HK1236552A1 - Anti-vasa antibodies, and methods of production and use thereof - Google Patents

Description

anti-VASA antibodies and methods of production and use thereof

This application claims priority to U.S. provisional application No. 62/051,130 filed on 16/9 2014 and U.S. provisional application No. 62/089,054 filed on 8/12 2014, which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates generally to antibodies, their preparation and use. In particular, the disclosure relates to antibodies that specifically bind to human VASA protein, methods of producing such antibodies, and diagnostic, therapeutic, and clinical methods of using such antibodies.

Background

VASA proteins were identified in Drosophila (Drosophila) as components of germplasm that encode DEAD family ATP-dependent RNA helicases (Liang et al (1994), Development,120: 1201-11; Lasko et al (1988), Nature 335: 611-17). The molecular functions of VASA are involved in binding to target mRNA in the production of germ cells, oogenesis and translation initiation (Gavis et al (1996), Development 110: 521-28). VASA is required for polar cell formation and is limited to the entire developing germ plasmic lineage.

Vasa isogenes have been isolated in various animal species, and VASA can be used as a molecular marker of germ Cell lineages in most animal species (Noce et al (2001), Cell Structure and Function 26: 131-36). For example, Castrillon et al (2000), Proc. Natl. Acad. Sci (USA)97(17):958590-9590 indicates that the human Vasa gene is expressed in the ovary and testis, but is undetectable in somatic tissues.

The presence of mammalian female germline stem cells (also known as oocyte stem cells or Ovarian Stem Cells (OSCs) or egg precursor cells) in the somatic tissue of the mammalian ovary was first described in Johnson et al (2004), Nature428:145-50, and has been demonstrated by other research groups (e.g., Zou et al (2009), Nature CellBiology, published on-line DOI:10.1038/ncb 1869; Telfer & Alberti (2012), Nature medicine18(3): 353-4). The potential use of OSCs for the generation of oocytes for artificial propagation techniques (ART), including In Vitro Fertilization (IVF), or as a source of highly functional mitochondria for mitochondrial transfer to oocytes, and the use of OSCs for the treatment of various symptoms of menopause, have been described in the scientific and patent literature (e.g., Tilly & Telfer (2009), mol. Hum. Repro.15(7): 393-8; Zou et al (2009), supra; Telfer & Albertini (2012), supra; White et al (2012), Nature Medicine18(3): 413-21; WO 2005/121321; U.S. Pat. No. 7,955,846; U.S. Pat. No. 8,652,840; WO 2012/142500; U.S. Pat. No. 8,642,329 and U.S. Pat. No. 8,647,869).

When OSC was first characterized by Johnson et al (2004) (supra), it was demonstrated that cells express VASA protein, and antibodies to VASA protein have been used to isolate OSC from ovarian tissue homogenates (e.g., zuo et al (2009), supra; White et al (2012), supra). Furthermore, White et al (2012) (supra) demonstrated that antibodies directed against the N-terminal domain of VASA cannot be used to isolate live VASA-expressing OSCs, whereas antibodies directed against the C-terminal domain can efficiently isolate cells, indicating that the C-terminal domain, rather than the N-terminal domain, is extracellular and thus accessible to antibodies.

The production of polyclonal antibodies against VASA was first described in Castrillon et al (2000) (supra) and WO 01/36445. Polyclonal antibodies directed against the C-terminal portion of human VASA protein are commercially available from Abcam plc (Cambridge, UK; product code AB13840) and R & D Systems, Inc. (Minneapolis, MN; catalog No. AF2030), and monoclonal antibodies directed against the N-terminal portion of human VASA are also commercially available from R & D Systems, Inc. (Minneapolis, MN; catalog No. AF 2030).

However, there remains a need for high affinity antibodies directed against the C-terminal extracellular domain of VASA for use in identifying (e.g., by immunohistochemistry or labeled antibodies) and isolating (e.g., by magnetic or fluorescence activated cell sorting) cells expressing VASA, including but not limited to OSC.

Summary of The Invention

The present invention discloses anti-VASA antibodies (mabs), particularly humanized mabs that specifically bind VASA with high affinity. The invention provides the amino acid sequences of the CDRs of the light and heavy chains of these anti-VASA mabs and consensus sequences of these CDRs. The disclosure also provides nucleic acid molecules encoding anti-VASA mabs, expression vectors, host cells, methods for making anti-VASA mabs, and methods for expressing anti-VASA mabs. Finally, the invention discloses methods of isolating and/or purifying cells expressing VASA using anti-VASA mabs.

These and other aspects and embodiments of the disclosure are shown and described below. Other systems, processes, and features will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description of the invention. It is intended that all such additional systems, processes, and features be included within this description, be within the scope of the present invention, and be protected by the accompanying claims.

