject, comprising: (a) contacting a sample obtained from the mother's body and comprising fetal cells and maternal cells with a first antibody conjugate, wherein said first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof; and (ii) a colloidal magnetic particle, wherein said first antibody is conjugated to said colloidal magnetic particle, and (b) isolating cells associated with said first antibody conjugate by exposing the sample obtained from the mother's body to a magnetic field, thereby obtaining a fetal cell sample.

[0157] Also described herein is a method for obtaining a fetal cell sample from a sample obtained from a pregnant subject, comprising: (a) contacting a sample obtained from the mother and comprising fetal cells and maternal cells with a conjugate of a first antibody and a second exogenous aggregation-enhancing factor (EAEF), wherein said first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof; (ii) a colloidal magnetic particle, and (iii) a first EAEF, wherein said first antibody is conjugated to said colloidal magnetic particle, and said first EAEF is conjugated to said colloidal magnetic particle, and (b) isolating cells associated with said first antibody conjugate by exposing the sample obtained from the mother to a magnetic field, thereby obtaining a fetal cell sample. In some embodiments, the first EAEF comprises a first member of a specific binding pair, and the second EAEF comprises a second member of a specific binding pair, wherein the specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-Fc antibody, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0158] Also described herein is a method for obtaining a fetal cell sample from a sample obtained from a pregnant subject, comprising: (a) contacting a sample obtained from the mother and comprising fetal cells and maternal cells with a first antibody conjugate, wherein said first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof; and (ii) a colloidal magnetic particle, wherein said first antibody is conjugated to said colloidal magnetic particle and said first antibody is an anti-TREML2 antibody, and (b) isolating cells bound to said first antibody conjugate by exposing the sample obtained from the mother to a magnetic field, thereby obtaining a fetal cell sample.

[0159] Also described herein is a method for obtaining a fetal cell sample from a sample obtained from a pregnant subject, comprising: (a) contacting a sample obtained from the mother and comprising fetal cells and maternal cells with a conjugate of a first antibody and a second exogenous aggregation-enhancing factor (EAEF), wherein said first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof; and (ii) a colloidal magnetic particle, wherein said first antibody is conjugated to said colloidal magnetic particle and said colloidal magnetic particle is conjugated to said first EAEF, and (b) isolating cells associated with said first antibody conjugate by exposing the sample obtained from the mother to a magnetic field, thereby obtaining a fetal cell sample. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of a specific binding pair, wherein the specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-Fc antibody and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin and iminobiotin-avidin.

[0160] In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC). In some embodiments, the fetal cell is an erythroblast. In some embodiments, the fetal cell is a trophoblast cell.

[0161] In some embodiments, any of the methods described herein further comprises isolating cells bound to the anti-TREML2 antibody or the first antibody, wherein the isolating cells occurs prior to identifying the cells.

[0162] In some embodiments, any of the methods described herein includes using a first antibody. 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 magnetic particles are ferrofluid magnetic particles.

[0163] In some embodiments, any of the methods described herein includes isolating cells bound to the first antibody or antigen-binding fragment thereof. In some embodiments, isolating the cells includes applying a magnetic field to the sample.

[0164] In some embodiments, the magnetic particles are attached to a first exogenous aggregation enhancing factor (EAEF), wherein said first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0165] In some embodiments, any of the methods described herein comprises adding a second EAEF to induce aggregation of magnetic particles, wherein said second EAEF comprises another member of a specific binding pair.

[0166] In some embodiments, isolating cells bound to the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of adding a member of a specific binding pair to the enriched sample to eliminate aggregation of magnetic particles in the enriched sample.

[0167] In some embodiments, any of the methods described herein comprises adding to the sample at least one aggregation inhibiting agent selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibiting agent is a chelating agent. In some embodiments, the chelating agent is EDTA. The reducing agent can be mercaptoethanesulfonic acid. The aggregation inhibitor can be bovine serum albumin (BSA).

[0168] In some embodiments, any of the methods described herein utilizes a second antibody. In some embodiments, the second antibody is an antibody that binds to a TREML2 protein, or comprises, consists of, or consists essentially of an antigen-binding fragment that binds to a TREML2 protein.

[0169] In some embodiments, any of the methods described herein includes isolating single fetal cells. In some embodiments, isolating single fetal cells is accomplished by isolating single fetal cells bound to a second antibody.

[0170] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the isolation of single fetal cells is based on immunofluorescence technology. In some embodiments, the isolation of single fetal cells is performed by fluorescence-activated cell sorting (FACS). In some embodiments, the isolation of single fetal cells is performed using a DEPArray.

[0171] In some embodiments, any of the methods described herein includes performing a sequencing-based analysis of one or more nucleic acid molecules isolated from a fetal cell. In some embodiments, the sequencing-based analysis includes a short tandem repeat (STR) analysis.

[0172] In some embodiments, any of the methods described herein includes analyzing a fetal cell. In some embodiments, analyzing a fetal cell includes performing a genomic or genetic analysis. In some embodiments, performing a genetic analysis includes detecting the presence or absence of one or more genetic abnormalities in a fetal cell.

[0173] In some embodiments, said first antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein.

[0174] In some embodiments, an antibody that binds to a TREML2 protein or an antigen-binding fragment that binds to a TREML2 protein comprises 1, 2, 3, 4, 5, or 6 CDRs selected from (a) a complementarity determining region (CDR) 1 of a heavy chain variable region (HCVR) comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) CDR2 of LCVR comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) CDR3 of LCVR comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID Nos: 6-11 independently comprise one or more substitutions, additions, or deletions.

[0175] In some embodiments, the anti-TREML2 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, isolating the cells comprises placing the cells in a magnetic separator. In some embodiments, isolating the cells comprises applying a magnetic field to the sample.

[0176] In some embodiments, the anti-TREML2 antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, isolating the cells comprises flow cytometry. In some embodiments, the flow cytometry is fluorescence-activated cell sorting (FACS).

[0177] In some embodiments, isolating cells includes executing a DEP Array.

[0178] In some embodiments, identifying cells comprises performing a sequencing reaction.

[0179] In some embodiments, the sample is a sample enriched in fetal cells prior to contacting the sample with an anti-TREML2 antibody. In some embodiments, the sample is enriched in fetal cells by contacting the sample with a ferrofluid reagent, wherein the ferrofluid comprises an antibody attached to the ferrofluid.

[0180] In some embodiments, the antibody binds to a protein selected from EpCAM, CD 105, and CD71.

[0181] In some embodiments, the methods described herein further comprise isolating cells bound to the antibody attached to the ferrofluid, thereby producing a sample enriched in fetal cells.

[0182] In some embodiments, any of the methods described herein includes a sequencing-based analysis. In some embodiments, the sequencing-based analysis includes a short tandem repeat (STR) analysis.

[0183] In some embodiments, any of the methods described herein further comprises analyzing a fetal cell. In some embodiments, analyzing a fetal cell comprises detecting the presence or absence of one or more abnormalities. In some embodiments, analyzing a fetal cell comprises performing genomic analysis. In some embodiments, analyzing a fetal cell comprises performing genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in a fetal cell. In some embodiments, performing genetic analysis comprises detecting the presence or absence of a chromosomal aberration in a fetal cell. In some embodiments, the chromosomal aberration is trisomy 21, trisomy 18, or trisomy 13.

[0184] In some embodiments, any of the methods described herein further includes performing genetic testing of the fetal cell. In some embodiments, performing genetic testing of the fetal cell includes detecting the presence or absence of one or more fetal abnormalities. In some embodiments, performing genetic testing of the fetal cell includes performing genomic analysis. In some embodiments, performing genetic testing of the fetal cell includes performing genetic analysis. In some embodiments, performing genetic analysis includes detecting the presence or absence of a chromosomal aberration in the fetal cell. In some embodiments, the chromosomal aberration is trisomy 21, trisomy 18, or trisomy 13.

[0185] In some embodiments, any of the methods described herein further comprises providing treatment recommendations based on the results of the analysis of the fetal cell. In some embodiments, any of the methods described herein further comprises providing treatment recommendations based on the results of the genetic testing of the fetal cell.

[0186] In some embodiments, any of the methods described herein further comprises administering a therapeutic agent to the subject based on the results of the analysis of the fetal cell. In some embodiments, any of the methods described herein further comprises administering a therapeutic agent to the subject based on the results of the genetic testing of the fetal cell.

[0187] In some embodiments, any of the methods described herein further includes recommending additional monitoring of the subject or fetus based on the results of the analysis of the fetal cell. In some embodiments, any of the methods described herein further includes recommending additional monitoring of the subject or fetus based on the genetic testing of the fetal cell.

[0188] Figure 1 depicts an exemplary method for isolating, detecting, and/or analyzing rare cells. The methods described herein may comprise, consist of, or consist essentially of one or more of the steps shown in Figure 1. In some embodiments, the method comprises, consists of, or consists essentially of (a) obtaining a sample comprising a plurality of cells from a subject (101); and (b) isolating the rare cells (110). In some embodiments, the method comprises, consists of, or consists essentially of (a) obtaining a sample comprising a plurality of cells from a subject (101); (b) isolating the rare cells (110); and (c) analyzing the rare cells (120). In some embodiments, the method comprises, consists of, or consists essentially of (a) obtaining a sample comprising a plurality of cells from a subject (101); (b) isolating the rare cells (110); (c) analyzing the rare cells (120); and (d) generating one or more reports based on the analysis of the rare cells (106).

[0189] As shown in Fig. 1, in some embodiments, the method comprises, consists of, or consists essentially of (a) obtaining a sample from a subject comprising a plurality of cells (101); (b) isolating rare cells (110) by (i) extracting non-rare cells from the sample to obtain a rare cell-enriched sample (102); and (ii) isolating rare cells from the rare cell-enriched sample (103); (c) analyzing the rare cells (120) by (i) purifying nucleic acid molecules from the rare cells (104); and (ii) sequencing one or more nucleic acid molecules (105); and (f) generating one or more reports (106). In some embodiments, the rare cells are fetal cells. In some embodiments, enriching for rare cells (102) comprises, consists of, or consists essentially of contacting a sample with a ferrofluid, wherein said ferrofluid comprises an antibody attached to a magnetic particle, and said antibody binds to a marker on rare cells. In some embodiments, the marker on rare cells is any of the markers described herein. In some embodiments, the marker on rare cells is a TREML2 protein. In some embodiments, said antibody is any of the antibodies described herein. In some embodiments, said antibody is an anti-TREML2 antibody. In some embodiments, said antibody is any of the anti-TREML2 antibodies described herein. In some embodiments, the ferrofluid comprises, consists of, or consists essentially of the ferrofluid structure depicted in Fig. 2. In some embodiments, enriching for rare cells (102) further comprises, consists of, or essentially consists of using an external gradient magnetic separator with respect to the sample to remove cells not associated with the ferrofluid. In some embodiments, the method further comprises, consists of, or essentially consists of contacting the rare cells with one or more additional antibodies, wherein the one or more additional antibodies are conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the rare cells are contacted with one or more additional antibodies prior to isolating the rare cells (103). In some embodiments, isolating the rare cells (103) comprises, consists of, or essentially consists of selecting single cells associated with an antibody that binds to a marker on the rare cells. In some embodiments, the antibody is an anti-TREML2 antibody. In some embodiments, the anti-TREML2 antibody is any of the anti-TREML2 antibodies described herein. In some embodiments, an anti-TREML2 antibody comprises (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a CDR3 LCVR comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, isolating rare cells (103) comprises, consists of, or consists essentially of sorting rare cells from an enriched cell sample. In some embodiments, isolating rare cells (103) comprises, consists of, or consists essentially of performing fluorescence activated cell sorting (FACS). In some embodiments, isolating rare cells (103) comprises, consists of, or consists essentially of performing DEPArray. In some embodiments, purifying nucleic acid molecules from rare cells (104) comprises, consists of, or consists essentially of performing nucleic acid amplification. In some embodiments, purifying nucleic acid molecules from rare cells (104) comprises, consists of, or consists essentially of producing a nucleic acid library.

