WO2013123084A1 - Method for detecting circulating fetal cells - Google Patents

Abstract

The present application discloses a method of identifying, isolating or selecting fetal cells from a mixed pool of adult and fetal cells, comprising (a) obtaining a sample of mixed pool of adult and fetal cells; (b) contacting the sample with an antibody that binds to MUCl*, NM23 or NME7, and (c) identifying, isolating or selecting the cells to which the antibody has bound.

Description

METHOD FOR DETECTING CIRCULATING FETAL CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 61/598,206, filed February 13, 2012, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention:

[0003] The invention relates to a method for detecting a population of MUC1 expressing cells. In particular, the invention relates to a method for detecting and/or enriching for circulating fetal cells.

[0004] 2. General Background and State of the Art:

[0005] Normal stem cells are characterized by their ability to self-renew indefinitely and to differentiate to become adult cells of distinct tissue types. Progenitor cells have the ability to further differentiate into distinct cell types but have lost the ability to differentiate into any type of cell. The identification of the growth factors and their receptors that drive the growth of stem cells could provide the key to understanding how to grow and manipulate stem cells and progenitor cells for research, therapeutic and other uses.

[0006] MUC1 (mucin 1) is a transmembrane mucin glycoprotein that is expressed on a number of epithelial cell types (Molecular cloning and expression of the human tumor associated polymorphic epithelial mucin, PEM. Gendler Sj, Lancaster CA, Taylor-Papadimitriou J, Dhuig, Τ, Peat, N, Burchell, J, Pemberton, L, Lalani, E-N and Wilson D. (1990) J. Biol. Chem. 265, 15286-15293; Episialin, a carcinoma associated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini. Ligtenberg MJL, Vos HL, Genissen, AMC and Hilkens J. (1990) J. Biol. Chem. 265, 15573-15578), on haematopoietic cells (Evaluation of MUC1 and EGP40 in Bone marrow and Peripheral Blood as a Marker for Occult breast cancer. (2001). Zhong XY, Kaul S, Bastert G, Arch Gynecol Obstet 264: 177-181), and on progenitor cells as well (Epithelial Progenitors in the Normal Human mammary Gland. Stingl J, Raouf A, Emerman J, and Eaves C. (2005). Journal of Mamary Gland Biology and Neoplasia, Vol. 10, No. 1, 49-59).

[0007] The cell surface receptor MUC 1 is present at the apical border of healthy epithelium, but is aberrantly expressed (spread over the entire cell surface) in stem and progenitor cells. MUC1 protein can be cleaved or "shed" from the cell surface. The MUC1 ectodomain is actually comprised of three distinct regions: 1) the tandem repeats; 2) an interchain binding region that self-aggregates; and 3) the portion of the receptor that remains attached to the cell surface following proteolysis, called MGFR herein. The portion of the MUCl receptor that remains attached to the cell surface after cleavage, consisting primarily of PSMGFR, is the major growth factor receptor that mediates the growth of stem or progenitor cells in vitro.

[0008] In further detail, MUCl comprises several regions termed herein as follows, recited in an order starting from the region closest to the cell surface and progressing away from the cell. The basic structure of the MUCl receptor is illustrated in FIG. l. The receptor, as illustrated comprises: 1) cytoplasmic tail; 2) transmembrane section; 3) MGFR; 4) IBR, 5) Unique Region, 6) repeats, and N-terminus region comprising a signal peptide. For a detailed description of MUCl and its function in normal and tumor cells, see PCT/US2005/032821, which is incorporated by reference herein, in its entirety for its description of the function and activity of cleaved MUCl on the cell surface.

[0009] Because amniocentesis is an invasive procedure, carries risk of miscarriage, and cannot be performed until several weeks into a pregnancy, there is a need for a non-invasive method for detecting and isolating embryonic and fetal cells. Such cells may be analyzed by a number of techniques for screening for birth defects in the unborn fetus. DNA, and particularly fluorescence in situ hybridization (FISH) technology, a cytogenetic technique that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes, is sufficient for determining whether or not genetic abnormalities are present in the fetus. The problem is how to access sufficient numbers of fetal cells to do the analysis. It is known that some fetal cells enter the mother's circulation. But another problem is how to select the fetal cells from the mother's cells. One method involves selecting cells based on presence of Y or X chromosome. Other markers of fetal cells are needed to enable accurate selection of fetal cell and to enrich for fetal cells.

SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention is directed to a method for detecting and enriching for a population of stem or progenitor cells by detecting MUCl receptor on the cells. The detecting may be carried out by contacting the cells with (i) an agent that recognizes the MGFR portion of MUCl . The cells may be non-tumorous cells, preferably immature cells, such as stem cells, or progenitor cells, or fetal cells, even more preferably circulating or floating fetal cells in the blood of its mother. Further, in this method, the MUCl receptor may be a cell surface attached cleavage product. The MUCl cleavage product may be MGFR. Further, the MGFR may include PSMGFR. Further, the detecting agent may be a bivalent agent. The bivalent agent may recognize a portion of the MGFR. Further, the bivalent agent may be a synthetic compound. The bivalent agent may be a dimeric ligand of MUC 1. And still further, the bivalent agent may be an antibody, which may be monoclonal or polyclonal antibody. The cells may be enriched and concentrated by employing techniques such as FACS. Further, once the cells are enriched, the DNA of the fetal cells can be analyzed for abnormalities in the DNA sequence.

[0011] In one aspect, the present is directed to a method of identifying, isolating or selecting fetal cells from a mixed pool of adult and fetal cells, comprising: (a) obtaining a sample of mixed pool of adult and fetal cells; (b) contacting the sample with an antibody that binds to MUC1*, NM23 or NME7, and (c) identifying, isolating or selecting the cells to which the antibody has bound. Preferably, the antibody may bind to the N-terminal 35 amino acids of the PSMGFR peptide. The adult cells may be a pregnant woman's cells. The sample obtained from the pregnant woman may be blood, urine, saliva, amniotic fluid, colostrum or placenta of a pregnant woman. Further, the fetal cells may be fetal liver cells or fetal blood cells.