Brief description of the drawings

FIG. 1 provides the amino acid sequence of human VASA protein isoform 1 from GenBank accession NP-077726 (SEQ ID NO: 1).

FIG. 2 provides the amino acid sequence of mouse VASA homologous protein isoform 1 from GenBank accession NP-001139357 (SEQ ID NO: 2).

FIG. 3 provides an amino acid alignment between the C-terminal part of the human VASA protein (residues 690-724 of SEQ ID NO: 1) and the mouse VASA homologue (residues 691-728 of SEQ ID NO: 2).

Fig. 4A shows the region of the C-terminal domain of VASA/DDX4 polypeptide that reacts with the antibodies of the invention and control antibodies (AB13840, Abcam plc, Cambridge, UK), and fig. 4B shows the binding of control antibodies to VASA protein and V1 and V2 polypeptides.

Figure 5A shows dose-response binding curves for affinity of 1E9 and 1a12 for VASA; and fig. 5B shows the results of ELISA assays using VASA, V1 and V2 peptides, which indicate that 1E9 binds to the same epitope as the commercially available rabbit polyclonal antibody (AB13840, Abcam plc, Cambridge, UK). NC ═ negative control; VASA ═ SEQ ID NO:1 residue 700-724; VASA-1 ═ V1 or SEQ ID NO:1 residue 712-721; VASA-2 ═ V2 or SEQ ID NO:1 residue 700-.

FIG. 6A shows a dose-response binding curve of the IgG and scFv-Fc format of 1E9 for affinity for VASA; and figure 6B shows the results of an ELISA assay of binding of IgG and scFv-Fc versions of 1E9 to VASA, V1, and V2 peptides. NC ═ negative control; VASA ═ SEQ ID NO:1 residue 700-724; VASA-1 ═ V1 or SEQ ID NO:1 residue 712-721; VASA-2 ═ V2 or SEQ ID NO:1 residue 700-.

FIG. 7A shows the results of binding experiments using 3 anti-VASA hybridoma antibodies (2M1/1K3, 2M1/1K23, and 2M1/1L5) and two negative controls that are not VASA-specific (2M1/1F5 and 2M1/1H 5); FIG. 7B shows dose response curves of 4 VASA-specific hybridoma antibodies (2M1/1K3, 2M1/1K23, and 2M1/1L5) compared to 1E 9-lambda; and figure 7C shows a dose response curve for VASA-specific hybridoma antibody 2M1/2K4 compared to 1E9- λ.

FIG. 8 shows the results of subtype analysis of anti-VASA antibodies from 8 hybridomas (2M1/1L20, 2M1/1J20, 1M1/1C9, 2M1/1N3, 2M1/1K23, 1M1/1L5, and 2M1/2K 4).

FIGS. 9A-9B show an alignment of some VL sequences of the anti-VASA invention. The figure indicates the approximate positions of the three CDR regions (bold, underlined) and the SEQ ID NOs corresponding to each sequence.

FIGS. 10A-10B show an alignment of some VH sequences of the anti-VASA invention. The figure indicates the approximate positions of the three CDR regions (bold, underlined) and the SEQ ID NOs corresponding to each sequence.

FIG. 11 shows an alignment of the unique CDR sequences of the VL region of FIG. 9.

FIG. 12 shows an alignment of the unique CDR sequences of the VH region of FIG. 10.

Detailed Description

The present disclosure relates to isolated antibodies (abs), particularly abs that specifically bind VASA with high affinity. In certain embodiments, the anti-VASA Ab is derived from specific heavy and light chain sequences and/or comprises specific structural features, such as CDR regions comprising specific amino acid sequences. The present disclosure provides isolated anti-VASA abs, methods of making such anti-VASA abs, immunoconjugates and bispecific molecules comprising such anti-VASA abs, and methods of expressing such anti-VASA abs. The figure indicates the approximate positions of the three CDR regions (bold, underlined) and the SEQ ID NOs corresponding to each sequence. Also relates to methods of using the anti-VASA abs to isolate and/or purify VASA-expressing cells, including mammalian female germline stem cells or egg stem cells (OSCs) or egg precursor cells and progenitors thereof.

In order that the disclosure may be more readily understood, certain terms are defined. Other definitions are set forth throughout the detailed description of the invention.

Definition of

The term "antibody" or the abbreviation "Ab" as used herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof, with or without native glycosylation. An intact "antibody" refers to a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable region (V)_H) Heavy and heavyA chain constant region. The heavy chain constant region is composed of three domains C_H1、C_H2And C_H3And (4) forming. Each light chain includes a light chain variable region (V)_L) And has a domain C_LThe light chain constant region of (1). V_HAnd V_LThe regions may be further subdivided into Complementarity Determining Regions (CDRs) and Framework Regions (FRs). V_HAnd V_LEach region includes three CDRs, designated CDR1, CDR2, and CDR3, which interact with an antigen (e.g., VASA).