[0190] Figure 3 depicts an exemplary method for detecting rare cells (e.g., fetal cells). In some embodiments, the method comprises, consists of, or consists essentially of (a) contacting a sample (301) comprising a plurality of cells (302, 303) with a first antibody or antigen-binding fragment thereof (304); and (b) identifying the cells (303) bound to the first antibody or antigen-binding fragment thereof (304) as fetal cells. In some embodiments, the first antibody (304) is an antibody that binds to a TREML2 protein. In some embodiments, the antigen-binding fragment (304) binds to a TREML2 protein. In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more of the TREML2 antibodies described herein. In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of (a) a complementarity determining region (CDR) 1 of a heavy chain variable region (HCVR) comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) CDR3 of LCVR comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. The first antibody or antigen-binding fragment thereof can be attached to a magnetic particle. For example, the first antibody or antigen-binding fragment thereof can be in the form of a ferrofluid. Alternatively, the first antibody or antigen-binding fragment thereof can be conjugated to a label. The label can be any of the labels described herein. For example, the first antibody or antigen-binding fragment thereof can be conjugated to a fluorescent label. The cells can be identified by any of the identification techniques described herein. In some embodiments, identifying the cells bound to the first antibody or antigen-binding fragment thereof comprises isolating the cells bound to the first antibody or antigen-binding fragment thereof. Isolating the cells can comprise any of the cell isolation techniques described herein. In some embodiments, isolating the cells comprises magnetic separation. In some embodiments, identifying cells may include using a microscope. Isolating cells may include fluorescence microscopy. In some embodiments, identifying cells includes or is based on FACS. Alternatively or additionally, identifying cells includes or is based on DEP Array.

[0191] Figure 4 depicts an exemplary method for isolating and detecting rare cells. In some embodiments, the method for detecting rare cells comprises, consists of, or essentially consists of (a) contacting a sample (401) comprising a plurality of cells (402, 403) with an antibody conjugate (406), wherein said antibody conjugate (406) comprises a first antibody or antigen-binding fragment (404) attached to a magnetic particle (405); (b) enriching for rare cells (403) by applying a magnetic field to the sample (407) and removing cells (402) not bound to the antibody conjugate, thereby obtaining a sample enriched for rare cells (411); (c) contacting said rare cell enriched sample (411) with an antibody conjugate (410), wherein said antibody conjugate (410) comprises a second antibody or antigen binding fragment (408) conjugated to a label (409); and (d) identifying a cell bound to the antibody conjugate as a rare cell (403). In some embodiments, the rare cell is a fetal cell.

In some embodiments, the fetal cell is a fetal nucleated red blood cell (fhRBC). In some embodiments, the rare cell enriched sample (411) comprises a rare cell (403) bound to an antibody conjugate (406), wherein the antibody conjugate comprises a first antibody (404) or an antigen-binding fragment thereof (404) conjugated to a magnetic particle (405). Alternatively or in addition, the rare cell enriched sample (411) is further processed to detach the rare cells (403) from the antibody conjugate (406). In some embodiments, the first antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the first second antibody or antigen-binding fragment thereof binds to the TREML2 protein. In some embodiments, both said first antibody or antigen-binding fragment and said second antibody or antigen-binding fragment bind to a TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment or (b) the second antibody or antigen-binding fragment binds to a TREML2 protein. In some embodiments, (a) said first antibody or antigen-binding fragment binds to a protein selected from EpCAM, CD105, and CD71, and (b) said second antibody or antigen-binding fragment binds to a TREML2 protein. In some embodiments, (a) said first antibody or antigen-binding fragment binds to EpCAM, and (b) said second antibody or antigen-binding fragment binds to a TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to CD105 and (b) the second antibody or antigen-binding fragment binds to TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to CD71 and (b) the second antibody or antigen-binding fragment binds to TREML2 protein. In some embodiments, the antibody or antigen-binding fragment that binds to TREML2 is any of the TREML2 antibodies or antigen-binding fragments described herein. In some embodiments, the TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, 3, 4, 5, or 6 CDRs selected from (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the second antibody is an antibody that binds the first antibody. For example, if the first antibody is a goat IgG antibody, the second antibody can be a mouse anti-goat IgG antibody. In some embodiments, the label is any of the labels described herein. In some embodiments, the label is a fluorescent label. In some embodiments, the magnetic particle is a colloidal magnetic particle. In some embodiments, the colloidal magnetic particle is a ferrofluid magnetic particle. In some embodiments, the magnetic particle is further conjugated with a first exogenous aggregation-enhancing factor (EAEF). In some embodiments, the method, prior to step (A), further comprises contacting the sample (401) with a second EAEF capable of binding to the first EAEF. In some embodiments, adding the second EAEF induces aggregation of the antibody conjugate (406). In some embodiments, the method further comprises adding a third EAEF capable of binding to the first or second exogenous aggregation-enhancing factor. In some embodiments, adding the third EAEF eliminates aggregation of the first EAEF. In some embodiments, the method, prior to step (a), further comprises adding an aggregation-inhibiting agent to the sample. The cells may be identified by any of the identification techniques described herein. In some embodiments, identifying the cell may include using a microscope. Identifying the cell may include fluorescence microscopy. In some embodiments, identifying the cell includes or is based on FACS. Alternatively or additionally, identifying the cell includes or is based on DEP Array.

[0192] Figure 20 depicts another exemplary method for isolating and detecting rare cells. As shown in Figure 20, in some embodiments, the method for detecting rare cells comprises, consists of, or essentially consists of the step (A1): contacting a sample (2001) comprising a plurality of cells (2002, 2003) with a conjugate of a first antibody (2006) and a second exogenous aggregation enhancing factor (EAEF) (2011), wherein said first antibody conjugate (2006) comprises a first antibody or antigen-binding fragment (2004) attached to a magnetic particle (2005), wherein said magnetic particle (2005) is further conjugated to a first EAEF (2012); and a step (B1) of enriching rare cells (2003) by applying a magnetic field to the sample (2007) and removing cells (2002) not associated with the antibody conjugate (2006)-second EAEF (2011) complex, thereby obtaining a sample (2011) enriched in rare cells. As shown in step (A2), the addition of the second EAEF (2011) induces aggregation of the first antibody conjugate (2006). In some embodiments, the method further comprises adding a third EAEF (2013) to the sample (2011) enriched in rare cells before step (B2). As shown in step (B3), the addition of the third EAEF (2013) eliminates the aggregation of the first antibody conjugate (2006). In some embodiments, the method further comprises the step (C) of contacting the rare cell enriched sample (2011) with a second antibody conjugate (2010), wherein said second antibody conjugate comprises a second antibody or antigen binding fragment (2008) conjugated to a label (2009). In some embodiments, said method further comprises identifying a cell bound to the first antibody conjugate (2006) as a rare cell (2003) before step (D). In some embodiments, said method further comprises identifying a cell bound to the second antibody conjugate (2010) as a rare cell (2003) before step (D). In some embodiments, the rare cell is a fetal cell. In some embodiments, the fetal cell is a fetal nected red blood cell (fnRBC). In some embodiments, the rare cell enriched sample (2011) comprises a rare cell (2003) bound to a first antibody conjugate (2006), wherein said first antibody conjugate comprises a first antibody (2004) or an antigen-binding fragment thereof (2004) conjugated to a magnetic particle (2005), wherein said magnetic particle (2005) is further conjugated to a first EAEF (2012). Alternatively or in addition, the rare cell enriched sample (2011) is further processed to detach the rare cells (2003) from the antibody conjugate (2006). In some embodiments, the first antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the first second antibody or antigen-binding fragment thereof binds to the TREML2 protein. In some embodiments, both said first antibody or antigen-binding fragment and said second antibody or antigen-binding fragment bind to a TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment or (b) the second antibody or antigen-binding fragment binds to a TREML2 protein. In some embodiments, (a) said first antibody or antigen-binding fragment binds to a protein selected from EpCAM, CD105, and CD71, and (b) said second antibody or antigen-binding fragment binds to a TREML2 protein. In some embodiments, (a) said first antibody or antigen-binding fragment binds to EpCAM, and (b) said second antibody or antigen-binding fragment binds to a TREML2 protein. In some embodiments, (a) a first antibody or antigen-binding fragment binds to CD105 and (b) a second antibody or antigen-binding fragment binds to a TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to CD71 and (b) the second antibody or antigen-binding fragment binds to a TREML2 protein. In some embodiments, the antibody or antigen-binding fragment that binds to TREML2 is any of the TREML2 antibodies or antigen-binding fragments described herein. In some embodiments, the TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, 3, 4, 5, or 6 CDRs selected from (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the second antibody is an antibody that binds the first antibody. For example, if the first antibody is a goat IgG antibody, the second antibody can be a mouse anti-goat IgG antibody. In some embodiments, the label is any of the labels described herein. In some embodiments, the label is a fluorescent label. In some embodiments, the magnetic particle is a colloidal magnetic particle. In some embodiments, the colloidal magnetic particle is a ferrofluid magnetic particle. In some embodiments, the method further comprises adding an aggregation inhibiting agent to the sample before step (A1). In some embodiments, the first EAEF (2012) is dethiobiotin. In some embodiments, the second EAEF (2011) is streptavidin. In some embodiments, the third EAEF (2013) is biotin. The cells may be identified by any of the identification techniques described herein. In some embodiments, identifying the cell may comprise using a microscope. Identifying the cell may comprise fluorescence microscopy. In some embodiments, identifying the cell comprises or is based on FACS. Alternatively or additionally, the cell identification comprises or is based on a DEP Array. In some embodiments, the cell identification comprises or is based on an immunoassay.

[0193] Although the methods described herein may reduce the use of an anti-TREML2 antibody or antigen-binding fragment thereof or antibody conjugates comprising an anti-TREML2 antibody, any of these methods can be performed using any agent capable of binding to a TREML2 protein or conjugates comprising an agent capable of binding to a TREML2 protein. Accordingly, the methods described herein are not limited to the use of an anti-TREML2 antibody or antigen-binding fragment thereof or antibody conjugates comprising an anti-TREML2 antibody.

[0194] Cell-based methods for fetal genetic testing

[0195] Identification of a new fetal cell marker, such as TREML-2, allows for the isolation and/or detection of fetal cells, and then analysis of such cells. Accordingly, methods for cell-based fetal genetic testing are described herein. In some embodiments, the methods comprise (a) using an anti-TREML2 antibody to isolate fetal cells from a sample obtained from a pregnant subject; and (b) analyzing one or more nucleic acid molecules from the fetal cells to determine the likelihood of the fetus having one or more genetic abnormalities. Alternatively, the methods comprise isolating fetal cells using any of the methods for isolating or detecting fetal cells described herein; and analyzing one or more nucleic acid molecules from the isolated or detected fetal cells to determine the likelihood of the fetus having one or more genetic abnormalities. In some embodiments, the methods comprise analyzing fetal cells isolated and/or detected by any of the methods described herein. In some embodiments, the methods comprise analyzing fetal cells obtained by any of the methods described herein.

[0196] Described herein is a cell-based method for genetic testing of a fetus, comprising: (a) contacting a sample obtained from a pregnant subject with an anti-TREML2 antibody or antigen-binding fragment thereof, wherein said sample comprises a plurality of cells; (b) isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of said one or more nucleic acid molecules, wherein said report provides a probability of the presence of one or more genetic abnormalities in the fetus.

[0197] Described herein is a cell-based method for genetic testing of a fetus, comprising: (a) contacting a sample obtained from a pregnant subject with a first antibody or antigen-binding fragment thereof, wherein said sample comprises a plurality of cells, said first antibody or antigen-binding fragment is conjugated to a colloidal magnetic particle, and said first antibody or antigen-binding fragment thereof binds to a marker on a cell of the fetus; (b) isolating cells bound to the first antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells bound to the first antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of said one or more nucleic acid molecules, wherein said report provides a probability of the presence of one or more genetic abnormalities in the fetus.