[0012] In another aspect, the present invention is directed to a method for enriching for circulating fetal cells from a mixed pool of adult and fetal cells, comprising: (a) obtaining a sample of mixed pool of adult and fetal cells; (b) contacting the sample with an antibody that binds to MUC1*, wherein the antibody selectively binds to embryonic or fetal cells, and (c) enriching for the cells bound with the antibody. Preferably, the antibody may bind to the N- terminal 35 amino acids of the PSMGFR peptide. The adult cells may be a pregnant woman's cells. The sample obtained from the pregnant woman may be blood, urine, saliva, amniotic fluid, colostrum or placenta of a pregnant woman. Further, the fetal cells may be fetal liver cells or fetal blood cells.

[0013] In yet another aspect, the present invention is directed to a method for generating biologically useful cells comprising: (a) obtaining a sample of mixed pool of adult and fetal cells; (b) contacting the sample with an antibody that binds to MUC1*, wherein the antibody selectively binds to embryonic or fetal cells; and (c) isolating the cells bound with antibody. The method may further include expanding the isolated cells in (c) above to obtain expanded cells. Even further the method may include inducing the isolated cells in (c) to a less mature state before expanding the cells. And further, the method may include inducing differentiation of the expanded cells.

[0014] In another aspect, the isolated cells or expanded isolated cells may be banked for future use.

[0015] These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;

[0017] Figure 1 is a schematic of the full length MUC1 receptor and the growth factor receptor cleavage product, MGFR.

[0018] Figure 2 shows confocal microscope images of human embryonic stem cells that are either undifferentiated or differentiated and stained with anti-MUCl* antibody, anti-OCT4 antibody, and MUCl-full length antibody that binds to tandem repeats, then visualized with fluorescent secondary antibodies.

[0019] Figure 3 shows confocal microscope images of human embryonic stem cells that are either undifferentiated or differentiated and stained with anti-MUCl* antibody, anti-OCT4 antibody, and anti-NM23 antibody, then visualized with fluorescent secondary antibodies.

[0020] Figure 4 shows FACS (fluorescence activated cell sorting) scans of umbilical cord blood cells that were sorted by their ability to bind to an anti-MUC 1 * antibody or to an antibody that binds to full-length MUC1.

[0021] Figure 5 shows FACS scans of fetal cells and umbilical cord cells that were sorted by their ability to bind to an anti-MUCl* antibody or to an antibody that binds to full-length MUC1.

[0022] Figure 6 shows a FACS scan of liver cells derived from a fetus that were then expanded by culturing the cells in a media that contained a ligand that dimerized the extra cellular domain of MUC1*: in this case an anti-MUCl* antibody.

[0023] Figure 7 shows magnified photos of cells from umbilical cord blood that were captured and then expanded by contacting the cells with an antibody that dimerized the extra cellular domain of MUC1*. Liquid assay, 15% serum, no growth factors other than anti-MUCl* antibody; 10,000 cells per well; 10 days; compared to cells grown in lOOng/mL SCF, FLT-3L, TPO, and defined lipid concentrate; those cells grew more rapidly and were very large; many dendritic-like cells adhering to plate is seen; also note large cells in "no antibody" control.

[0024] Figure 8 shows a FACS scan of starting cell population of umbilical cord cells and a graph summarizing subsequent FACS analysis of the starting cells that were either cultured in the presence or absence of NM23-H1 dimers. No other growth factor or cytokines were added. CD34-positive Human Cord Blood Cells (AllCells) contain HSCs (CD34+/CD38-/CD90+) as well as progenitors (CD34+/CD38+/CD90-). Cells were defrosted, pelleted, washed in SFEM (Serum-Free Expansion Medium) from StemSpan, and resuspended with SFEM with no growth factors added. Approximately 4000 cells were plated in wells of a 96 well plate covered with poly-HEMA to prevent adhesion. Anti-MUCl * antibody was added to each of 5 wells to a final concentration of 0, 250, or 2000ng/ml. 5 days later, antibody was re-added to cells. 6 days later, cells were stained with anti-CD34-FITC and anti-CD38-PE-Cy5. MUC1* stimulation results in more CD34+/38- cells (HSC) and fewer CD34+/38+ (progenitor) cells than unstimulated cells.

[0025] Figure 9 is a collection of photos of Western blots showing that NME7 and NM23-H1 are expressed in the cytoplasm and nucleus of embryonic and iPS stem cells.

[0026] Figure 10 is a collection of photos of Western blots showing that NME7 and NM23- Hl are secreted from embryonic and iPS stem cells.

[0027] Figure 11 is an ELISA sandwich assay that shows that NME7 has two binding sites for the MUC1 * PSMGFR peptide and that it dimerizes the MUC1* receptor.

[0028] Figure 12 is a schematic of a preferred embodiment in which monoclonal anti-

MUC1* antibodies capture fetal or embryonic cells and are then expanded by culturing in a media containing NM23-H1 dimers or NME7. Differentiation is synchronized by the addition of the PSMGFR peptide that corresponds to the extra cellular domain of MUC1*.

[0029] Figure 13 is a collection of Day 1 photos of embryonic stem cells that were cultured on a C3 monoclonal antibody surface in a minimal media containing either NME7 or NM23-H1 dimers. The C3 monoclonal antibody binds to the N-terminal 35 amino acids of the PSMGFR sequence.

[0030] Figure 14 is a collection of Day 2 photos of embryonic stem cells that were cultured on a C3 monoclonal antibody surface in a minimal media containing either NME7 or NM23-H1 dimers. The C3 monoclonal antibody binds to the N-terminal 35 amino acids of the PSMGFR sequence.

[0031] Figure 15 is a collection of Day 3 photos of embryonic stem cells that were cultured on a C3 monoclonal antibody surface in a minimal media containing either NME7 or NM23-H1 dimers. The C3 monoclonal antibody binds to the N-terminal 35 amino acids of the PSMGFR sequence. The data in Figures 13-15 lead to the conclusion that both NME7 and NM23-H1 are MUC1* ligands and bind to MUC1 * as it exists on embryonic cells.

[0032] Figure 16 is a collection of photos of confocal microscope images of stem cells cultured in either bFGF or NM23-H1 dimers and transitioning to a less mature naive state after culture in NM23-H1 dimers.