The term "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., VASA). Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include Fab fragments, F (ab')₂Fragments, Fab' fragments, Fd fragments, Fv fragments, scFv fragments, dAb fragments, and isolated CDRs.

The term "monoclonal antibody" or "monoclonal antibody preparation" as used herein refers to a preparation of antibody molecules consisting essentially of antibodies having single heavy chain amino acid sequences and single light chain amino acid sequences (although which may have heterogeneous glycosylation).

The term "humanized antibody" as used herein includes antibodies having constant and variable Framework Regions (FRs) but no CDRs derived from human germline immunoglobulin sequences.

The term "recombinant antibody" as used herein includes all antibodies prepared, expressed, produced or isolated by recombinant means. In certain embodiments, the recombinant antibody is isolated from a host cell transformed to express the antibody (e.g., from a transfectoma). In other embodiments, recombinant antibodies are isolated from recombinant combinatorial antibody libraries (e.g., phage display libraries). Recombinant antibodies can also be prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences.

The term "isotype" as used herein refers to the heavy chain class encoded by the constant region gene (e.g., IgA, IgD, IgE, IgG and IgG of human antibodiesIgM) or light chain class (e.g., kappa or lambda in humans). The term "subtype" refers to a subclass within a subtype (e.g., IgA in humans₁、IgA₂、IgG₁、IgG₂、IgG₃、IgG₄)。

The phrase "antibody specific for a particular antigen" is used interchangeably herein with the phrase "antibody that specifically binds to a particular antigen". The term "K" as used herein_a"refers to the rate of binding, the term" K_d"is the off-rate of a particular antibody-antigen complex. The term "K_D"refers to the dissociation constant, which is defined by K_dAnd K_aAccording to some embodiments, an antibody that specifically binds human VASA is meant to be at 5 × 10^-8M or less, more preferably 1 × 10^-8K of M or less_DAn antibody that binds human VASA.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

anti-VASA antibodies

The present invention provides a variety of novel antibodies with high affinity for human VASA proteins, particularly the C-terminal region. The antibody may comprise the entire VH and VL regions disclosed herein, or may comprise only the CDR sequences disclosed herein. Furthermore, based on the CDR sequences disclosed herein, sequence motifs for the CDR sequences are provided, and the antibody may comprise CDR sequences defined by the motifs.

The CDR sequences of the invention (including the CDRs disclosed in fig. 11 and 12 and defined by the sequence motifs disclosed herein) can be combined with other immunoglobulin sequences according to methods well known in the art to produce immunoglobulin molecules having the antigen binding specificity determined by the CDRs of the invention.

In some embodiments, the CDRs of the invention are combined with Framework Region (FR) and constant domain (CH or CL) sequences from other antibodies. For example, although some of the CDRs disclosed herein are derived from murine hybridomas and have murine FR and constant domain sequences, they can be recombined with human or other mammalian FR and constant domain sequences to produce humanized or other recombinant antibodies. The production of such recombinant antibodies is well known to those skilled in the art and requires only routine experimentation.

The type of constant region included in such recombinant antibodies may be selected according to its intended use. For example, if the antibody is intended for therapeutic use to target VASA-expressing cells for destruction, heavy chain constant domains of the IgG subtype (i.e., Fc regions) may be used. If the antibody is used only as an agent for labeling cells (e.g., for Fluorescence Activated Cell Sorting (FACS)), an intact antibody, an antigen binding fragment (Fab), a single chain variable fragment (Fsc), a single domain antibody (sdAb), or even a non-antibody immunoglobulin molecule (e.g., MHC receptor extracellular domain) can be used with the CDRs of the invention.

The CDRs of the invention may be independently selected such that the CDR1, CDR2, and CDR3 sequences of a given variable light chain (VL) or variable heavy chain (VH) may be selected from different original VL and VH chains, from different VL and VH CDR motifs, or from a combination of the disclosed CDRs and motifs. However, the sequence of the light chain CDRs should be selected from published VL CDRs or VL CDR motifs, and the sequence of the heavy chain CDRs should be selected from published VH CDRs or VH CDR motifs. Similarly, for VL or VH chains, where appropriate, the sequence of the CDR1 region should be selected from the published CDR1 or CDR1 motif sequences, the sequence of the CDR2 region should be selected from the published CDR2 or CDR2 motif sequences, and the sequence of the CDR3 region should be selected from the published CDR3 or CDR3 motif sequences.