[0198] Disclosed herein is a cell-based method for genetic testing of a fetus, comprising: (a) contacting a sample obtained from a pregnant subject with a first antibody or antigen-binding fragment thereof and a second exogenous aggregation-enhancing factor (EAEF), wherein said sample comprises a plurality of cells, said first antibody or antigen-binding fragment is conjugated to a colloidal magnetic particle, said colloidal magnetic particle is conjugated to a first EAEF, and the first antibody or antigen-binding fragment thereof binds to a marker on a cell of the fetus; (b) isolating the cells bound to the first antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells bound to the first antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of said one or more nucleic acid molecules, wherein said report provides a probability of the presence of one or more genetic abnormalities in the fetus. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of a specific binding pair, wherein said specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0199] In some embodiments, the first antibody is an anti-TREML2 antibody. In some embodiments, the first antibody is an anti-CD71 antibody. In some embodiments, the first antibody is an anti-EpCAM antibody. In some embodiments, the first antibody is an anti-CD105 antibody. In some embodiments, if the first antibody is an anti-ECAM antibody or an anti-CD105 antibody, the method further comprises contacting the isolated cells with a second antibody or antigen-binding fragment thereof, wherein the second antibody binds to a marker on the fetal cells. In some embodiments, the second antibody is an anti-TREML2 antibody. In some embodiments, the second antibody is an anti-CD71 antibody. In some embodiments, the second antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the method further comprises isolating the cells bound to the second antibody. In some embodiments, the method further comprises analyzing nucleic acid molecules of cells bound to the second antibody or antigen-binding fragment thereof.

[0200] In some embodiments, the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof are fetal cells. In some embodiments, the fetal cells are fetal erythroblasts. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the fetal cells are fetal trophoblast cells.

[0201] In some embodiments, analyzing said one or more nucleic acid molecules comprises performing a karyotype analysis. Karyotype analysis can be performed using any technique known in the art.

[0202] In some embodiments, analyzing the one or more nucleic acid molecules comprises performing a sequencing-based analysis. The sequencing-based analysis can be performed using any technique known in the art. In some embodiments, the sequencing-based analysis comprises a short tandem repeat (STR) analysis.

[0203] In some embodiments, analyzing the one or more nucleic acid molecules comprises performing one or more amplification reactions. The nucleic acid amplification can be performed by any technique known in the art. In some embodiments, the nucleic acid amplification is performed by polymerase chain reaction (PCR).

[0204] In some embodiments, the one or more genetic abnormalities are selected from a trisomy, a sex chromosome abnormality, and a structural abnormality. In some embodiments, the genetic abnormality is a trisomy. In some embodiments, the trisomy is selected from trisomy 3, trisomy 4, trisomy 6, trisomy 7, trisomy 8, trisomy 9, trisomy 10, trisomy 11, trisomy 12, trisomy 13, trisomy 16, trisomy 17, trisomy 18, trisomy 20, trisomy 21, and trisomy 22. In some embodiments, the genetic abnormality is a sex chromosome abnormality. In some embodiments, the sex chromosome abnormality is selected from monosomy X, trisomy X, and Klinefelter syndrome. In some embodiments, the genetic abnormality is a structural abnormality. In some embodiments, the structural abnormality is a copy number variation (CNV). In some embodiments, the structural abnormality is a CNV deletion or a CNV duplication.

[0205] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a colloidal magnetic particle.

[0206] In some embodiments, isolation of cells bound to an anti-TREML2 antibody or antigen-binding fragment thereof is accomplished by applying a magnetic field to the sample.

[0207] In some embodiments, the methods described herein, prior to contacting the sample with the anti-TREML2 antibody, further comprise contacting the sample with a first antibody, wherein the first antibody binds to a protein selected from EpCAM, CD105, and CD71. In some embodiments, the methods described herein, prior to contacting the sample with the anti-TREML2 antibody, further comprise isolating cells bound to the first antibody. In some embodiments, the first antibody is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a colloidal magnetic particle. In some embodiments, isolating cells bound to the first antibody is accomplished by exposing the sample to a magnetic field.

[0208] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the isolation of cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is based on immunofluorescence technology. In some embodiments, the isolation of cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is accomplished by fluorescence-activated cell sorting (FACS). In some embodiments, the isolation of cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof is accomplished by DEP Array.

[0209] In some embodiments, the methods described herein further comprise contacting the cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof with a second antibody or antigen-binding fragment thereof. In some embodiments, the second antibody is an anti-TREML2 antibody or antigen-binding fragment thereof. In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the methods described herein further comprise isolating the cells bound to the second antibody or antigen-binding fragment thereof. In some embodiments, isolating the cells bound to the second antibody or antigen-binding fragment thereof is based on immunofluorescence technology. In some embodiments, isolating the cells bound to the second antibody or antigen-binding fragment thereof is accomplished by fluorescence-activated cell sorting (FACS). In some embodiments, isolation of cells bound to the second antibody or antigen-binding fragment thereof is performed using a DEP Array.

[0210] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABFN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661. Alternatively or additionally, the TREML2 antibody or antigen binding fragment thereof comprises, consists of, or essentially consists of 1, 2, 3, 4, 5, or 6 CDRs selected from (a) a complementarity determining region (CDR) 1 of a heavy chain variable region (HCVR) comprising, consisting of, or essentially consisting of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or essentially consisting of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or essentially consisting of the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR CDR1 comprising, consisting of, or essentially consisting of the amino acid sequence of SEQ ID NO: 9; (e) a CDR2 of LCVR comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a CDR3 of LCVR comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID NOS: 6-11 independently comprise one or more amino acid substitutions, additions, or deletions. In some embodiments, any of SEQ ID NOS: 6-11 independently comprise two or more amino acid substitutions, additions, or deletions.

[0211] In some embodiments, any of the methods described herein further comprises providing treatment recommendations based on the results of genetic testing of the fetal cell.

[0212] In some embodiments, any of the methods described herein further comprises administering a therapeutic agent to the subject based on the results of genetic testing of the fetal cell.

[0213] In some embodiments, any of the methods described herein further includes recommending additional monitoring of the subject or fetus based on genetic testing of a cell of the fetus.

[0214] Although the methods described herein may reduce the use of an anti-TREML2 antibody or antigen-binding fragment thereof or antibody conjugates comprising an anti-TREML2 antibody, any of these methods can be performed using any agent capable of binding to a TREML2 protein or conjugates comprising an agent capable of binding to a TREML2 protein. Accordingly, the methods described herein are not limited to the use of an anti-TREML2 antibody or antigen-binding fragment thereof or antibody conjugates comprising an anti-TREML2 antibody.

[0215] Rare cell marker binding agents

[0216] Disclosed herein are agents that bind a rare cell marker. As used herein, a "rare cell marker" is a marker (i.e., a cell surface protein) on a rare cell (e.g., a fetal cell). The rare cell marker may be a cell surface protein that is expressed more highly on a rare cell than another cell type in a sample. The rare cell marker may be a triggering receptor analog 2 expressed on myeloid cells (TREML2) protein. The rare cell marker may be a human TREML2 protein. The human TREML2 protein may have the amino acid sequence of SEQ ID NO: 1. Alternatively, the rare cell marker may be CD71. In some embodiments, the rare cell marker is not CD71.

[0217] As used herein, the terms "TREML2" and "TLS1" refer to the same protein and are used interchangeably. TLS1 and TREML2 refer to the same marker, have identical sequences corresponding to the amino acid sequence of SEQ ID NO: 1, and contain domains and fragments having the amino acid sequences of SEQ ID No: 2-5.

[0218] As used herein, a "rare cell" refers to a cell that is present in a sample obtained from a subject at a concentration of less than 10% of the total cell population, wherein the sample is an unpurified or unenriched sample. In some embodiments, the rare cell is present in the sample at a concentration of less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the total cell population. In some embodiments, the rare cell is present in the sample at a concentration of less than 1% of the total cell population. In some embodiments, the rare cell is a fetal cell and the sample is obtained from a pregnant subject.

[0219] As used herein, the term "raw sample" or "unenriched sample" may be used interchangeably and refer to a sample obtained from a subject that has not been processed to remove or isolate cells from the sample. Alternatively or additionally, a crude sample or unenriched sample refers to a sample obtained from a subject that is not depleted of one or more cells. Alternatively or additionally, a crude sample or unenriched sample refers to a sample obtained from a subject that contains a plurality of different cell types.

[0220] In some embodiments, the rare cell marker binding agent is selected from an antibody, an antibody fragment, a receptor, and a ligand. In some embodiments, the antibody fragment comprises an antigen-binding domain of an antibody. In some embodiments, the antibody fragment is selected from a monovalent antigen-binding fragment (Fab or Fab'), a bivalent antigen-binding fragment ((Fab)2 or (Fab')2), a variable fragment (Fv), a single-chain variable fragment (scFv), a divalent antibody, a triabody, a tetrabody, a minibody, and a bispecific scFv (bis-scFv).

[0221] In general, monovalent Fab fragments contain a single antigen-binding site, while bivalent (Fab)2 fragments contain two antigen-binding regions joined by disulfide bonds. Fab fragments are composed of the variable regions of the heavy (V _H ) and light chains (V _L ) and the constant regions 1 of the heavy ( _CH ¹ ) and light chains ( _CL ¹ ) of the antibody. Fv fragments contain an antigen-binding site composed of the variable regions of the heavy (V _H ) and light chains (V _L ), but do not contain the Fab constant regions ( _CH ¹ and _CL ). V _H and V _L are joined in Fv fragments by non-covalent interactions. Fab can be a dimer (Fab ₂ ) or a trimer (Fab ₃ ), which allows binding of 2 or 3 different antigens, respectively.

[0222] The orientation of the V domains and the linker can vary, resulting in different shapes of Fv molecules. In general, if the linker is at least 12 residues long, scFv fragments are mostly monomeric. Linkers of 3-11 residues in length result in scFv molecules that are unable to fold into a functional Fv domain. These molecules associate with a second scFv molecule, resulting in a bivalent diabody. Triabodies or tetrabodies can be formed with linker lengths of less than three residues. Minibodies are scFv- _CH3 fusion proteins that assemble into bivalent dimers. Bis-scFv fragments consist of scFv fragments with two different variable domains and are capable of simultaneously binding two different epitopes.

[0223] The antibody may be a polyclonal antibody. Alternatively or in addition, the antibody may be a monoclonal antibody. The antibody may be an immunoglobulin gamma (IgG antibody). The IgG antibody may be an IgG1 antibody. The IgG antibody may be an IgG2 antibody. The IgG antibody may be an IgG3 antibody. The IgG antibody may be an IgG4 antibody. The antibody may be an immunoglobulin mu (IgM antibody). The antibody may be an immunoglobulin epsilon (IgE antibody). The antibody may be an immunoglobulin delta (IgD antibody). The antibody may be an immunoglobulin alpha (IgA antibody). The IgGA antibody may be an IgGA1 antibody. Alternatively, the IgG antibody is an IgGA2 antibody

[0224] In some embodiments, the agent is an antibody or antibody fragment that binds to a TREML2 protein. In some embodiments, the antibody or antibody fragment binds to an extracellular domain of a TREML2 protein. In some embodiments, the extracellular domain comprises the amino acid sequence of SEQ ID NO: 2. Alternatively, the antibody or antibody fragment can bind to a fragment of the extracellular domain of TREML2. The fragment of the extracellular domain comprises the amino acid sequence of SEQ ID NO: 3-4. The antibody or antibody fragment can bind to the N-terminal domain of a TREML2 protein.

[0225] In some embodiments, the anti-TREML2 antibody is a polyclonal antibody. The polyclonal antibody may be selected from an anti-TREML2 antibody selected from sc-109096 (Santa Cruz Biotechnology, Inc.), ARP49877 P050 (Aviva Systems Biology), OACA04996 (Aviva Systems Biology), AF3259 (R&D Systems), PA5-47471 (Thermo Fisher), ABIN634968 (Antibodies-online.com), ABIN928294 (Antibodies-online.com), 30-552 (ProSci), ABIN2463297 (antibodies-online.com), ABIN749888 (antibodies-online.com), bs-2737r (Bioss), ABIN1999045 (antibodies-online.com), 11655-rp02 (Sino Biological), ABIN293207 (antibodies-online.com), ABIN2387613 (antibodies-online.com), t8282-40 (USBio), ABIN4249314 (antibodies-online.com) and nbpl-70737-20ul (Novus Biologicals).