[0033] Figure 17 shows magnified photos of infant fibroblast cells that were reverted to iPS cells by ectopically expressing Oct4, Sox2, and Klf4 in the presence of NM23-H1 dimers and in the absence of bFGF. [0034] Figure 18 shows magnified photos of infant fibroblast cells that were reverted to iPS cells by ectopically expressing Oct4, Sox2, Klf4 and c-Myc in the presence of NM23-H1 dimers and in the absence of bFGF.

[0035] Figure 19 shows magnified photos of infant fibroblast cells that were reverted to iPS cells by ectopically expressing Oct4, Sox2, Klf4 and c-Myc in bFGF.

[0036] Figure 20 shows FACS scans of adult breast cancer cells that are recognized by the monoclonal anti-MUCl* antibody C2 that binds to the C-terminal 35 amino acids of the PSMGFR peptide and recognizes cancer cells, but not stem cells.

[0037] Figure 21 shows FACS scans that show that the monoclonal anti-MUCl* antibody C3 that binds to the N-terminal 35 amino acids of the PSMGFR peptide recognize embryonic cells but not adult cells or cancer cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] In the present application, "a" and "an" are used to refer to both single and a plurality of objects.

[0039] The term "MUCl Growth Factor Receptor" (MGFR) is a functional definition meaning that portion of the MUCl receptor that interacts with an activating ligand, such as a growth factor or a modifying enzyme such as a cleavage enzyme, to promote cell proliferation. The MGFR region of MUCl is that extracellular portion that is closest to the cell surface and is defined by most or all of the PSMGFR, as defined below. The MGFR is inclusive of both unmodified peptides and peptides that have undergone enzyme modifications, such as, for example, phosphorylation, glycosylation, etc. Results of the invention are consistent with a mechanism in which this portion is made accessible to the ligand upon MUCl cleavage at a site associated with tumorigenesis that causes release of the some or all of the IBR from the cell.

[0040] As used herein, "anti-PSMGFR" refers to any antibody that recognizes a region of the MGFR and optionally any portion of PSMGFR. Antibody to nat-PSMGFR is exemplified and preferred in the application, but is not meant to be limited to an antibody made against this specific sequence, as other fragments of MGFR and PSMGFR are also contemplated.

[0041] The term "Interchain Binding Region" (IBR) is a functional definition meaning that portion of the MUC 1 receptor that binds strongly to identical regions of other MUC 1 molecules giving MUCl the ability to aggregate (i.e. self-aggregate) with other MUCl receptors via the IBRs of the respective receptors. This self-aggregation may contribute to MUCl receptor clustering, observed in healthy cells. In a preferred embodiment, the IBR may be approximately defined as a stretch of at least 12 to 18 amino acid sequence within the region of the full-length human MUCl receptor defined as comprising amino acids 507 to 549 of the extracellular sequence of the MUC1 receptor (SEQ ID NO: l), with amino acids 525 through 540 and 525 through 549 especially preferred (numbers refer to Andrew Spicer et al., J. Biol. Chem Vol 266 No. 23, 1991 pgs. 15099-15109; these amino acid numbers correspond to numbers 1067, 1109, 1085, 1100, 1085, 1109 of Genbank accession number PI 5941 ; PID G547937, SEQ ID NO: 1) or fragments, functional variants or conservative substitutions thereof, as defined in more detail below.

[0042] The term "cleaved IBR" means the IBR (or a portion thereof) that has been released from the receptor molecule segment which remains attached to the cell surface. The release may be due to enzymatic or other cleavage of the IBR. As used herein, when the IBR is "at the surface of a cell", it means the IBR is attached to the portion of the cell surface receptor that has not been shed, or cleaved. The cleaved IBR of interest is a "disease-associated cleavage", i.e. that type of cleavage that can result in cancer.

[0043] The term "Constant Region" (CR) is any non-repeating sequence of MUC 1 that exists in a 1 : 1 ratio with the IBR and forms part of the portion of MUC 1 that is shed upon cleavage in healthy and tumorigenic cells.

[0044] The term "Repeats" is given its normal meaning in the art.

[0045] The term "Primary Sequence of the MUC1 Growth Factor Receptor" (PS MGFR) is a peptide sequence that defines most or all of the MGFR in some cases, and functional variants and fragments of the peptide sequence, as defined below. The PSMGFR is defined as SEQ ID NO: 10 listed below in Table 1, and all functional variants and fragments thereof having any integer value of amino acid substitutions up to 20 (i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and/or any integer value of amino acid additions or deletions up to 20 at its N- terminus and/or C-terminus. A "functional variant or fragment" in the above context refers to such variant or fragment having the ability to specifically bind to, or otherwise specifically interact with, ligands that specifically bind to, or otherwise specifically interact with, the peptide of SEQ ID NO: 10. One example of a PSMGFR that is a functional variant of the PSMGFR peptide of SEQ NO: 10 (referred to as nat-PSMGFR - for "native") is SEQ NO: 12 (referred to as var-PSMGFR), which differs from nat-PSMGFR by including an -SPY- sequence instead of the native -SRY- (see bold text in sequence listings). Var-PSMGFR may have enhanced conformational stability, when compared to the native form, which may be important for certain applications such as for antibody production. The PSMGFR is inclusive of both unmodified peptides and peptides that have undergone enzyme modifications, such as, for example, phosphorylation, glycosylation, etc. [0046] The term "Extended Sequence of the MUC1 Growth Factor Receptor" (ESMGFR) is a peptide sequence, defined below (See Table 1 - SEQ ID NO: 15), that defines all of His-var- PSMGFR plus 9 amino acids of the proximal end of PSIBR.

[0047] The term "Tumor- Specific Extended Sequence of the MUC1 Growth Factor Receptor" (TSESMGFR) is a peptide sequence (See, as an example, Table 1 - SEQ ID NO: 16) that defines a MUC1 cleavage product found in tumor cells that remains attached to the cell surface and is able to interact with activating ligands in a manner similar to the PSMGFR.

[0048] PSIBR is a peptide sequence, defined below (See Table 1 - SEQ ID NO: 17), that defines most or all of the IBR.

[0049] "Truncated Interchain Binding Region" (TPSIBR) is a peptide sequence defined below (See Table 1 - SEQ ID NO: 18), that defines a smaller portion of the IBR that is released from the cell surface after receptor cleavage in some tumor cells.