Detecting or isolating cells using anti-VASA antibodiesMethod of producing a composite material

The anti-VASA antibodies of the invention can be used in standard methods of immunoaffinity purification, immunohistochemistry, and immunotherapy, but are specifically applied to cells and tissues expressing VASA protein.

For example, the anti-VASA antibodies of the invention can be used to isolate VASA-expressing cells from a mixed population of cells that contains only a portion of VASA-expressing cells. For example, it has been found that female germ line stem cells or egg cell stem cells or precursors thereof are present in very low proportions in ovarian tissue. Ovarian tissue (e.g., ovarian surface epithelium and/or cortex) can be excised, dissociated into individual cells, and subjected to techniques such as FAC using fluorescently labeled anti-VASA antibodies or immunoaffinity purification using immobilized anti-VASA antibodies. As noted above, isolated VASA-expressing cells have a variety of uses in assisted reproduction techniques.

Alternatively, immunohistochemistry may be performed using the anti-VASA antibodies of the invention to identify cells or tissues expressing VASA and/or to quantify VASA expression in such cells.

In addition, the anti-VASA antibodies of the invention can be used therapeutically to target VASA-expressing cells for destruction by antibody-dependent cell-mediated cytotoxicity (ADCC) or immunotoxins comprising the anti-VASA antibodies of the invention conjugated to a radioactive or chemotoxic moiety. The antibody-drug conjugates of the anti-VASA antibodies of the invention may also be used to deliver therapeutic agents to cells expressing VASA.

Nucleic acid molecules encoding anti-VASA antibodies

The invention also provides nucleic acid molecules encoding the anti-VASA antibodies of the invention. Standard tables of the universal genetic code may be used to select codons that will encode the desired amino acid sequence, or specialized codon tables that reflect codon preference characteristics of different organisms may be used to design such nucleic acids. Thus, for example, to optimize expression of the anti-VASA antibodies of the invention in CHO cells, nucleic acids encoding the desired antibodies can be designed using a codon table optimized for CHO cells.

Nucleic acids encoding the anti-VASA antibodies of the invention can be included in a variety of vectors known in the art, including cloning vectors (e.g., bacterial or mammalian cloning vectors), transformation vectors (e.g., homologous recombination, viral integration, or autonomously replicating vectors), and expression vectors (e.g., high copy number, inducible or constitutive mammalian expression vectors).

Cells expressing anti-VASA antibodies

The invention also provides host cells expressing heterologous sequences encoding the anti-VASA antibodies of the invention. Such host cells are useful for the commercial production of anti-VASA antibodies of the invention, and may be produced by transforming appropriate host cells with the above-described expression vectors.

In some embodiments, the invention provides mammalian cells, including CHO cells, expressing an anti-VASA antibody of the invention. However, one skilled in the art can express antibodies in a variety of host cells, including bacterial, yeast, insect, and mammalian systems. See, e.g., Verma et al (1998), J.Immunol.methods 216(1-2):165-81, the entire contents of which are incorporated herein by reference.

Examples

Immunogenic peptides

The following peptides were used as immunogens to generate antibodies against the C-terminal domain of human VASA and to screen for antibodies that bind VASA with high affinity:

VASA-1(V1) immunogen SQAPNPVDDE (SEQ ID NO:1 residue 712-721)

VASA-2(V2) immunogen GKSTLNTAGF (SEQ ID NO:1 residue 700-

As shown in figure 3, these immunogens comprise the amino acid sequence from the C-terminal domain of VASA, which is highly conserved between human VASA protein and mouse VASA homologues.

Generation of hybridomas

Hybridomas were formed in separate experiments using VASA peptide immunogens V1 and V2 (above). The peptide is conjugated to the carrier protein by standard methods. Mice were immunized with the conjugated peptide and the immune response was increased by boosting with the conjugated peptide. After the period of increasing antibody titer in serum, animals were sacrificed and spleens removed. Splenic B cells were fused with a mouse fusion partner cell line (SP2-0) for isolation and cloning. Hybridomas were formed by limiting dilution growth (outgrowth) and clones were generated by clonal titration experiments. The presence of VASA-reactive antibodies was checked by ELISA assay. Hybridomas are derived by growth and stabilization of plated cells under limiting dilution cell clones.

Binding of VASA-reactive antibodies in the C-terminal domain region of VASA/DDX4 polypeptides was compared to binding of control antibodies (AB13840, Abcam plc, Cambridge, UK) to delineate similar epitopes of binding. Exemplary results are shown in fig. 4.

Analysis of hybridomas

The hybridomas were injected intraperitoneally into mice, and after allowing growth phase, ascites fluid was collected and purified, all using standard procedures, and then analyzed by ELISA.