[0226] The anti-TREML2 antibody may be a monoclonal antibody. The monoclonal antibody may be selected from MA5-30973 (Thermo Fisher), ABIN19999041 (antibodies-online.com), 11655-r001 (Sino Biological), and BD563661 (Fisher Scientific).

[0227] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, 3, 4, 5, or 6 CDRs selected from (a) a complementarity determining region (CDR) 1 of a heavy chain variable region (HCVR) comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) CDR2 of LCVR comprising, consisting of, or essentially consisting of the amino acid sequence of SEQ ID NO: 10; and (f) CDR3 of LCVR comprising, consisting of, or essentially consisting of the amino acid sequence of SEQ ID NO: 11.

[0228] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, or 3 CDRs selected from (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; and (c) an HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8.

[0229] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, or 3 CDRs selected from (a) a light chain variable region (LCVR) complementarity determining region CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (b) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (c) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11.

[0230] In some embodiments, an anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of (a) a complementarity determining region (CDR) 1 of a heavy chain variable region (HCVR) comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8); (d) a light chain variable region (LCVR CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; and (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) CDR3 LCVR comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11.

[0231] In some embodiments, any of SEQ ID NOS: 6-11 independently comprise one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 6 comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 7 comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 8 comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 9 comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 10 comprises one amino acid substitution, addition, or deletion. In some embodiments, SEQ ID NO: 11 comprises one, two, or three or more amino acid substitutions, additions, or deletions.

[0232] In some embodiments, the anti-TREML2 antibody is conjugated to a label to form a conjugated antibody. In some embodiments, the label is selected from a fluorescent label, a radionuclide, an enzymatic label, a chemiluminescent label, and a hapten. In some embodiments, the detectable label is a hapten. In some embodiments, the hapten is selected from DCC, biotin, nitropyrazole, thiazolesulfonamide, benzofurazan, and 2-hydroxyquinoxaline. In some embodiments, the detectable label is biotin. In some embodiments, the label is a fluorescent molecule. In some embodiments, the fluorescent molecule is selected from a fluorophore, a cyanine dye, and a near infrared (NIR) dye. In some embodiments, the fluorescent molecule is fluorescein. In some embodiments, the fluorescent molecule is fluorescein isothiocyanate (FITC). In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin. In some embodiments, the conjugated antibody is selected from ABIN6070559 (antibodies-online.com), abx307664 (Abbexa polyclonal), ABIN6070561 (antibodies-online.com), abx307665 (Abbexa, polyclonal), ABIN2662892 (antibodies-online.com), bld-351203 (BioLegend), ABIN2662891 (antibodies-online.com), bld-351204 (BioLegend), ABIN2662890 (antibodies-online.com, monoclonal), and bld-351104 (BioLegend).

[0233] Magnetic Particles

[0234] The methods, compositions, and kits described herein may include or use magnetic particles. For example, any of the antibodies (or, more generally, any rare cell marker binding agents) described herein may be conjugated to a magnetic particle. In some embodiments, a rare cell marker binding agent (e.g., TREML2) is conjugated to a magnetic particle. The magnetic particles may be colloidal magnetic particles. The colloidal magnetic particles may be a ferrofluid.

[0235] As used herein, the term "magnetic particle" refers to a particle that can be manipulated using a magnetic field. The magnetic particle comprises a metal. Examples of metals include, but are not limited to, iron, nickel, cobalt, and copper. [0236] As used herein, the term "colloidal magnetic particle" refers to a magnetic particle coated with a non-magnetic material. An example of a non-magnetic particle is bovine serum albumin (BSA).

[0237] As used herein, the term "ferrofluid magnetic particle" refers to a colloidal magnetic particle containing iron.

[0238] In some embodiments, the magnetic particles are submicron in size. In some embodiments, the particle diameter is generally less than about 300 nanometers (nm), 275 nm, 250 nm, 225 nm, 200 nm, 190 nm, 180 nm, 170 nm, 160 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, or 100 nm. In some embodiments, the particle diameter is generally at least 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, or 120 nm or more. In some embodiments, the particle diameter is between about 40 nm and 250 nm, 40 nm and 200 nm, 50 nm and 200 nm, 50 nm and 190 nm, 50 nm and 180 nm, 50 nm and 170 nm, 60 nm and 200 nm, 70 nm and 200 nm, 80 nm and 200 nm, 90 nm and 200 nm, 90 nm and 175 nm, or 90 nm and 150 nm.

[0239] In some embodiments, the magnetic mass of the particle is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% or more. In some embodiments, the magnetic mass of the particle is between about 40% and 95%, 45% and 95%, 50% and 90%, 55% and 90%, 60% and 90%, or 70% and 90%.

[0240] In some embodiments, particles in the range of 90-150 nm and with a magnetic mass of 70 to 90% can be used.

[0241] In some embodiments, the particles are characterized by resistance to gravitational separation from the solution. The particles may be resistant to gravitational separation for an extended period of time. The particles may be resistant to gravity separation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105 or 120 or more minutes. The particles may be resistant to gravity separation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105 or 120 or more hours. The particles can be resistant to gravitational separation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105 or 120 or more days. [0242] In some embodiments, the magnetic particles consist of a crystalline core of a superparamagnetic material surrounded by coating molecules associated with the magnetic core, such as physically adsorbed or covalently attached, which impart stabilizing colloidal properties thereto. The coating material can be applied in an amount effective to prevent non-specific interactions between biological macromolecules present in the sample and the magnetic cores. Such biological macromolecules may include sialic acid residues on the surface of non-specific cells, lectins, glycoproteins and other membrane components. In addition, the coating material can provide the highest possible magnetic mass/nanoparticle ratio. The size of the magnetic crystals that make up the core is small enough and they do not contain a complete magnetic domain. The size of the nanoparticles is such that their Brownian energy exceeds their magnetic moment. As a consequence, the alignment of the north and south poles and the subsequent mutual attraction/repulsion of these colloidal magnetic particles does not seem to occur even in a moderate magnetic field, which contributes to the stability of their solution.

[0243] Magnetic particles can be separated in separators with a significant external magnetic field gradient. These characteristics facilitate sample handling and provide economic advantages over more complex internal gradient columns loaded with ferromagnetic beads or steel wool.

[0244] Magnetic particles can be obtained by modifying the base material as described in EP 0 842 042, which is incorporated herein by reference in its entirety.

[0245] The magnetic particles can be coated with Abs (or more generally, any agents) capable of recognizing differentially expressed proteins corresponding to the most promising candidates identified in Example 1. In some embodiments, the magnetic particles can be coated with an agent that binds to a rare cell marker (e.g., TREML2). The magnetic particles can be coated with any of the antibodies or agents described herein.

[0246] The magnetic particles can be coated by any method known in the art. For example, the magnetic particles can be coated with an antibody described in US 6,365,362 B1, which is incorporated herein by reference in its entirety.

[0247] Figure 2 shows an exemplary structure of a magnetic particle of a ferrofluid. The magnetic particle of a ferrofluid described herein may include, consist of, or consist essentially of the structure of a magnetic particle of a ferrofluid shown in Figure 2. In some embodiments, the magnetic particle of a ferrofluid described herein has the structure of a magnetic particle of a ferrofluid shown in Figure 2. As shown in Figure 2, the exemplary structure of a magnetic particle of a ferrofluid includes, consists of, or consists essentially of an iron atom surrounded by bovine serum albumin (BSA). BSA is attached to streptavidin (SA), which is attached to biotin (BT). BT may be attached to another BSA molecule, which is attached to an exogenous aggregation-enhancing factor (e.g., dethiobiotin (Dt-BT)). The BT may also be linked to an antibody (Y) that binds to a marker on a rare cell. In some embodiments, the rare cell is a fetal cell. In some embodiments, the marker is TREML2. Alternatively, the marker is EpCAM, CD105, or CD71.

[0248] Figure 7 depicts a schematic of the aggregation of magnetic particles by controlled aggregation. As shown in Figure 7, a magnetic particle, such as the ferrofluid magnetic particle depicted in Figure 2, is attached to an exogenous aggregation-enhancing factor (e.g., dethiobiotin (Dt-BT)). The addition of a second EAEF (e.g., streptavidin (SA)) capable of binding to the first EAEF promotes the aggregation of the antibody-magnetic particle conjugates. In some embodiments, the aggregation of the antibody-magnetic particle conjugates is eliminated by adding a third EAEF, wherein the third EAEF is capable of binding to the first EAEF or the second EAEF. In some embodiments, the third EAEF is identical to the first EAEF. Alternatively, the third EAEF is identical to the second EAEF. In another embodiment, the third EAEF is a binding partner of the first EAEF or a second EAEF (e.g., biotin).

[0249] Compositions and sets

[0250] Described herein are compositions and kits comprising any of the anti-TREML2 antibodies described herein or an antigen-binding fragment thereof. The compositions and kits may further comprise one or more components selected from a magnetic reagent, one or more additional antibodies, an aggregation inhibitor, and an aggregation factor.

[0251] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof; and (b) a magnetic reagent.

[0252] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof; and (b) a colloidal magnetic particle.

[0253] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof; and (b) one or more additional antibodies or antigen-binding fragments thereof.

[0254] Also described herein is a kit comprising (a) an anti-TREML2 antibody or antigen-binding fragment thereof; and (b) a second antibody or antigen-binding fragment thereof, wherein said second antibody binds to a protein expressed on the surface of a fetal nucleated red blood cell (fhRBC).

[0255] Also described herein is a kit comprising (a) an anti-TREML2 antibody or antigen-binding fragment thereof; and (b) a second antibody or antigen-binding fragment thereof, wherein said second antibody is conjugated to a label.

[0256] Also described herein is a kit comprising (a) a first anti-TREML2 antibody or antigen-binding fragment thereof, wherein said first anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a magnetic particle; and (b) a second anti-TREML2 antibody or antigen-binding fragment thereof, wherein said second anti-TREML2 antibody is conjugated to a label.

[0257] In some embodiments, the composition or kit comprises an anti-TREML2 antibody or antigen-binding fragment thereof, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region (HCVR) comprising, consisting of, or consisting essentially of (i) a complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (ii) a CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; and (iii) a CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8); and (b) a light chain variable region (LCVR) comprising, consisting of, or consisting essentially of (i) a CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (ii) CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (iii) CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID Nos: 6-11 independently comprise one or more amino acid substitutions, additions, or deletions.

[0258] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof; and (b) a buffer comprising an aggregation inhibitor.

[0259] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof; and (b) an exogenous aggregation-enhancing factor.

[0260] Any of the compositions, kits, or methods described herein may include one or more magnetic reagents. The magnetic reagent may comprise one or more magnetic particles. The magnetic reagent may comprise one ferromagnetic particle, a supraparamagnetic particle. The magnetic reagent may comprise a ferrofluid reagent.

[0261] As used herein, the term "ferrofluid particle" refers to a particle that can be magnetized to have the properties of a permanent magnet.

[0262] The magnetic reagent may comprise a supraparamagnetic particle. As used herein, the term "supraparamagnetic particle" may refer to a particle that is responsive to a magnetic field. A supraparamagnetic particle is a particle that exhibits magnetic properties only when exposed to a magnetic field. In some embodiments, the colloidal magnetic particle is a supraparamagnetic particle.

[0263] In some embodiments, the magnetic reagent comprises a magnetic particle. In some embodiments, the magnetic particle has a size of about 1.5 to about 50 microns, 0.7-1.5 microns, or less than 200 nm. In some embodiments, the magnetic particle has a size of less than 200 nm. In some embodiments, the magnetic reagent comprises a magnetic particle conjugated to an antibody. In some embodiments, the magnetic particle conjugated antibody is an antibody that binds to a protein selected from epithelial cell adhesion molecule (EpCAM) and endoglin (CD105). Alternatively, the magnetic particle conjugated antibody binds to CD147. In a further embodiment, the magnetic particle conjugated antibody binds to CD45. In another embodiment, such an antibody conjugated to magnetic particles binds to a protein expressed on the surface of a fetal cell.