[0050] PSMGFRTC is a truncated MUC1 receptor isoform comprising PSMGFR and truncated at or within about up to 30 (i.e. within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) amino acids of its N-terminus and comprising the transmembrane and cytoplasmic sequences of full-length MUC1 receptor. As used herein, the phrase "at its N-terminus" referring to the location of a recited sequence within a larger molecule, such as a polypeptide or receptor, refers to such a sequence being no more than 30 amino acids from the N- terminal amino acid of the molecule. Optionally the PSMGFRTC, as well as the other truncated MUC1 receptor isoforms discussed below, can include a MUC1 N- terminal signaling sequence (Table 1- SEQ ID NOS: 2, 3, or 4), typically between 20 and 30 amino acids in length, or a functional fragment or variant thereof. Such a sequence is typically encoded by the nucleic acid constructs encoding the truncated MUC1 receptor isoform and is translated but is typically cleaved prior to or upon insertion of the receptor in the membrane of the cell. Such a PSMGFRTC, i.e. including the optional signal sequence, would still be a peptide or protein "having a PSMGFR" sequence "at its N-terminus" by the above definition. An example is nat-PSMGFRTC (SEQ ID NO: 5, with or without the signal peptide of SEQ ID NOS: 2, 3, or 4 at the extreme N-terminus) having nat-PSMGFR (SEQ NO: 10) at its N-terminus (i.e. at the extreme N-terminal end or within 30 amino acids thereof).

[0051] A "ligand" to a cell surface receptor, refers to any substance that can interact with the receptor to temporarily or permanently alter its structure and/or function. Examples include, but are not limited to binding partners of the receptor, (e.g. antibodies or antigen-binding fragments thereof), and agents able to alter the chemical structure of the receptor (e.g. modifying enzymes). [0052] A "growth factor" refers to a species that may or may not fall into a class of previously-identified growth factors, but which acts as a growth factor in that it acts as an activating ligand.

[0053] A "MUC1 presenting cell" refers to cells expressing MUC1 and/or MGFRs on the surface.

[0054] The term "immature cell" is used herein to refer to cells that are in various stages of differentiation from undifferentiated stem cells to progenitor cells and other cells such as various pre-cursor cells and neutrophils, which are partially differentiated, and in particular fetal cells and circulating or floating fetal cells in its mother's blood, and excludes cells that are fully differentiated.

[0055] The term, "stem cell" refers to a cell with capability of multi-lineage differentiation and self -renewal, as well as the capability to regenerate tissue. Stem cells may originate from but not limited to umbilical cord blood, liver stem cells, pancreatic stem cells, neuronal stem cells, bone marrow stem cells, peripheral blood stem cells, or a mixture thereof. Further, the invention is not limited to transplantation of any particular stem cell obtained from any particular source, but may include stem cells from "multiple stem cell sources" in mixture with one another. Thus, expanded mesenchymal stromal cells may be used in cotransplantation of the stem cells obtained from single or multiple stem cell sources to increase the amount of engraftment.

[0056] A subject, as used herein, refers to any mammal (preferably, a human), and preferably a mammal that has a disease that may be treated by administering stem cells or progenitor cells to a site within the subject. Examples include a human, non-human primate, cow, horse, pig, sheep, goat, dog, or cat. Generally, the invention is directed toward use with humans.

[0057] The samples used herein are any body tissue or body fluid sample obtained from a subject, such as amniotic fluid, colostrum or placenta of a pregnant woman. Preferred are body fluids, for example lymph, saliva, blood, urine, milk and breast secretions, and the like. Blood is most preferred. Samples of tissue and/or cells for use in the various methods described herein can be obtained through standard methods including, but not limited to: tissue biopsy, including punch biopsy and cell scraping, needle biopsy, and collection of blood or other bodily fluids by aspiration or other methods.

[0058] Fetal Cell Detection Through MUC1 Receptor

[0059] MUC1 is known to be cleaved to the MUC1* form on embryonic stem cells and some progenitor cells. In particular, MUC1* is present on fetal liver cells and some fetal blood cells. The antibodies that bind to the PSMGFR sequence of MUC1* can be used to sort cells, by FACS for example to select for cells on which MUC1 is clipped to the MUC1* form. [0060] Therefore, to identify and isolate fetal cells from the mother's cells, antibodies that bind to MUC1 * or more specifically, that bind to the PSMGFR peptide can be used in conjunction with standard selection methods including fluorescence activated cell sorting (FACS), magnetic cell sorting and the like.

[0061] For instance, using FACS, if it is desired to enrich for a population of fetal cells from the blood sample of the mother, this can be done by tagging the fetal cells with an antibody linked to a fluorescent dye. The antibody is bound to the MUC1* protein on the fetal cell. The laser light excites the dye which emits a color of light that is detected by the photomultiplier tube, or light detector. By collecting the information from the light (scatter and fluorescence) a computer can determine which cells are to be separated and collected.

[0062] The MUC1 receptor is expressed on normal epithelium, wherein the receptor is typically clustered at the apical border of adult luminal cells. MUC1 is also expressed on intestinal mucosa, pluri-potent bone marrow stem cells, neutrophil pre-cursors and neutrophils. However, Applicant has observed that Embryonic stem cells are MUC1* positive. Figure 2 shows that embryonic stem cells are MUCl *-positive. The immunofluorescent images show that undifferentiated embryonic stem cells stained positive when contacted with a rabbit polyclonal antibody directed to the PSMGFR sequence and are devoid of the tandem repeat domain of MUC1-FL (full length). The antibody used to probe the embryonic cells for the presence of full- length MUC1 was VU4H5 a mouse monoclonal antibody or HMPV another antibody that binds to full-length MUCl 's tandem repeats. NM23-H1 is secreted by human embryonic stem cells (Figure 3). It then binds to MUC1 * on the cell surface. Anti-NM23-Hl antibodies and anti- MUC1 * antibodies were used in these immunocytochemistry experiments. As can be seen, NM23-H1 and MUC1* co-localize on undifferentiated embryonic stem cells.

[0063] Early progenitor cells of a developing embryo or fetus are also MUC1* positive. MUC1 cleavage is turned on at critical progenitor stages in all three germlines (Figure 4). Cells were obtained from umbilical cord blood. FACS was used to sort cells that were MUC1 * positive using a rabbit polyclonal antibody that was raised by immunizing animals with the PSMGFR peptide ("MUC1 * SRY"). Cells that stained positive with the HMPV antibody that recognizes the tandem repeats of the full-length MUC1 were not selected. The isolated MUC1* -positive cells were then expanded by stimulating MUC1 * with a dimerizing ligand: bivalent anti-MUCl * antibody, NM23-H1 dimers or NME7.