Binding of ascites-derived antibodies to VASA, VASA-1 and VASA-2 polypeptides was used to select antibodies for further analysis. For example, as shown in FIG. 7, binding of four anti-VASA hybridoma antibodies (2M1/1K3, 2M1/1K23, 2M1/1L5, and 2M1/2K4) was compared to binding of two negative controls (2M1/1F5 and 2M1/1H5) that were not VASA-specific, and/or binding of the 1E 9-lambda antibody (described below).

Panning of recombinant library

As an alternative to hybridoma technology, phage display technology was used to generate antibodies against amino acid residues 700-724 of human VASA/DDX 4. Phage display libraries were formed from a pool of normal B cells from about 40 blood donors. The phage was used to display scFv chains of antibodies.

Panning human naive against VASA/DDX4700-724 peptideThe results of the scFv library are shown in table 1 below:

TABLE 1

The ELISA results of the single colonies identified after 3 and 4 rounds of selection are shown in tables 2-4 below. Two clones were notable: "1A 12" (panel 1, row A, column 12) and "1E 9" (panel 1, row E, column 9).

TABLE 2

TABLE 3

TABLE 4

The ELISA results of the single colonies identified after 5 rounds of selection are shown in tables 5-7 below. Notable clones include 1a11, 1B4, 1B7, 1D4, 1D5, 1E2, 1E3, 1F7, 1G3, 1G12, 2B8, 2C7, 2E11, 2F1, 2G8, 2G10, 2H9, 3B2, 3B5, 3B7, 3D11, 3E5, 3E12, 3F6, and 3H 11.

TABLE 5

TABLE 6

TABLE 7

The clones shown in bold were PCR amplified.

Conversion to scFv-Fc fusions and expression in mammalian cells

After 5 rounds of panning, the DNA digestion pattern showed that many clones from the 5 th round of panning were identical, indicating that no additional rounds of selection and ELISA analysis were required.

Two unique clones (1a12, 1E9) were selected for conversion to scFv-Fc fusions for expression in mammalian cells and for ELISA and FACS analysis. Fig. 5A shows a dose-response binding curve, which indicates that 1E9 has an EC50 of 0.02779nM, while 1a12 has an EC50 of 0.2156 nM. Furthermore, fig. 5B shows the results of ELISA assays using V1 and V2VASA peptides, which indicate that 1E9 binds to the same epitope as the commercially available rabbit polyclonal antibody (AB13840, Abcam plc, Cambridge, UK).

Two different forms of the 1E9 antibody were compared: IgG and scFv-Fc. As shown in FIG. 6A, the 1E9IgG had an EC50 of 0.08919nM, and the 1E9scFv-Fc had an EC50 of 0.3072 nM. Furthermore, as shown in FIG. 6B, both forms are specific for the VASA-1 epitope.

Synthetic antibody gene production

Synthetic antibody genes were generated using the following steps:

(1)subtype determination of hybridoma antibodies. The IgG subtype of hybridoma antibodies was determined using a commercially available Kit according to the manufacturer's protocol (e.g., Mouse Monoclonal Antibody Isotyping Kit, cat # MMT1, AbDSerotech, Kidlington, UK). FIG. 8 shows the results of subtype analysis of anti-VASA antibodies from 8 hybridomas (2M1/1L20, 2M1/1J20, 1M1/1C9, 2M1/1N3, 2M1/1K23, 1M1/1L5, and 2M1/2K 4). All antibodies were IgG1, IgG2a, or IgG2 b.

(2)Degenerate primer synthesis. Based on the subtype Information of the 8 hybridoma antibodies tested, a mouse IgG database (International Immunogenetics Information) was usedOr an IMGT database; see Lefranc et al (2003), leukamia 17: 260-; 882:569-604) to design degenerate primers for mouse IgG VH and VL. 10 degenerate forward primers for VH chain and 10 degenerate forward primers for VL chain were designed and synthesized (9 for kappa chain and 1 for lambda chain). In addition, 2 degenerate reverse primers for VH chain (1 for IgG1 and IgG2b subtypes, 1 for IgG2a subtype) and 5 for VL chain (4 for kappa chain, 1 for lambda chain) were designed and synthesized.

(3)RNA extraction, amplification, cloning and sequencing. RNA was extracted from hybridoma cells by standard techniques, first strand cDNA synthesis was performed by standard techniques using gene-specific and oligo (dT) primers, and cDNA was amplified using gene-specific primers. The amplified DNA was then ligated into a commercially available bacterial cloning vector (pMD18-T, Sino Biological, Inc., Beijing, China). Standard methods were performed to ligate the productsTransformed into e.coli (e.coli) DH5 α and positive clones were sequenced.