[0264] In some embodiments, the magnetic reagent comprises a ferrofluid reagent. As used herein, the term "ferrofluid reagent" refers to a liquid suspension comprising magnetic particles. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising a magnetic particle conjugated to an anti-TREML2 antibody. Alternatively, the ferrofluid reagent comprises a liquid suspension comprising a magnetic particle conjugated to one or more antibodies described herein. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising a magnetic particle conjugated to an anti-EpCAM antibody. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising a magnetic particle conjugated to an anti-CD105 antibody. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising a magnetic particle conjugated to an antibody that binds to a protein expressed on the surface of a fetal cell. In some embodiments, the ferrofluid reagent comprises a liquid suspension comprising a magnetic particle conjugated to an anti-CD147 antibody.

[0265] In some embodiments, the kits further comprise one or more dye reagents. In some embodiments, one or more dye reagents comprises one or more antibody conjugates. In some embodiments, the antibody conjugate of the one or more antibody conjugates is an antibody conjugated to a label. In some embodiments, the antibody binds a protein selected from CD71, glycophorin A (GPA), and CD45.

[0266] In some embodiments, any of the antibodies described herein (e.g., an anti-TREML2 antibody or one or more additional antibodies) further comprises a label. In some embodiments, the label is conjugated to the antibody. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0267] Any of the kits described herein may include one or more antibodies or fragments thereof. The one or more antibodies may bind to a protein expressed on the surface of a cell of the fetus. Alternatively or additionally, the one or more antibodies may bind to a protein expressed on the surface of a cell of the mother. The one or more antibodies may bind to a protein selected from EpCAM, CD105, CD147, CD15, CD71, GPA, and CD45. The one or more antibodies may bind to a protein selected from CD15, CD71, GPAhCD45.

[0268] Any of the kits described herein may include one or more antibodies or fragments thereof, wherein said one or more antibodies bind to a protein expressed on the surface of a fetal nucleated red blood cell (fnRBC) or trophoblast cells. Said antibody may bind to a protein selected from EpCAM, CD105, CD71, and CD45.

[0269] Any of the kits described herein may include one or more aggregation inhibitors. The kits described herein may include 1, 2, 3, 4, or 5 or more aggregation inhibitors. The aggregation inhibitor may inhibit endogenous aggregation factors of the ferrofluid. In some embodiments, the aggregation inhibitor is selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. The reducing agent may be mercaptoethanesulfonic acid. The aggregation inhibitor may be bovine serum albumin (BSA). The chelating agent may be EDTA.

[0270] The aggregation inhibitor may comprise an antibody or fragment thereof, wherein said antibody is of the same isotype as the anti-TREML2 antibody. Said antibody may be a non-specific antibody. In some embodiments, the antibody is a murine antibody.

[0271] Any of the kits described herein may comprise an anti-TREML2 antibody, wherein said anti-TREML2 antibody may be attached to a ferrofluid. Any of the kits described herein may comprise an anti-TREML2 antibody, wherein said anti-TREML2 antibody is conjugated to a magnetic particle. The magnetic particle may be a colloidal magnetic particle. The magnetic particle may be a ferrofluid magnetic particle.

[0272] Any of the kits described herein may include an exogenous aggregation enhancing factor (EAEF). In some embodiments, the kits described herein include 1, 2, 3, 4, or 5 or more EAEFs. In some embodiments, the magnetic particles described herein are attached to one or more EAEFs. In some embodiments, the EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, antibody protein A-Fc, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0273] In some embodiments, the kits described herein comprise two or more EAEFs. In some embodiments, the first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-Fc antibody, and avidin-biotin, biotin analog-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin, and the second EAEF comprises the other member of the specific binding pair.

[0274] In some embodiments, the kits described herein include a third EAEF. In some embodiments, the third EAEF is identical to the first EAEF. Alternatively, the third EAEF is identical to the second EAEF. In some embodiments, the third EAEF is capable of interacting with the first EAEF. In some embodiments, the third EAEF is capable of interacting with the second EAEF. When a third EAEF is present that is identical to the first or second EAEF or capable of interacting with the first or second EAEF, the addition of the third EAEF results in the elimination of aggregation of magnetic particles.

[0275] In some embodiments, the kits described herein include two or more aggregation inhibiting agents. In some embodiments, the aggregation inhibiting agent is selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibiting agent is a chelating agent. In some embodiments, the chelating agent is EDTA. The reducing agent can be mercaptoethanesulfonic acid. The aggregation inhibitor can be bovine serum albumin (BSA).

[0276] Examples

[0277] Example 1: Identification of new fetal cell markers

[0278] This example describes the identification of new fetal cell markers.

[0279] Isolation of Nucleated Red Blood Cells (nRBC)

[0280] Fetal whole blood (n=5) was obtained through ultrasound-guided procedures from pregnant women (at 10 ⁺ 0 and 15 ⁺⁶ weeks of gestation) undergoing elective surgical termination of pregnancy.

[0281] 20 ml of peripheral blood were collected from pregnant women at delivery (n=2) or before surgical termination of pregnancy (n=1).

[0282] After collection, fetal and maternal blood was diluted with an equal volume of phosphate-buffered saline (PBS) and slowly layered onto a Percoll gradient. Samples were centrifuged at 1800 rpm for 10 minutes at room temperature. The layer containing fetal or adult erythroblasts in interphase was collected and washed twice with PBS.

[0283] For maternal blood, a depletion step for CD45/CD15-positive cells was performed by labeling the cells with anti-CD45 antibody and anti-CD15 antibody beads (Miltenyi Biotec) using an LD Column (Miltenyi Biotec).

[0284] A microfluidic device was also used to remove extraneous red blood cells from maternal blood and enrich it with adult erythroblasts.

[0285] Cell enrichment and sorting by flow cytometry

[0286] To obtain samples for FACS sorting, enriched fetal and maternal blood cells were stained with anti-CD71 antibody (Miltenyi Biotec), anti-GPA antibody (BD Bioscience), anti-CD45 antibody (Miltenyi Biotec), Hoechst (nuclear staining), and Sytox Green dye (live/dead cells) at room temperature for 30 minutes.

[0287] FACS sorting

[0288] Erythroblasts were gated and sorted as shown in Figures 5A-5E. Figure 5B: FSC-H/W gate and double cell exclusion. Figure 5C: Sytox Green-negative live cell gate. Figure 5D: GPA/Hoechst double-positive gate. Figure 5E: CD71+/CD45- cells gate.

[0289] For fetal blood samples, less than 200,000 target erythroblasts were sorted.

[0290] For maternal blood samples, the maternal erythroblast count never exceeded 1000.

[0291] Isolation of RNA from a sorted population

[0292] Treated sorted cells were used to isolate total RNA.

[0293] Total RNA was isolated from sorted cells using the Picopure RNA Isolation Kit (Applied Biosystems), quantified using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher), and analyzed for quality using the RNA 6000 Pico kit on an Agilent 2100 Bio analyzer.

[0294] Preparation for RNA sequencing and library preparation

[0295] The cDNA library was generated by Illumina sequencing (Appendix A; RNA-sequencing protocol). Sequencing was performed on a HiSeq 2000 at 20 million reads/sample.

[0296] Sequencing

[0297] Reads obtained from next-generation sequencing (Illumina) were first quality checked using standard procedures using the FastQC protocol, then aligned to the reference genome and quantified using STAR version 2.5 software. The resulting read count matrices (i.e., read count tables for all detected features - coding or non-coding RNAs in each sample) were condensed in a data reduction step, retaining only genes with at least a single value in a single sample.

[0298] Data Analysis Using Bioinformatics Tools

[0299] The obtained data were further processed using the DESeq2 R/Biocondutor package for differential expression in order to find genes whose expression was significantly increased or decreased in the samples of the two types being compared.

[0300] The default DESeq2 differential expression analysis consisted of the following steps: estimating the size factors in each sample using the "median ratio method" (Anders and Huber, 2010), finding the estimated variance for each gene using a fitting procedure that optimizes the variance of data fitting a negative binomial distribution, and finally using the estimated size and variance factors to perform significance analysis of the fitted distribution coefficients.

[0301] From the table obtained from DESeq2 analysis, baseline sample means, log2-fold changes, standard errors, statistical tests, p-values, and adjusted p-values were finally extracted for 20,205 features (genes) with non-zero total read counts. Differentially expressed genes were filtered according to the adjusted p-value (Benjamini-Hochberg/FDR method) with a cutoff of 0.01 and a 2-fold change threshold. With these parameters, 3,233 genes were selected as differentially expressed, with the majority of them (2,961) upregulated (regardless of the fold change threshold) in fetal blood.

[0302] The resulting gene table was supplemented with annotations and functional descriptions extracted from the Ensembl database. Genes associated with the annotation "plasma membrane" according to the gene ontology term GO:0005886 were flagged and additionally tagged "transmembrane" using data from the Uniprot database. Among them, 366 genes were differentially expressed, and the expression of most of them (336) was increased (regardless of the threshold fold change) in fetal cells.

[0303] In parallel with the differential expression analysis, read counts were normalized and transformed using DESeq2 to select for more stringent expression criteria: The first selection was performed starting from the list of genes expressed exclusively in the fetal samples, i.e. genes with zero read counts in all three tissue samples (12187 genes, "ALL DATA" list), according to the following criteria: 1) selection of genes found (i.e. expressed) in all fetal samples; 2) selection of genes with a mean/SD expression ratio greater than 1; 3) selection of genes associated with both the GO tag "plasma membrane" and the tag "predicted transmembrane" according to the Uniprot database. See the selection scheme below. The resulting 77 genes were then ranked by descending mean expression in the fetus ("RANKED" list). Final selection with manual curation was performed taking into account known biological function, stability of expression level across samples and rough estimate of absolute expression level (comparison of reads with gene length), antibody availability and other biological considerations (16 genes, “Selection” list).

[0304] Selection scheme

[0305] The following is the selection scheme used to identify potential new fetal cell markers:

[0306] 1) genes NOT expressed in any maternal sample: 12187

[0307] 2) genes expressed in ALL fetal samples, and only in them: 2079

[0308] 2.1) genes annotated as associated with the GO term "Plasma membrane": 213

[0309] 2.2) genes predicted to be trans membrane based on UniProt: 305

[0310] 3) genes associated with the GO term "Plasma membrane" and predicted to be trans membrane: 89

[0311] 4) genes with a mean/SD ratio greater than 1:77

[0312] Ranking of differentially expressed genes from a gene list

[0313] Further final manual curated selection and ranking procedure was performed taking into account transcript length, read count and other biologically relevant criteria, yielding the following top-scoring candidates:

[0314] Detection of antibodies specific to selected target molecules

[0315] Testing Am candidates against erythroblasts by FACS analysis

[0316] To determine the effect of differential expression of RNA levels on the levels of corresponding proteins, immunostaining with commercially available antibodies (n=13) was performed for flow cytometry and DEPArray analysis.

[0317] Isotypically similar Abs conjugated to the same fluorochromes and present at the same concentrations as commercially available antibodies were used as negative controls.

[0318] All 13 antibodies shown in Table 2 were first tested on frozen fetal blood.

[0319] Only antibodies positively expressed on fetal erythroblasts were tested on maternal frozen blood samples.

[0320] In addition to the specific antibodies to be tested or isotype controls, antibodies used for staining included anti-CD71 Ab (Miltenyi Biotec), anti-GPA Ab (BD Bioscience), anti-CD45 Ab (Miltenyi Biotec), Hoechst, for detection of erythroblasts. Briefly, cells (2.5 - 5 x 10 ⁵ ) were incubated with Ab in the presence of FcR blocking reagent (Miltenyi Biotec) at room temperature for 30 min. After washing off unbound Ab, the cell pellet was resuspended with AutoMACS electrode buffer (Miltenyi) containing Sytox Green dye.