[0064] Fetal cells were obtained from a fetus, and from umbilical cord blood (Figure 5). FACS was used to sort cells that were MUC1 * positive using a rabbit polyclonal antibody that was raised by immunizing animals with the PSMGFR peptide ("MUC1* SRY"). Cells that stained positive with the HMPV antibody that recognizes the tandem repeats of the full-length MUC1, but negative for MUC1 *, were not selected. The isolated MUCl *-positive cells were then expanded by stimulating MUC1 * with a dimerizing ligand: bivalent anti-MUCl * antibody, NM23-H1 dimers or NME7.

[0065] Therefore, cells from a developing embryo or fetus, can be detected and or isolated by contacting a pool of cells with an agent, such as an antibody, that binds to the extra cellular domain of MUC1 *. Fetal liver cells that were MUC1 * positive were captured and isolated using an antibody raised by immunizing animals with the PSMGFR peptide. The sorted cells were then expanded by treating the cells with either bivalent anti-MUCl* antibody, NM23-H1 dimers or NME7 (Figure 6).

[0066] Umbilical cord blood cells that were MUC1* positive were captured and isolated using an antibody raised by immunizing animals with the PSMGFR peptide. The sorted cells were then expanded by treating the cells with either bivalent anti-MUCl* antibody, NM23-H1 dimers or NME7 (Figure 7).

[0067] Stimulation of MUC1 * on hematopoietic stem cells (HSCs), from cord blood, expands cells and inhibits progression from the 34+/38- HSC state to the next 34+/38+ progenitor stage. No other growth factors were added (Figure 8).

[0068] Both embryonic stem (ES) cells and iPS cells express NME7 and NM23-H1. Western blots show that human breast cancer cells (T47D), human embryonic stem cells (BGOIV and HES-3) as well as human induced pluripotent stem (iPS) cells express NME7 and NME1 (NM23-H1) in their cytoplasm and nucleus (Figure 9). ES and iPS cells also secrete NME7 and NM23-H1. Western blots of the conditioned media of human embryonic stem (BGOIV and HES-3) and induced pluripotent stem (iPS) cells secrete NME7 (Figure 10). Therefore, in addition to anti-MUCl* antibodies, agents or antibodies that bind to NME7 or NM23-H1 can be used to capture and isolate embryonic or fetal cells from a sample containing a mixed pool of cells. Such a mixed pool may be a sample taken from a pregnant woman and the cells collected may be derived from the mother as well as the developing embryo or fetus.

[0069] Some of these embryonic or fetal cells will be in samples taken from the mother, including blood, urine, amniotic fluid, placenta, breast secretions including milk and colostrum, as well as other maternal bodily fluids.

[0070] To enhance detection or isolation of embryonic or fetal cells, the pool of cells may be contacted with agents that bind to other stem cell markers, including but not limited to NM23- Hl, NM23-H2, NME7, Tra 1-60, Tra 1-81, SSEA1, SSEA3, SSEA4, CD34, CD38, c-Kit, CD90. A pool of cells may be contacted simultaneously or sequentially with agents such as peptides, small molecules or antibodies that bind to one or more of these other markers in addition to agents that bind to MUC1*. [0071] NME7 is secreted by cells in the very early stages of embryogenesis. NME7 binds to the extra cellular domain of MUC1*. The sandwich assay of Figure 11 shows that NME7 has two binding sites for the MUC1* PSMGFR peptide. In the first step a strep tag PSMGFR peptide is passively coated onto the plate surface. In a second step (A), NME7 is added at the indicated concentrations. After typical washes and rinse steps, a second histidine tagged PSMGFR peptide is added. In a third step (B) labeled secondary antibody to the histidine tag is added to quantify the binding of the second PSMGFR peptide. Results show that the NME7 growth factor has two binding sites for the MUC1* extra cellular domain peptide and that it dimerizes the receptor. Therefore, agents that bind to NME7 or the detection of NME7 in a captured cell identify it as an embryonic or fetal cell.

[0072] At later stages of embryonic and fetal development, the NME family protein that is expressed is NME1 (also called NM23-H1) in dimeric form. NM23-H1 dimers bind to the extra cellular domain of MUC1* also. At still later stage of embryonic development NM23-H1 hexamers are secreted by the embryonic or fetal cells, which then bind to a cell surface protein. Thus, agents such as antibodies that bind to NME7, NM23-H1, NM23-H1 dimers, NM23-H1 hexamers and/or NM23-H2 can be used to detect or isolate embryonic or fetal cells.

[0073] Binding agents capable of capturing embryonic or fetal cells can be immobilized on surfaces that may be particle like in nature. A single type of binding agent may be attached to one surface or particle or a combination of binding agents may be attached to the same surface or particle. Surfaces may be planar substrates, beads, particles, nanoparticles, magnetic beads or particles and the like. Surfaces need not be solid. For example, the binding agents can be immobilized on a filter or polymer that allows the sample fluid to pass through while collecting the embryonic or fetal cells.

[0074] There are several reasons why detecting or isolating embryonic or fetal cells would be advantageous. First, having isolated embryonic or fetal cells would enable their characterization by any of the available methods of genetic or molecular analysis. For example, the cells could be screened for genetic defects, to determine sex, to bank cells for future use or to expand the isolated cells.

[0075] Immature Cell Expansion

[0076] Another reason for detecting and isolating embryonic or fetal cells is to revert the captured cells to a less mature state, such as iPS cells or naive stem cells, and if desired then expanding those cells. The cells can then be banked for future use, or directly used. Isolated cells may be reverted to a less mature state or to pluripotent stem cells, then used for therapeutic uses, for research, for drug screening or drug development. [0077] In a preferred embodiment, embryonic or fetal cells are isolated then cultured on a surface of anti-MUCl* antibodies and expanded in a media containing NM23-H1 dimers or NME7. Differentiation can be enhanced by optionally adding the PSMGFR peptide to disrupt stem-like growth and synchronize differentiation (Figure 12). Figures 13-15 show that embryonic stem cells captured on a surface of anti-MUCl * monoclonal antibodies (C3) then cultured in a media containing no growth factor or cytokine except either NME7 or NM23-H1 dimers proliferate and remain pluripotent. See PCT/US 12/60684 for a description of C3 antibodies, the contents of which are incorporated by reference herein in its entirety regarding the description of C3 antibodies. Resultant cells not only express all the typical markers of pluripotency such as OCT4, Tra 1-60, Tra 1-81, SSEA3, SSEA4, KLF4 and Nanog, but they also express markers that show that they are in a true pluripotent state called the naive state. Embryonic stem cells can be reverted to a less mature state by culturing them in NM23-H1 dimers or in NME7.