Antibody sequence analysis

Clones producing potentially useful anti-Vasa antibodies were DNA sequenced and the corresponding amino acid sequences were deduced. Sequences derived from 8 antibodies of the above hybridomas (i.e., 1N23, 1K23, 2K4, 1C9, 1J20, 1L20, 1K3, 1L5), another 4 antibodies derived from hybridomas produced under the same (i.e., CTA4/5, CTB4/11, CTC2/6, CTD2/6) and 2 antibodies derived from phage display (i.e., 1a12 and 1E9) are disclosed.

Variable light chain sequences

VL of 1N23. Positive VL clones from the 1N23 hybridoma were sequenced and found to encode 6 functional VL chains. These 6 clones were designated 1N23VL5-5, 1N23VL5-8_0816, 1N23VL1-8, 1N23VL1-2_0820, 1N23VL1-4_0820 and 1N23VL 1-2.

VL of 1K23. Positive VL clones from the 1K23 hybridoma were sequenced and found to encode 4 functional VL chains. These 4 clones were designated 1K23VL2-5, 1K23VL2-6, 1K23VL2-8_0822 and 1K23VL2-3_ 0829.

VL of 2K4. Positive VL clones from the 2K4 hybridoma were sequenced and found to encode 8 functional VL chains. These 8 clones were designated 2K4VL1-3_0820, 2K4VL1-4, 2K4VL1-1, 2K4VL1-6_0820, 2K4VL2-5_0816, 2K4VL2-4, 2K4VL2-6_0816 and 2K4VL 2-5.

VL of 1C9. Positive VL clones from the 1C9 hybridoma were sequenced and found to encode 3 functional VL chains. These 3 clones were designated 1C9VL2-4, 1C9VL2-6 and 1C9VL2-3_ 0816.

VL of 1J20. Positive VL clones from the 1J20 hybridoma were sequenced and found to encode 3 functional VL chains. These 3 clones were designated 1J20VL5-2_0907, 1J20VL5-6_0907 and 1J20VL4-3_ 0907.

VL of 1L20. Positive for hybridoma from 1L20VL clones were sequenced and found to encode a functional VL chain. This clone was named 1L20VL5-0912_ 091.

VL of 1K3. Positive VL clones from the 1K3 hybridoma were sequenced and found to encode 4 functional VL chains. These 4 clones were designated 1K3VL2-5, 1K3VL2-5, 1K3VL2-3 and 1K3VL 2-4.

VL of 1L5. Positive VL clones from the 1L5 hybridoma were sequenced and 2 clones were found to encode functional VL chains. These 2 clones were designated 1L5VL2-4 and 1L5VL 3-1.

Additional VL. VL sequences were obtained for 4 additional hybridoma antibodies designated CTA4_ VL, CTB4_ VL, CTC6_ VL, CTD6_ VL.

Alignment of VL sequences. An alignment of all the VL sequences described above is shown in figure 9. The figure indicates the approximate positions of the three CDR regions (bold, underlined) and the SEQ ID NOs corresponding to each sequence.

Unique VL CDR sequences. An alignment of the unique CDR sequences of the VLs in fig. 9 is shown in fig. 11. Of the 34 VL sequences, there were only 5 unique CDR1 sequences, 6 unique CDR2 sequences, and 8 unique CDR3 sequences as shown in fig. 11.

VL CDR consensus sequences. Based on the sequences disclosed in fig. 11, as well as the structural/functional characteristics of the naturally occurring amino acids, consensus sequences for VL CDRs can be determined.

One consensus sequence is VL CDR1 motif 1:

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁(SEQ ID NO:132)

wherein X₁Is Q, N, K, R, S or T; x₂Is S, T, C, N or Q; x₃Is I, L, V, M or A; x₄V, L, I, M, A or absent; x₅H, K, R or absent; x₆S, T, C or absent; x₇Is N,Q or absent; x₈G, A or absent; x₉Is N or Q; x₁₀Is T, S, C, N or Q; and X₁₁Is Y, F or W. In some embodiments, X₁Limited to Q, K or S; and/or X₂Limited to S or N; and/or X₃Limited to I or L; and/or X₄Limited to V, L or absent; and/or X₅Limited to H or absent; and/or X₆Limited to S or absent; and/or X₇Limited to N or absent; and/or X₈Limited to G or absent; and/or X₉Limited to N; and/or X₁₀Limited to T, S or N; and/or X₁₁Limited to Y or F. In some embodiments, subsequence X₁X₂X₃Limited to QNI; in some embodiments, subsequence X₁X₂X₃Limited to Q S L; and in some embodiments, subsequence X₁X₂X₃Limited to ks L. Further, in some embodiments, when X is₁X₂X₃When Q S L or Q N I, then X₄Is V; and in other embodiments when X₁X₂X₃When is KS L, then X₄Is L. In some embodiments, when X₉X₁₀When it is N T, then X₁₁Is Y.