[0321] Table 2: FACS analysis results

[0322] Anti-TLT2 Ab 1 was used for TREML2 (this protein is also referred to herein as TLS-1)

[0323] Example 2: Ferrofluid-based technology for cell capture and selection

[0324] In this example, selected antibodies against markers expressed exclusively on fetal cells were used to conjugate with a ferrofluid. TREML2-FF (also called TLS1-FF) refers to an antibody capable of binding a protein expressed by the TLS1 gene and conjugated with a ferrofluid.

[0325] FF-Ab size was verified using a NanoBrook Zeta Plus particle size analyzer and concentration was verified using a spectrophotometer.

[0326] Controlled enrichment

[0327] The blood sample is pre-incubated with a buffer containing one or more inhibitors to inhibit endogenous ferrofluid aggregation factors (described in EP 1311820, incorporated herein by reference in its entirety) prior to adding the ferrofluid to the blood. One of the inhibitors may be a reducing agent, such as mercaptoethanesulfonic acid, at a concentration of 100 mM, which can inhibit IgM-induced aggregation without affecting the ligands used to label the cells. The reducing agent may be added to the blood as the only reagent. The second inhibitor may be bovine serum albumin, which may be included in the buffer at a concentration of 10 mg/mL, and will neutralize any HABAA. The third inhibitor may be a non-specific mouse antibody, in particular of an appropriate isotype that matches the isotype of the antibody in the ferrofluid. It may be included in the buffer at a concentration of 0.5-5 mg/ml to neutralize even the most potent HAMAs. The fourth inhibitor may be streptavidin, which should be included in the buffer if necessary to neutralize any anti-streptavidin antibody present in the plasma. Pretreatment of the blood with the above buffer and reducing agent may take from 15 to 30 minutes to neutralize all endogenous aggregation factors. After neutralization of all endogenous aggregation factors, an exogenous ferrofluid aggregation factor was added to the sample, followed by the ferrofluid. The ferrofluid was coupled to an antibody specific for the targets, as well as to another ligand specific for the exogenous aggregation factor. After optimal labeling of target cells with ferrofluid and induction of ferrofluid aggregation with an exogenous aggregation factor, the sample was subjected to magnetic separation to enrich the targets.

[0328] The sample was placed in a magnetic separator (Immunicon, catalog #QS-012) for 10 minutes. The sample was removed from the magnet, vortexed, and placed back in the magnetic separator for 10 minutes to collect the magnetically labeled cells. The uncollected sample was aspirated, and the cells collected by the magnet were resuspended in 0.75 ml wash-dilution buffer and re-separated in the magnetic separator for 10 minutes. The uncollected sample was discarded, and the collected cells were resuspended after removing the tube from the magnetic separator. After removal of all non-specific components, the magnetically labeled targets and free ferrofluid were resuspended in buffer. To be on the safe side, exogenously mediated ferrofluid aggregation was eliminated. This can be accomplished by resuspending the prepared sample in a buffer containing a disaggregation factor that binds to an exogenous aggregation factor. The disaggregation factor caused disaggregation of all ferrofluid aggregates, leaving the cells for further analysis.

[0329] Example 3: Detection and Analysis of Fetal Cells

[0330] This example describes the isolation and analysis of single fetal cells. DEPArray can be performed as described in EP 0842042, which is incorporated herein by reference in its entirety.

[0331] Pregnant women and healthy volunteers

[0332] Peripheral blood samples from 14 pregnant women at 12–17+2 weeks of gestation were collected via venipuncture into 10 mL CellSave Preservative Tubes (Menarini Silicon Biosystems, Huntington Valley, PA, CUIA). For enrichment experiments, peripheral blood was collected from healthy donors. All donors provided written informed consent, and the study protocol was approved by the Medical Ethics Committee of the San Gherardo Hospital, Monza, Italy. All samples were processed within 1–4 days.

[0333] Antibodies against epithelial cell adhesion antigen (EpCAM), vascular endothelial marker (CD105), and/or TREML 2 attached to a ferrofluid were used to enrich for fetal trophoblast cells relative to non-human cells. Enriched cells were labeled with a phycoerythrin (PE)-labeled monoclonal antibody (mAb) against TREML2. Enriched cells were also labeled with a fluorescent tag, allophycocyanin (APC)-labeled anti-cytokeratin C11 mAb, APC-labeled anti-HLA-G mAb, and fluorescein isothiocyanate (FITC)-labeled anti-CD45 mAb to recognize leukocytes.

[0334] These target cell enrichment procedures (e.g., trophoblast cells) were necessary because the frequency of such cells in maternal blood is known to be extremely low, amounting to only 1-10 cells in 1 ml of whole blood (which contains over a billion cells).

[0335] CVS and cord blood for enrichment

[0336] Fetal trophoblast cells obtained by chorionic villus sampling (CVS) or fetal erythroblasts obtained from umbilical cord blood were added to whole blood of healthy volunteers.

[0337] CVS culture was selected for CD105/EpCAM expression. Cells were grown at 37°C and 5% _CO2 in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Gibco), and L-glutamine (Gibco). Before addition, cells were detached from the flask, resuspended in 10 ml PBS (Gibco), and placed in a CellSave Preservative Tube for at least 1 day.

[0338] Cord blood samples obtained at San Gerardo Hospital were collected in a CellSave Preservative Tube.

[0339] Spike experiments were performed to demonstrate the specificity of the selection procedure using ferrofluid-conjugated antibodies to capture fetal cells.

[0340] Fetal blood and bone marrow samples

[0341] Fetal whole blood (n=3) was obtained through ultrasound-guided procedures from pregnant women (at 10 ⁺ 0 and 15 ⁺⁶ weeks of gestation) undergoing elective surgical termination of pregnancy.

[0342] All donors provided written informed consent and the study protocol was approved by the Medical Ethics Committee of KK Women's and Children's Hospital, Singapore.

[0343] After collection, fetal blood was diluted with an equal volume of PBS and slowly layered onto a Percoll gradient. Samples were centrifuged at 1800 rpm for 10 min at room temperature. The layer containing fetal erythroblasts in interphase was collected and washed twice with PBS.

[0344] Cryo-frozen adult erythroblast-containing bone marrow mononuclear cells were purchased (Lonza, Cat#2M-125C). Cells were thawed, treated with DNase I, and washed according to the manufacturer's instructions. Cells were then maintained in RPMI supplemented with 10% FBS, penicillin-streptomycin, and L-glutamine at 37°C for 1 hour and used as a negative control (3 different donors were tested). [0345] Four clones of commercially available anti-TREML2 antibodies were tested in samples by flow cytometry.

[0346] Isotype-matched Abs conjugated to the same fluorochromes as commercially available anti-TREML2 antibodies were used at the same concentrations. In addition to anti-TREML2 Ab or isotype control, Abs used for staining included anti-CD71 Ab (Miltenyi Biotec), anti-GPA Ab (BD Bioscience), anti-CD45 Ab (Miltenyi Biotec), Hoechst. Briefly, cells (2.5-5×10 ⁵ ) were incubated with Abs in the presence of FcR blocking reagents (Miltenyi Biotec) at room temperature for 30 min. After washing away unbound Abs, the cell pellet was resuspended with Sytox Green electrode buffer for gating of live cells for FACS analysis.

[0347] Preparation of antibodies with dethiobiotin and ferrofluid for controlled aggregation

[0348] In some embodiments, the ferrofluids in the practice of the present invention are particles that behave as colloids. Such particles are characterized by a submicron size, generally less than 200 nanometers (nm), and resistance to gravitational separation from solution over an extended period of time. Particles in the range of 90-150 nm and with a magnetic mass of 70 to 90% were used. Suitable magnetic particles consist of a crystalline core of a superparamagnetic material surrounded by coating molecules associated with the magnetic core, such as physically adsorbed or covalently attached, which impart stabilizing colloidal properties thereto. The coating material should preferably be applied in an amount effective to prevent non-specific interactions between biological macromolecules present in the sample and the magnetic cores. Such biological macromolecules may include sialic acid residues on the surface of non-specific cells, lectins, glycoproteins and other membrane components. In addition, the coating material should provide the highest possible magnetic mass/nanoparticle ratio. The size of the magnetic crystals constituting the core is quite small and they do not contain a complete magnetic domain. The size of the nanoparticles is such that their Brownian energy exceeds their magnetic moment. As a consequence, the alignment of the north and south poles and the subsequent mutual attraction/repulsion of these colloidal magnetic particles does not seem to occur even in a moderate magnetic field, which contributes to the stability of their solution. Finally, magnetic particles should be separated in separators with a significant gradient of the external magnetic field. These characteristics facilitate sample handling and provide economic advantages compared to more complex internal gradient columns loaded with ferromagnetic beads or steel wool. Magnetic particles with the above-described properties can be obtained by modifying the base materials described in EP 842 042. In a preferred embodiment of the present invention, magnetic particles coated with an antibody against CD105 were prepared as described in US 6,365,362 B1, which is incorporated herein by reference in its entirety.

[0349] Recombinant human antibody against the CD105 antigen was prepared from hybridoma cell number 166,707 (R&D Systems) and conjugated to the support material using standard coupling chemistry as described in U.S. Patent Publication No. 09/248,388. The anti-CD105 Ab-ferrofluid was then resuspended in 20 mM HEPES, pH 7.5 for conjugation to dethiobiotin using N-hydroxysuccinimide-DL-dethiobiotin (NHS-dethiobiotin) (Sigma, Cat. No. H-2134). NHS-dethiobiotin stock solution was prepared in DMSO at a concentration of 1 mg/mL. NHS-dethiobiotin (5 mg) was added to 1 mg of anti-CD105 Ab-ferrofluid and incubated at room temperature for 2 hours. Unreacted NHS dithiobiotin was removed by washing three times with 20 mM HEPES, pH 7.5, containing 1 mg/mL BSA, 0.05% Proclin 300 using a high field gradient magnet. After the final wash, dithiobiotin/anti-CD105 Ab-ferrofluid was resuspended in water/BSA/Proclin 300 and filtered through a 0.2 μm syringe filter. The iron concentration of anti-CD105 Ab-ferrofluid was determined by spectrophotometric analysis and adjusted to 0.22 mg/mL. Particle size determination was performed using a NanoBrook 90Plus Particle Size Analyzer (Brookhaven Instruments Corporation).

[0350] Anti-CD71, anti-TREML2, and anti-EpCAM antibodies were conjugated to the ferrofluid using a similar method.

[0351] Blood Processing

[0352] Blood aliquots of 7.5 ml were diluted with 6.5 ml dilution buffer (Menarini Silicon Biosystems).

[0353] The blood sample (7.5 mL aliquots) was pre-incubated with 6.5 mL of dilution buffer (Menarini Silicon Biosystems) containing one or more inhibitors for inhibiting endogenous ferrofluid aggregation factors (described in EP 1 311 820, incorporated herein by reference in its entirety) prior to adding the ferrofluid to the blood. One of the inhibitors may be a reducing agent, such as mercaptoethanesulfonic acid, at a concentration of 100 mM, which can inhibit IgM-induced aggregation without affecting the ligands used to label the cells. The reducing agent may be added to the blood as the only reagent. The second inhibitor may be bovine serum albumin, which may be included in the buffer at a concentration of 10 mg/mL, and will neutralize any HABAA. The third inhibitor may be a non-specific mouse antibody, particularly of the appropriate isotype to match the antibody in the ferrofluid. It may be included in the buffer at a concentration of 0.5-5 mg/ml to neutralize even the most potent HAMAs. The fourth inhibitor may be streptavidin, which should be included in the buffer if necessary to neutralize any anti-streptavidin antibody present in the plasma. Pretreatment of the blood with the above buffer and reducing agent may take 15 to 30 minutes to neutralize all endogenous aggregation factors. During this incubation, the diluted blood was centrifuged at 800 g for 10 min without braking at room temperature to remove plasma.