[0078] The immunofluorescent images in Figures 13 to 15 show staining using an antibody that binds to a tri-methylated Histone 3. When the target is stained as a discrete dot, the cells have already inactivated one X-chromosome (XaXi) and are more mature than truly pluripotent stem cells (naive) in which both X's are still active (XaXa). In the less mature, naive state when both X's are still active the tri-methylated Histone 3 antibody stains in a diffuse pattern across entire nucleus. This staining pattern is referred to as a cloud pattern. Figure 16 shows that the starting embryonic stem cells cultured in the standard bFGF media are 100% XaXi. After 10 passages in NM23-H1 ~ 50% of cells have reverted to a less mature state (XaXi). From this point, single cell clones were isolated that are 100% in the less mature XaXa state. The bottom row shows that when these cells were exposed to bFGF for only 4 passages, they are essentially 100% in the more mature XaXi state. After 2 passages in bFGF they were 85% in the more mature state. The expanded cells isolated from maternal samples can then be banked for future uses, which may include therapeutic uses, basic research, drug screening or drug discovery.

[0079] Figures 17 and 18 show that infant fibroblast cells were reverted to iPS cells by ectopically expressing Oct4, SOX2 and Klf4 (+/- c-Myc) and culturing in NME7 or NM23-H1 dimers. NM23 media was used to make fibroblast cells revert to a less mature state using the iPS generation using methods of Yamanaka with only three Yamanaka factors Oct4, Sox2, Klf4 (OSK) and omitting bFGF from the protocol. As can be seen by immunofluorescence, stem specific surface protein Tra 1-60 is present on the vast majority of cells that underwent reversion to a less mature state. Figure 19 shows that the efficiency of iPS generation using standard methods and culturing in bFGF-containing media was reduced by several orders of magnitude. [0080] In another aspect of the invention, the isolated cells, which may be expanded or reverted to a less mature state then expanded, are differentiated to a more mature state such as cardiomyocytes, beta cells for diabetes, neuronal cells, vascularizing cells, blood cells, blood cell precursors or any cell type. The differentiated cells can then be banked for future uses or used directly. Uses include but are not limited to screening for genetic defects, drug screening for identification of new drugs or toxicity profiling of candidate drugs, for therapeutic use, generating stem, progenitor and adult cells for self-use or use in others. In addition, the isolated cells may be typed according to HLA in order to match HLA-compatible future recipients who would benefite from therapies derived from stem cells. Thus a library of HLA-typed and disease associated stem cells could be generated for any of the uses described above. In a preferred embodiment, the isolated embryonic or fetal cells are expanded by contacting the cells with NM23-H1 dimers or with NME7.

[0081] Antibodies

[0082] MUC1 is a transmembrane protein with its C -terminus inside the cell and its N- terminus at the distal portion of its extra cellular domain. The precise location of the N-terminus can vary. The term MUC1* refers to the MUC1 protein in which much of its extra cellular domain is missing, either because it has been cleaved by an enzyme or because it is an alternate splice isoform that is devoid of the tandem repeat domain, also called the "core". In this and previous patent applications filed by the inventors, MUC1* was described as the MGFR (MUC1 growth factor receptor) and described as being free of enough of the self-aggregation domain that its ligand, NM23 or NME7, could be able to bind to it. The amino acid sequence of MUC1* is comprised essentially of the PSMGFR. However, we have discovered that MUC1* on embryonic and fetal cells is longer than MUC1* as it exists on cancer cells and on other more mature tissues. This difference could be due to different cleavage enzymes or alternate splice variants. Binding agents, such as antibodies, that will capture embryonic or fetal cells should bind to the N-terminal portion of the PSMGFR sequence and even further into the C-terminal end of the IBR (inter chain binding region). Monoclonal antibodies, such as C3 and C8 (see PCT/US 12/60684, for a description of C3 and C8 antibodies, the contents of which are incorporated by reference herein in its entirety regarding the description of C3 and C8 antibodies) that bind to the last 35 amino acids of the N-terminus of the PSMGFR peptide (devoid of the 10 amino acids proximal to the cell surface) were able to capture embryonic and fetal cells. Conversely, monoclonal antibodies, such as C2 and E6 (see WO/2010/042562, for a description of C2 and E6 antibodies, the contents of which are incorporated by reference herein in its entirety regarding the description of C2 and E6 antibodies) that bind to the C-terminal 35 amino acids of the PSMGFR sequence capture cancer cells but not embryonic or fetal cells. Figure 20 shows that by FACS analysis live Mucl * (+) breast cancer cells (T47D cells) are detected and isolated by the C2 monoclonal antibody but not by the C3 monoclonal. Figure 21 shows that by FACS, the C3 antibody detected and isolated embryonic cells but not the breast cancer cells.

[0083] Peptides used for antibody production may or may not be glycosylated prior to immunizing animals. The sequence of these peptides need not exactly reflect the sequence of MUC 1 receptor as it exists in the general population. For example, the inventors observed that antibodies raised against the PSMGFR peptide variant var-PSMGFR (SEQ ID NO: 12), having an "-SPY-" motif have a higher affinity and greater specificity for the MUC1 protein than antibodies raised against the actual native sequence (i.e. nat-PSMGFR, SEQ ID NO: 10), having an "-SRY-" motif. One may also, in certain embodiments, introduce mutations into the PSMGFR peptide sequence to produce a more rigid peptide that may enhance antibody production. For example the R to P mutation in the var-PFMGFR sequence of SEQ ID NO: 12 may actually have provided a more rigid peptide and was thus more immunogenic. Another method for producing antibodies against regions of peptides that are not particularly immunogenic, such as the IBR or TPSIBR is to tag the specific peptide sequence with an irrelevant sequence in which the amino acids are of the D-form and thus act to stimulate the immune response of the host animal. Peptide sequences that are used to immunize animals for antibody production may also be glycosylated. The MUC1 peptide sequences that were used herein for drug screening and to generate cognate antibodies were derived from the human species of MUCl . Since there is considerable conservation across species for the PSMGFR and IBR and some portions of the UR, it is anticipated that MUCl peptides whose sequences are derived from other species can also be used in drug screens and to generate antibodies for these same purposes.