Of particular note are SEQ ID NOs: 86-88 are distinct from the other sequences in FIG. 11, and an alternative consensus sequence is VL CDR1 motif 2:

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁(SEQ ID NO:133)

wherein X₁Is Q, N, K or R; x₂Is S, T, C, N or Q; x₃Is I, L, V, M or A; x₄Is V, L, I, M or A; x₅Is H, K or R; x₆Is S, T or C; x₇Is N or Q; x₈Is G or A; x₉Is N or Q; x₁₀Is T, S or C; and X₁₁Is Y, F or W. In some embodiments, X₁Limited to Q or K; and/orX₂Limited to S or N; and/or X₃Limited to I or L; and/or X₄Limited to V or L; and/or X₅Limited to H; and/or X₆Limited to S; and/or X₇Limited to N; and/or X₈Limited to G; and/or X₉Limited to N; and/or X₁₀Limited to T; and/or X₁₁Limited to Y. In some embodiments, subsequence X₁X₂X₃Limited to QNI; in some embodiments, subsequence X₁X₂X₃Limited to Q S L; and in some embodiments, subsequence X₁X₂X₃Limited to ks L. Further, in some embodiments, when X is₁X₂X₃When Q SL or QNI is present, then X₄Is V; and in other embodiments when X₁X₂X₃When is KS L, then X₄Is L. In some embodiments, when X₉X₁₀When it is N T, then X₁₁Is Y.

For VL CDR2, one consensus sequence is VL CDR2 motif 1:

Y₁Y₂Y₃(SEQ ID NO:134)

wherein Y is₁Is K, R or H; y is₂Is V, I, L, M, A, T, S or C; and Y is₃Is S, T, C, N or Q. In some embodiments, Y is₂Limited to V, I, M or T; and/or Y₃Limited to S or N.

Of particular note is the VL CDR2 sequence of SEQ ID No. 94, which is distinct from the other sequences in fig. 11, and an alternative consensus sequence is VL CDR2 motif 2:

Y₁Y₂Y₃(SEQ ID NO:135)

wherein Y is₁Is D or E; y is₂Is N or Q; and Y is₃Is N or Q. In some embodiments, Y is₁Limited to D; and/or Y₂Limited to N; and/or Y₃Limited to N.

Similarly, with particular attention to the VL CDR2 sequence of SEQ ID NO 95 as distinct from the other sequences in FIG. 11, an alternative consensus sequence is VL CDR2 motif 3:

Y₁Y₂Y₃(SEQ ID NO:136)

wherein Y is₁Is Q or N; y is₂Is D or E; and Y is₃Is K, R or H. In some embodiments, Y is₁Limited to Q; and/or Y₂Limited to D; and/or Y₃Limited to K.

For VL CDR3, one consensus sequence is VL CDR3 motif 1:

Z₁Z₂Z₃Z₄Z₅Z₆Z₇Z₈Z₉Z₁₀(SEQ ID NO:137)

wherein Z₁Is S, T, C, F, Y, M, L, V, I or A; z₂Is Q, N, S, T or C; z₃Is S, T, C, G, A, H, K, R, Q, N, Y, F or W; z₄Is A, G, S, T, C, L, I, V, M, D or E; z₅Is H, K, R, E, D, S, T or C; z₆Is V, L, I, M, A, Y, F, W, S, T or C; z₇P, S, T, C or absent; z₈S, T, C or absent; z₉Is W, P, L, I, V, M, A, F or Y; and Z₁₀Is T, S, C, V, L, I, M, A. In some embodiments, Z₁Limited to S, F, M or L; and/or Z₂Limited to Q or S; and/or Z₃Limited to S, G, H, Q or Y; and/or Z₄Limited to A, S, T, L or D; and/or Z₅Limited to H, E, D or S; and/or Z₆Limited to V, Y, F or S; and/or Z₇Limited to P, S or absent; and/or Z₈Limited to S or absent; and/or Z₉Limited to W, P, L or F; and/or Z₁₀Limited to T or V.