[0354] After neutralization of all endogenous aggregation factors, an exogenous ferrofluid aggregation factor (streptavidin) was added to the sample, followed by the ferrofluid. The ferrofluid was coupled to an antibody specific for the targets, as well as to another ligand specific for the exogenous aggregation factor, such as dethiobiotin (dethiobiotin-streptavidin binding pair).

[0355] Anti-CD105 antibody-ferrofluid, anti-EpCAM antibody-ferrofluid, and/or anti-TREML2 antibody-ferrofluid were used to enrich for fetal trophoblast cells. Anti-CD71 antibody-ferrofluid and/or anti-TREML2 antibody-ferrofluid were used to enrich for fetal erythroblasts. After optimal labeling of target cells with ferrofluid and induction of ferrofluid aggregation with an exogenous aggregation factor, the sample was subjected to magnetic separation to enrich for targets.

[0356] The sample was placed in a magnetic separator (Immunicon, catalog #QS-012) for 10 minutes. The sample was removed from the magnet, vortexed, and placed back in the magnetic separator for an additional 10 minutes. The sample was removed from the magnet, vortexed again, and placed back in the magnetic separator for an additional 20 minutes to collect the magnetically labeled cells. The uncollected sample was aspirated, and the cells collected by the magnet were resuspended in 3 ml of wash-dilution buffer and re-separated in the magnetic separator for 10 minutes. The uncollected sample was discarded, and the collected cells were resuspended after removing the tube from the magnetic separator. After removal of all non-specific components, the magnetically labeled targets and free ferrofluids were resuspended in buffer. In some cases, ferrofluid aggregation mediated by an exogenous factor was eliminated. The elimination of aggregation can be accomplished by resuspending the prepared sample in a buffer containing a disaggregation factor that binds to the exogenous aggregation factor (in the case of the binding pair dethiobiotin-streptavidin, the exogenous agent that eliminates aggregation can be biotin). Regardless of theoretical concepts, the disaggregation factor caused disaggregation of all ferrofluid aggregates, leaving the cells for further analysis.

[0357] For trophoblast cells, enriched cells were labeled with a fluorescent label - a phycoerythrin (PE)-labeled anti-TREML2 monoclonal antibody (mAb). For trophoblast cells, enriched cells were also labeled with a fluorescent label - a nucleic acid stainer (Hoechst 33342) for DNA staining, allophycocyanin (APC)-labeled anti-cytokeratin C11 mAb, APC-labeled anti-HLA-G mAb, and/or fluorescein isothiocyanate (FITC)-labeled anti-CD45 mAb for leukocyte recognition.

[0358] For erythroblasts, enriched cells were labeled with a fluorescent label - a phycoerythrin (PE)-labeled anti-TREML2 monoclonal antibody (mAb). For erythroblasts, enriched cells were also labeled with a fluorescent label - a nucleic acid dye (Hoechst 33342) for DNA staining, a phycoerythrin (PE)-labeled anti-CD71 monoclonal antibody (mAb), and/or a fluorescein isothiocyanate (FITC)-labeled anti-CD45 mAb.

[0359] Stained cells were fixed with 2% paraformaldehyde (PFA) for 20 min at room temperature, then washed and resuspended in the appropriate volume of buffer for DEPArray™ NxT System (Menarini Silicon Biosystems) or FACS analysis.

[0360] DEPArray Analysis

[0361] DEPArray™ NxT is a solid-state technology for the precise isolation of pure single cells. It consists of a DEPArray™ control unit and a disposable cartridge that utilizes advanced microfluidic and silicon biochip technologies to precisely manipulate each single target cell in an enriched sample.

[0362] The phenomenon that allows manipulation of cells within the chip is called "dielectrophoresis" and is based on the ability to polarize a particle in a liquid suspension medium through the action of electric fields. The polarization creates a force field that can be used to trap each individual particle in a series of potential wells, allowing the positioning of the particles to be controlled. Each potential well can be controlled by changing the chip's program to move one or more particles from an initial position to a final destination for their isolation.

[0363] The DEP Array™ enables the selection and isolation of rare cells with extremely high resolution (down to single cells) and extremely high purity, with cell selection occurring through multiparametric analysis of fluorescent signals and morphological characteristics obtained from brightfield or fluorescence imaging.

[0364] This technology has already been used to isolate and select single circulating tumor cells from the blood of patients with tumors (as described in EP 1311820, incorporated herein by reference in its entirety).

[0365] Whole blood samples from healthy volunteers were spiked with chorionic villus culture containing trisomy 21 fetal trophoblast cells. Samples were enriched and stained as described previously.

[0366] Trophoblast cells were analyzed on the DEPArray™ NxT System. Trophoblast cells exhibited positive TREML2 staining. Additionally, cells exhibiting positive total cytokeratin (CK) staining, undetectable CD45 labeling, and positive nuclear staining were classified as fetal trophoblast cells and isolated as single cells.

[0367] Umbilical cord blood containing fetal erythroblasts pre-labeled with Draq5 nuclear dye was added to whole blood samples from healthy volunteers. Samples were enriched with anti-CD71-ferrofluid and anti-TREML2-ferrofluid and stained as described previously. Enriched erythroblasts were analyzed on the DEPArray™ NxT System. Cells exhibiting positive CD71 staining, undetectable CD45 labeling, and positive Hoechst/Draq5 nuclear staining were classified as fetal erythroblasts.

[0368] Demonstration of fetal cell origin by short tandem repeat (STR) analysis

[0369] Isolated cells were lysed using the DEPArray™ LysePrep kit (MSB, Italy) according to the manufacturer's instructions.

[0370] Single-cell DNA was amplified by PCR using the PowerPlex Fusion 6c Human DNA Amplification Kit (Promega TMD045), which consisted of a multiplex primer set targeting 27 loci of the human genome.

[0371] Genomic DNA was also isolated from 200 μl of maternal whole blood using the QIAgen DSP Blood Mini Kit (QIAgen) to serve as a control. When available, fetal genomic DNA obtained directly from CVS tissue or culture or amniotic fluid was also analyzed.

[0372] The CTP analysis was performed according to the manufacturer's recommendations, fragment analysis was performed on a ThermoFisher Scientific 3500 Genetic Analyzer (POP-4 and 36-cm capillary array); subsequent software analysis was performed using GeneMapper® ID-X version 1.4. The allele pattern in isolated single cells was then compared with the fetal and parental genomic DNA pattern to assess allele dropout and expected inheritance patterns.

[0373] ROS: Clinical study involving 20 pregnant women in the first trimester of pregnancy

[0374] Twenty-mL peripheral blood samples from 14 pregnant women at 12–17+2 weeks of gestation were collected via venipuncture into 10-mL CellSave Preservative Tubes (Menarini Silicon Biosystems, Huntington Valley, PA, CIIIA). All samples were processed after 1–4 days. Fetal trophoblast cells were successfully isolated from 14 pregnant women (Table X). An average of 1.4 fetal trophoblast cells were isolated from the 14 positive pregnant women.

[0375] Copy number variant (CNV) analysis

[0376] Whole blood samples from healthy volunteers were spiked with chorionic villus culture containing fetal trophoblast cells. Samples were enriched and stained as described previously.

[0377] Whole genome amplification (Amplil WGA, Menarini Silicon Biosystems) of single fetal trophoblast cells isolated using DEPArray™ NxT was performed.

[0378] 5 µl of Ampli1™ WGA product was purified using 1.8X SPRIselect Beads (Beckman Coulter) according to the manufacturer's instructions and eluted in 12.5 µl of TE library preparation buffer using the Ampl1™ Low pass kit (Menarini Silicon Biosystems).

[0379] FASTQ files generated using 13 Ampli1™ LowPass libraries were aligned to the hgl9 reference genome using BWA. Copy number profiles were calculated using Control-FREEC, without controls and with GC normalization. Copy number plots were generated using custom Python scripts. [0380] Results

[0381] Figure 8 shows a schematic of the fetal cell enrichment workflow. As shown in Figure 8, the workflow consists of 3 distinct steps: 1. Sample collection and target cell capture using magnetic fluid-conjugated antibodies that allowed for specific selection of target cells. 2. Target cells were labeled with the selected antibodies and loaded onto a DEPArray cartridge for screening and selection. The selected single cells were then sorted using the DEPArray instrument. 3. Sorted single cells were analyzed using short tandem repeat (STR) analysis technologies to demonstrate their fetal cell origin.

[0382] In this example, fetal cells were enriched and stained from whole blood of pregnant women. Pure single cells were isolated using DEPArray™ for whole genome amplification and genomic analysis.

[0383] Figures 9-10 demonstrate the specificity of the novel anti-TREML2 antibody by flow cytometric analysis of TREML2 (i.e., TLS) expression on erythroblasts isolated from fetal blood (FB) (Figure 9) and bone marrow (BM) (Figure 10) samples. As shown in Figures 9-10, erythroblasts isolated from fetal blood or bone marrow samples were gated with (1) FSC-A/SSC-A to gate the bulk cell population, (2) gate live cells negative for Sytox Green, (3) FSC-H/W to exclude double cells, (4) gate GPA/Hoechst double positive cells, (5) gate CD71+/CD45- cells, and (6) gate TLS1 and overlay isotype control to determine % TREML2 positive cells.

[0384] Figures 11A-11J show TLS1 expression on fetal erythroblasts isolated from various fetal blood (FB) samples of different clones. Figures 12A-12L show TLS1 expression on erythroblasts isolated from bone marrow (BM) samples of different clones. As shown in Figures 11A-11J, fetal erythroblasts from fetal blood showed positive staining with an anti-TREML2 antibody, while no expression was detected for adult erythroblasts isolated from bone marrow (Figures 12A-12L).

[0385] Table 3. Measurement of the size of the ferrofluid. The average diameter (nm) of CD105-FF was 128.70 nm.

[0386] Table 4: Measurement of the size of the ferrofluid. The average diameter (nm) of TREML-2-FF was 189.57. Both sizes were in the range characteristic of colloidal particles.

[0387] Trophoblast cells derived from CVS cultures were used to demonstrate the specificity of CD105-FF uptake and enrichment by EpCAM-FF.

[0388] Figure 13 shows a scatter plot analysis of TREML-2-positive trophoblast cells identified using DEPArray™ after addition and enrichment with CD105-FF and EpCAM-FF. Figure 14 shows a gallery of CellBrowser® images: Trophoblast cells showed positive staining with TREML-2-PE antibody, CK-APC, and nuclear staining.

[0389] Cord blood-derived trophoblast cells were used to demonstrate the specificity of CD71 or TREML-2 uptake and enrichment.

[0390] Figure 15A shows a scatter plot analysis of Draq5/Hoechst-positive erythroblasts spiked into healthy donor blood and enriched with CD71-FF. Figure 15B shows a gallery of CellBrowser® images: erythroblasts show positive staining with CD71-PE antibody, with Draq5 and Hoechst nuclear staining, and negative staining with CD45-FITC antibody.

[0391] Figure 16A shows a scatter plot analysis of Draq5/Hoechst-positive erythroblasts spiked into healthy donor blood and enriched using TREML-2-FF. Figure 16B shows a gallery of CellBrowser® images: erythroblasts show positive staining with CD71-PE antibody, with Draq5 and Hoechst nuclei stained, and negative staining with CD45-FITC antibody.

[0392] Sorted single cells were analyzed using short tandem repeat (STR) analysis technology to demonstrate their fetal origin (compared to maternal DNA as well as fetal LNC analysis obtained by amniocentesis). Identical locus profiles were found. Figure 17 shows STR analysis in fetal single cells.

[0393] A clinical trial was conducted in which 14 pregnant women were enrolled at various stages of pregnancy; fetal cells obtained from blood samples from these pregnant women were detected by positive CTP assay results.

[0394] Table 5. Summary results of the CTP analysis are shown

[0395] To demonstrate the feasibility of detecting trisomy 21 in single fetal cell extractions from chorionic villus (VK) samples, we performed copy number variation (CNV) analysis.

[0396] Figure 18 shows the results of CNV analysis in fetal cells. As shown in Figure 18, single cell extraction (e.g., extraction 1 (R1), extraction 3 (R3), and extraction 6 (R6)) by DEPArray confirmed the presence of trisomy 21 in the chorionic villus (VK) sample library.