[0084] In certain aspects, the invention provides antibodies or antigen-binding fragments thereof. In one embodiment, the invention provides an antibody or antigen-binding fragment that specifically binds to MGFR. In certain embodiments, the above-mentioned antibodies or antigen-binding fragments thereof specifically bind to PSMGFR. In certain such embodiments, the antibodies or antigen-binding fragments thereof can specifically bind to the amino acid sequence set forth in SEQ ID NO: 10 or a functional variant or fragment thereof comprising up to 15 amino acid additions or deletions at its N- terminus or comprising up to 20 amino acid substitutions; in other embodiments, it specifically binds to the amino acids set forth in SEQ ID NO: 10 or a functional variant or fragment thereof comprising up to 10 amino acid substitutions; in other embodiments, the antibodies or antigen-binding fragments thereof specifically bind to the amino acid set forth in SEQ ID NO: 10 or a functional variant or fragment thereof comprising up to 5 amino acid substitutions; and in yet another embodiments the antibodies or antigen- binding fragments thereof specifically bind to the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the antibody or antigen-binding fragment of the invention is a human, humanized, xenogenic or a chimeric human-non-human antibody or antigen-binding fragment thereof. In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise an intact antibody or an intact single-chain antibody. For antibodies or antigen-binding fragments that are monovalent, in certain embodiments, they may comprise a single-chain Fv fragment, a Fab' fragment, a Fab fragment, or a Fd fragment. For antibodies or antigen-binding fragments of the invention that are bivalent, certain embodiments comprise an antigen-binding fragment that is a F(ab')2. In certain such compositions, the antibody or antigen- binding fragment thereof can be polyclonal, while in other embodiments it can be monoclonal.

[0085] Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

[0086] As is now well known in the art, the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205, which are incorporated by reference herein in their entirety. Such antibodies, or fragments thereof are within the scope of the present invention.

[0087] In certain embodiments, fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.

[0088] In certain embodiments the present invention comprises methods for producing the inventive antibodies, or antigen-binding fragments thereof, that include any one of the step(s) of producing a chimeric antibody, humanized antibody, single-chain antibody, Fab-fragment, F(ab')2 fragment, bi-specific antibody, fusion antibody, labeled antibody or an analog of any one of those. Corresponding methods are known to the person skilled in the art and are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. The production of chimeric antibodies is described, for example, in WO89/09622. Methods for the production of humanized antibodies are described in, e.g., EP-A1 0 239 400 and WO90/07861. A further source of antibodies to be utilized in accordance with the present invention are so-called xenogeneic antibodies. The general principle for the production of xenogeneic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. As discussed below, the antibodies, of the invention may exist in a variety of forms (besides intact antibodies; including, for example, antigen binding fragments thereof, such as Fv, Fab and F(ab')2, as well as in single chains (i.e. as single chain antibodies); see e.g., WO88/09344.

[0089] Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides, in certain embodiments, for F(ab')₂, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')₂ fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies.

Table 1: Peptide sequences (listed from N-terminus to C-terminus):

Full-length MUC1 Receptor (Mucin 1 precursor, Genbank Accession number: PI 5941)

MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSAT QRSSVPSSTE KNAVSMTSSV LSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTS APDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS

APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDNRPALGS TAPPVHNVTS ASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHS SVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL

(SEQ ID NO:l)

N-terminal MUC-1 signaling sequence for directing MUC1 receptor and truncated isoforms to cell membrane surface. Up to 3 amino acid residues may be absent at C-terminal end as indicated by variants in SEQ ID NOS:2, 3 and 4.

MTPGTQSPFFLLLLLTVLT (SEQ ID NO:2).

MTPGTQSPFFLLLLLTVLT VVTA (SEQ ID NO:3)

MTPGTQSPFFLLLLLTVLT VVTG (SEQ ID NO:4)

A truncated MUC 1 receptor isoform having nat-PSMGFR at its N-terminus and including the transmembrane and cytoplasmic sequences of a full-length MUC 1 receptor ("nat-PSMGFRTC isoform" - An example of "PSMGFRTC" - shown excluding optional N-terminus signal sequence, which may be cleaved after translation and prior to expression of the receptor on the cell surface):

G TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL

(SEQ ID NO:5)

A truncated MUC 1 receptor isoform having nat-PSMGFR and PSIBR at its N-terminus and including the transmembrane and cytoplasmic sequences of a full-length MUC1 receptor ("CM isoform"- shown excluding optional N-terminus signal sequence, which may be cleaved after translation and prior to expression of the receptor on the cell surface):

GFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL

(SEQ ID NO:6)

A truncated MUC1 receptor isoform having nat-PSMGFR + PSIBR + Unique Region at its N- terminus and including the transmembrane and cytoplasmic sequences of a full-length MUC 1 receptor ("UR isoform"- shown excluding optional N-terminus signal sequences):

ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHS TVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQI YKQGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL (SEQ ID NO:7)

A truncated MUC 1 receptor isoform including the transmembrane and cytoplasmic sequences of a full-length MUC 1 receptor ("Y isoform"- shown excluding optional N-terminus signal sequence, which may be cleaved after translation and prior to expression of the receptor on the cell surface): GSGHASSTPG GEKETSATQR SSVPSSTEKN AFNSSLEDPS TDYYQELQRD

ISEMFLQIYK QGGFLGLSNI KFRPGSVVVQ LTLAFREGTI NVHDMETQFN QYKTEAASRY NLTISDVSVS DVPFPFSAQS GAGVPGWGIA LLVLVCVLVA LAIVYLIALA VCQCRRKNYG QLDIFPARDT YHPMSEYPTY HTHGRYVPPS STDRSPYEKV SAGNGGSSLS YTNPAVAATS ANL

(SEQ ID NO:8)