[0397] Figure 19 shows the results of CNV analysis of a healthy donor. As shown in Figure 19, the healthy donor (HD) exhibited a flat copy number profile similar to the profiles obtained from single PBMCs isolated using DEP Array.

[0398] Example 4: Workflow procedures for the collection of nRBCs from maternal blood

[0399] This example describes one method for collecting nucleated red blood cells (nRBCs) from a blood sample of a pregnant subject. As shown in Fig. 6, a blood sample was obtained from a pregnant subject (601). The blood sample was collected in CellSave tubes (601). The nRBCs can be enriched using magnetic separation (602, such as enrichment using a ferrofluid). Alternatively or in addition, the sample can be processed using the CELLTRACKS® AUTOPREP® System (603). Single cells can be imaged and isolated using the image-based technology of the DEPArray™ NxT control unit (604). After cell isolation, purified nucleic acids were obtained from the isolated cells (605). Genomic and/or genetic analysis was performed (606). For example, the nucleic acid molecules were sequenced to detect chromosomal aberrations.

[0400] Example 5: RNA Sequencing Protocol

[0401] Nucleic acid molecules such as RNA can be isolated from rare cells (e.g., fetal cells). This example shows a typical method for sequencing RNA from fetal cells.

[0402] SMART-Seq V2

[0403] Smartseq version 2 was adapted for RT-PCR with some modifications. [0404] For fetal erythroblasts (FE), 2 ng of total RNA was used as input to the reverse transcriptase reaction. Due to the limited number of maternal FE that could be sorted, all maternal FE RNA was concentrated and used for the reverse transcriptase reaction.

[0405] (1) Reverse transcription

[0406] 1 µl of oligo-dT 30VN primer (10 µM) and 1 µl of dNTP mixture (10 mM each) were added to sample tubes.

[0407] Samples were incubated at 72°C for 3 min and immediately placed on ice.

[0408] The reverse transcription mixture was prepared on ice as follows and 5.7 µl was added to each sample.

[0409] The reaction mixture was incubated in a thermal cycler as follows:

[0410] (2) Before PCR amplification

[0411] The PCR mixture was prepared on ice as described below and 15 µl was added to each sample.

[0412] Samples were placed in a thermal cycler and the following program was run.

[0413] The number of PCR cycles depended on the cell type and could be increased (for cells with a small amount of RNA) or decreased (for cells with a large amount of RNA).

[0414] (3) Purification of PCR products

[0415] Amplified cDNA products were purified twice using AMPure XP PCR beads (Beckman Coulter) at 0.5X the reaction volume. Purified cDNA was quantified using the High Sensitivity DNA Kit on an Agilent 2100 Bioanalyzer.

[0416] (4) Preparing DNA samples for Illumina Nextera XT

[0417] To obtain libraries, the Illumina NEXTERA XT DNA Kit was used with modifications (the volume of cDNA sample, reagents and reaction mixture was optimized as 1/4 of the volume specified in the manufacturer's instructions)

[0418] cDNA was diluted accordingly and adjusted to 300 pg.

[0419] 1.25 µl cDNA (300 pg) was transferred into a 0.2 ml PCR tube.

[0420] 2.5 µl Tagment DNA Buffer and 1.25 µl Amplicon Tagment Miz were added.

[0421] The tagmentation reaction mixture was incubated in a thermal cycler at 55 degrees for 5 min.

[0422] 1.25 µl NT was added immediately and incubated at room temperature for 5 min.

[0423] 1.25 µl of index 1, 1.25 µl of index 2, and 3.75 µl of Nextera PCR Master Mix (NPM) were added to the tagged DNA.

[0424] Amplification was performed using the following program:

[0425] (5) Purification of DNA libraries:

[0426] AMPure XP Beads were added to the DNA library (0.6X reaction volume).

[0427] The beads were discarded and the supernatant from the first purification was saved. [0428] For the second purification, AMPure XP Beads were added (0.7X the volume of the reaction mixture).

[0429] The beads were retained and DNA fragments were eluted.

[0430] Successfully obtained libraries (average length 400 bp) were quantified using the High Sensitivity DNA Kit on an Agilent 2100 Bioanalyzer.

[0431] To pool libraries, the concentration of each library sample was adjusted to 19 nM and pooled by volume.

[0432] (6) Sequencing DNA libraries:

[0433] Libraries were transferred to the sequencing machine and sequenced paired-end (2×101 bp) on an Illumina HiSeq™ High Output v3 system.

[0434] Example 6: Detection of trophoblast cells

[0435] This example describes a method for detecting trophoblast cells. This example utilized ferrofluid-based technology to capture and select cells and DEPArray technology to sort cells.

[0436] Trophoblast cells were captured using a ferrofluid-based, controlled aggregation technology. A blood sample from a pregnant subject was contacted with a ferrofluid containing colloidal magnetic particles conjugated with an anti-EpCAM antibody (EpCAM-FF) or colloidal magnetic particles conjugated with an anti-CD105 antibody (CD105-FF). The blood sample contained multiple cells (fetal cells and maternal cells). A first exogenous aggregation-enhancing factor, such as dethiobiotin, was conjugated to the colloidal magnetic particle. A second exogenous aggregation-enhancing factor, such as streptavidin, was added to the sample. Without regard to theoretical considerations, the addition of a second exogenous aggregation-enhancing factor induced aggregation of the colloidal magnetic particles, thereby facilitating the isolation of fetal cells and reducing non-fetal cell contamination. The sample was applied to a magnetic separator and cells bound to EpCAM-FF or CD105-FF were isolated.

[0437] To facilitate further analysis of cells bound to EpCAM-FF or CD105-FF, a third exogenous aggregation-enhancing factor, such as biotin, was added to the isolated cells. Without regard to theoretical considerations, the addition of a third exogenous aggregation-enhancing factor eliminated the aggregation of the colloidal magnetic particles, thereby facilitating single-cell analysis.

[0438] DEPArray cell sorting technology: TLS1 (i.e., TREML2) was used as a candidate for trophoblast cell staining. Isolated cells were stained with anti-TLS antibody (i.e., anti-TREML2 antibody), anti-HLA-G antibody, and cytokeratin labeled with a fluorescent tag. The sample with isolated and stained cells was loaded onto a DEPArray cartridge and analyzed using the DEPArray instrument. Cells were identified as trophoblasts if they stained positive for TLS, HLA-G, and cytokeratin.

[0439] Example 7: Diagnosis of fetal abnormalities

[0440] Fetal cells isolated or identified by any of the methods described herein were further analyzed to diagnose fetal abnormalities. Karyotype analysis was performed on the fetal cells to detect chromosomal aberrations. If a chromosomal aberration was detected in the fetus, the corresponding disorder was diagnosed. For example, if three copies of chromosome 21 were detected in the fetus, Down syndrome was diagnosed. In another example, if three copies of chromosome 18 were detected in the fetus, Edwards syndrome was diagnosed.

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SEQUENCE LIST

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<120> COMPOSITIONS AND METHODS FOR ISOLATION, DETECTION AND ANALYSIS

FETAL CELLS

<130> 111346-0203

<140> PCT/IB2020/056632

<141> 2020-07-14

<150> US 62/874 306

<151> 2019-07-15

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<---

Claims (37)

1. A method for detecting fetal cells in a blood sample obtained from a pregnant subject containing fetal cells and non-fetal cells, said method comprising: (a) contacting the sample with a first antibody conjugated to magnetic particles, wherein said first antibody binds to an endothelial marker selected from CD105 and CD71; (b) isolating the cells bound to the first antibody conjugated to magnetic particles to obtain an enriched sample; (c) contacting said enriched sample with a second antibody conjugated to a label that binds to a fetal cell marker selected from trigger receptor analogue 2 expressed on myeloid cells (TREML2) and cytokeratin (CK); and (d) identifying the cell bound to the second antibody conjugated to the label as a fetal cell, wherein the said fetal cell is a fetal trophoblast or a fetal erythroblast. 2. The method according to paragraph 1, characterized in that the magnetic particles are colloidal magnetic particles. 3. The method according to paragraph 2, characterized in that said colloidal magnetic particles are magnetic particles of a ferromagnetic fluid. 4. The method according to any one of paragraphs 1-3, characterized in that step (b) includes exposure of the sample to a magnetic field. 5. The method according to claim 4, characterized in that said magnetic particles are additionally attached to a first exogenous aggregation-enhancing factor (EAEF), wherein said first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-Fc of an antibody and avidin-biotin, biotin analogue-avidin, dethiobiotin-streptavidin, dethiobiotin-avidin, iminobiotin-streptavidin and iminobiotin-avidin. 6. The method of claim 5, wherein step (a) comprises adding a second EAEF to induce aggregation of magnetic particles, wherein said second EAEF comprises another member of the specific binding pair. 7. The method of claim 6, wherein step (b) comprises adding a member of a specific binding pair to the enriched sample in order to eliminate aggregation of magnetic particles in the enriched sample. 8. The method according to any one of the preceding claims, further comprising, prior to step (a), adding to the sample at least one aggregation inhibiting agent selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. 9. The method according to claim 8, characterized in that the aggregation inhibiting agent is a chelating agent, and wherein said chelating agent is EDTA. 10. The method according to any of the preceding claims, characterized in that said second antibody conjugated to the label binds to TREML2. 11. The method of claim 10, further comprising isolating single fetal cells prior to step (d). 12. The method according to claim 11, characterized in that the isolation of single fetal cells is performed by isolating single fetal cells associated with a second antibody conjugated to a label. 13. The method according to claim 1, characterized in that the label is a fluorescent label. 14. The method according to claim 13, characterized in that the isolation of single fetal cells is based on immunofluorescence technology. 15. The method according to claim 14, characterized in that the isolation of single fetal cells is performed by fluorescence-activated cell sorting (FACS). 16. The method according to claim 14, characterized in that the isolation of single fetal cells is performed using DEP Array. 17. The method according to any of the preceding claims, characterized in that step (d) comprises performing a sequencing-based analysis. 18. The method according to claim 17, characterized in that the sequencing-based analysis includes analysis of short tandem repeats (STR). 19. The method according to any of the preceding claims, further comprising analyzing fetal cells, wherein analyzing the fetal cells comprises performing genomic or genetic analysis. 20. The method according to claim 19, characterized in that performing genomic or genetic analysis includes detecting the presence or absence of one or more genetic abnormalities in a fetal cell. 21. The method according to any one of the preceding claims, characterized in that the second antibody conjugated to the label binds to TREML2 and comprises a heavy chain variable region (HCVR) comprising: (i) the complementarity determining region (CDR) 1 of HCVR comprising the amino acid sequence of SEQ ID NO: 6; (ii) CDR2 of HCVR comprising the amino acid sequence of SEQ ID NO: 7; (iii) CDR3 of HCVR comprising the amino acid sequence of SEQ ID NO: 8; and/or a light chain variable region (LCVR) comprising: (iv) CDR1 LCVR comprising the amino acid sequence of SEQ ID NO: 9; (v) CDR2 LCVR comprising the amino acid sequence of SEQ ID NO: 10; and (vi) CDR3 LCVR comprising the amino acid sequence of SEQ ID NO: 11. 22. The method according to any one of the preceding claims, characterized in that the second antibody conjugated to the label binds to TREML2 and is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661. 23. A method for detecting fetal cells in a blood sample obtained from a pregnant subject containing fetal cells and non-fetal cells, said method comprising: (a) contacting the sample with a first antibody or antigen-binding fragment thereof conjugated to magnetic particles, wherein the first antibody or antigen-binding fragment thereof binds to trigger receptor analogue 2 expressed on myeloid cells (TREML2); and (b) identifying a cell bound to the first antibody or antigen-binding fragment thereof conjugated to magnetic particles as a fetal cell, wherein said fetal cell is a fetal trophoblast or a fetal erythroblast. 24. The method of claim 23, further comprising contacting the sample with a second antibody conjugated to a label that binds to cytokeratin (CK) and identifying a cell that binds to the second antibody conjugated to a label as a fetal trophoblast.