A truncated MUCl receptor isoform having nat-PSMGFR + PSIBR + Unique Region + Repeats at its N-terminus and including the transmembrane and cytoplasmic sequences of a full-length MUC 1 receptor ("Rep isoform"- shown excluding optional N-terminus signal sequence, which may be cleaved after translation and prior to expression of the receptor on the cell surface): LDPRVRTSAP DTRPAPGSTA PQAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DTRPAPGSTA PPAHGVTSAP DNRPALGSTA PPVHNVTSAS GSASGSASTL VHNGTSARAT TTPASKSTPF SIPSHHSDTP TTLASHSTKT DASSTHHSSV PPLTSSNHST SPQLSTGVSF FFLSFHISNL QFNSSLEDPS TDYYQELQRD ISEMFLQIYK QGGFLGLSNI KFRPGSVVVQ LTLAFREGTI NVHDVETQFN QYKTEAASRY NLTISDVSVS DVPFPFSAQS GAGVPGWGIA LLVLVCVLVA LAIVYLIALA VCQCRRKNYG QLDIFPARDT YHPMSEYPTY HTHGRYVPPS STDRSPYEKV SAGNGGSSLS YTNPAVAAAS ANL

(SEQ ID NO:9)

Native Primary Sequence of the MUCl Growth Factor Receptor (nat-PSMGFR - an example of "PSMGFR"):

GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 10)

Native Primary Sequence of the MUCl Growth Factor Receptor (nat-PSMGFR - An example of "PSMGFR"), having a single amino acid deletion at the N-terminus of SEQ ID NO: 10):

TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 11)

"SPY" functional variant of the native Primary Sequence of the MUCl Growth Factor Receptor having enhanced stability (var-PSMGFR - An example of "PSMGFR"):

GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 12)

"SPY" functional variant of the native Primary Sequence of the MUCl Growth Factor Receptor having enhanced stability (var-PSMGFR - An example of "PSMGFR"), having a single amino acid deletion at the C-terminus of SEQ ID NO: 12):

TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 13)

Truncated PSMGFR receptor (TR) (having "SPY" sequence of var-PSMGFR):

GTINVHDVETQFNQYKTEAASPYNLTISDVSVS (SEQ ID NO: 14)

Extended Sequence of MUC 1 Growth Factor Receptor (ESMGFR) (having "SPY" sequence of var-PSMGFR):

VQLTLAFREGTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPF (SEQ ID NO: 15) Tumor-Specific Extended Sequence of MUC 1 Growth Factor Receptor (TSESMGFR) (having "SPY" sequence of var-PSMGFR):

SVVVQLTLAFREGTINVHDVETQFNQYKTEAASPYNLTISDVSVS DVPFPFSAQSGA (SEQ ID NO:16)

Primary Sequence of the Interchain Binding Region) (PSIBR):

GFLGLSNIKFRPGSVVVQLTLAFRE (SEQ ID NO: 17)

Truncated Interchain Binding Region) (TPSIBR):

SVVVQLTLAFREG (SEQ ID NO: 18)

Repeat Motif 2 (RM2):

PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 19)

[0090] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.

EXAMPLES

[0091] EXAMPLE 1 - Antibody production

[0092] Antibodies that bind to the MGFR portion of the MUC1 receptor, referred to herein as anti-PSMGFR are described in detail in PCT Application No. PCT/US2004/027954 (WO 2005/019269), in particular in Example 8 of the PCT Application. Antibody production is also described in PCT Application No. PCT/US2005/032821, in particular in Example 2 of the PCT Application. Inventive antibodies were raised against the PSMGFR portion of the MUC1 receptor, in particular nat-PSMGFR or var-PSMGFR shown in Table 1 using standard methods of antibody production. Rabbit polyclonal antibodies were produced and purified by column chromatography in which the immunizing peptide was attached to the chromatography column beads. The antibodies, anti-nat-PSMGFR and anti-var-PSMGFR, were shown to specifically and sensitively bind to the MGFR portion of the MUC1 receptor.

[0093] All of the references cited herein are incorporated by reference in their entirety.

[0094] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.

Claims

WHAT IS CLAIMED IS :

1. A method of identifying, isolating or selecting fetal cells from a mixed pool of adult and fetal cells, comprising

(a) obtaining a sample of mixed pool of adult and fetal cells;

(b) contacting the sample with an antibody that binds to MUC1*, NM23 or NME7, and

(c) identifying, isolating or selecting the cells to which the antibody has bound.

2. The method according to claim 1, wherein the antibody binds to the N-terminal 35 amino acids of the PSMGFR peptide.

3. The method according to claim 1, wherein the adult cells are pregnant woman's cells.

4. The method according to claim 1, wherein the sample is blood, urine, saliva, amniotic fluid, colostrum or placenta of a pregnant woman.

5. The method according to claim 1, wherein the fetal cells are fetal liver cells or fetal blood cells.

6. A method for enriching for circulating fetal cells from a mixed pool of adult and fetal cells, comprising

(a) obtaining a sample of mixed pool of adult and fetal cells;

(b) contacting the sample with an antibody that binds to MUC1*, wherein the antibody selectively binds to embryonic or fetal cells, and

(c) enriching for the cells bound with the antibody.

7. The method according to claim 6, wherein the antibody binds to the N-terminal 35 amino acids of the PSMGFR peptide.

8. The method according to claim 6, wherein the adult cells are pregnant woman's cells.

9. The method according to claim 6, wherein the sample is blood of a pregnant woman.

10. The method according to claim 6, wherein the fetal cells are fetal liver cells or fetal blood cells.

11. A method for generating biologically useful cells comprising,

(a) obtaining a sample of mixed pool of adult and fetal cells;

(b) contacting the sample with an antibody that binds to MUCl*, wherein the antibody selectively binds to embryonic or fetal cells; and

(c) isolating the cells bound with antibody.

12. The method according to claim 11, comprising expanding the isolated cells in (c) to obtain expanded cells.

13. The method according to claim 12, comprising inducing the isolated cells in (c) to a less mature state before expanding the cells.

14. The method according to claim 12, comprising inducing differentiation of the expanded cells.

15. The method according to claim 11, wherein the isolated cells are banked for future use.

16. The method according to claim 12, wherein the expanded isolated cells are banked for future use.