Of particular note are the VL CDR3 sequences of SEQ ID NOS 96-98 at position Z₅Where it is positively charged and not the other sequences in figure 11, an alternative consensus sequence is VL CDR3 motif 2:

Z₁Z₂Z₃Z₄Z₅Z₆Z₇Z₈Z₉Z₁₀(SEQ ID NO:138)

wherein Z₁Is S, T, C, F or Y; z₂Is Q or N; z₃Is S, T, C, G or A; z₄Is A, G, S, T or C; z₅Is H, K or R; z₆Is V, L, I, M or A; z₇Is P or absent; z₈Is absent; z₉Is W, P, L, I, V, M, A, F or Y; and Z₁₀Is T, S or C. In some embodiments, Z₁Limited to S or F; and/or Z₂Limited to Q; and/or Z₃Limited to S or G; and/or Z₄Limited to A, S or T; and/or Z₅Limited to H; and/or Z₆Limited to V; and/or Z₇Limited to P or absent; and/or Z₈Limited to the absence; and/or Z₉Limited to W, P, L or F; and/or Z₁₀Limited to T.

Particular attention is drawn to the VL CDR3 sequence of SEQ ID NO 99-102 at position Z₅Where it is negatively charged and not the other sequences in figure 11, an alternative consensus sequence is the VL CDR3 motif 3:

Z₁Z₂Z₃Z₄Z₅Z₆Z₇Z₈Z₉Z₁₀(SEQ ID NO:139)

wherein Z₁Is M, C, L, I, V, A; z₂Is Q or N; z₃Is H, K, R, Q, N, G, A, Y or F; z₄Is L, I, V, M, A, D or E; z₅Is E or D; z₆Is Y or F; z₇Is P; z₈Is absent; z₉Is W, P, L, I, V, M, A, F or Y; and Z₁₀Is T, S or C. In some embodiments, Z₁Limited to M or L; and/or Z₂Limited to Q; and/or Z₃Limited to H, Q, G or Y; and/or Z₄Limited to L or D; and/or Z₅Limited to E or D; and/or Z₆Limited to Y or F; and/or Z₇Limited to P; and/or Z₈Limited toIs absent; and/or Z₉Limited to W, P, L or F; and/or Z₁₀Limited to T.

It is specifically noted that the VL CDR3 sequence of SEQ ID NO. 103 is distinct from the other sequences in FIG. 11, and an alternative consensus sequence is VL CDR3 motif 4:

Z₁Z₂Z₃Z₄Z₅Z₆Z₇Z₈Z₉Z₁₀(SEQ ID NO:140)

wherein Z₁Is S, T or C; z₂Is S, T or C; z₃Is Y or F; z₄Is T, S, or C; z₅Is S, T or C; z₆Is S, T or C; z₇Is S, T or C; z₈Is S, T or C; z₉Is W, P, F or Y; and Z₁₀Is V, L, I, M, A, T, S or C. In some embodiments, Z₁Limited to S or T; and/or Z₂Limited to S or T; and/or Z₃Limited to Y; and/or Z₄Limited to T or S; and/or Z₅Limited to S or T; and/or Z₆Limited to S or T; and/or Z₇Limited to S or T; and/or Z₈Limited to S or T; and/or Z₉Limited to W, P or F; and/or Z₁₀Limited to V, L, I, T or S. In some embodiments, Z₁Limited to S; and/or Z₂Limited to S; and/or Z₃Limited to Y; and/or Z₄Limited to T; and/or Z₅Limited to S; and/or Z₆Limited to S; and/or Z₇Limited to S; and/or Z₈Limited to S; and/or Z₉Limited to W; and/or Z₁₀Limited to V.

Finally, it is specifically noted that the VL CDR3 sequence of SEQ ID NO 104 is distinct from the other sequences in FIG. 11, and an alternative consensus sequence is VL CDR3 motif 5:

Z₁Z₂Z₃Z₄Z₅Z₆Z₇Z₈Z₉Z₁₀(SEQ ID NO:141)

wherein Z₁Is Q or N; z₂Is A or G; z₃Is W, Y or F; z₄Is D or E; z₅Is S, T or C; z₆Is R, K or H; z₇Is T, S or C; z₈Is V, I, L, M or A; z₉Is V, I, L, M or A; and Z₁₀Is I, L, V, M or A. In some embodiments, Z₁Limited to Q; and/or Z₂Limited to A; and/or Z₃Limited to W; and/or Z₄Limited to D; and/or Z₅Limited to S; and/or Z₆Limited to R; and/or Z₇Limited to T; and/or Z₈Limited to V; and/or Z₉Limited to V; and/or Z₁₀Limited to I.

Variable heavy chain sequence

VH of 1N23. Positive VH clones from the 1N23 hybridoma were sequenced and found to encode 4 functional VH chains. These 4 clones were designated 1N23VH3-5, 1N23VH3-7, 1N23VH2-1 and 1N23VH 1-5.

VH of 1K23. Positive VH clones from the 1K23 hybridoma were sequenced and found to encode 6 functional VH chains. These 6 clones were named 1K23VH2-1_0910, 1K23VH1-4_0907, 1K23VH1-10_0907, 1K23VH8-4_0907, 1K23VH8-5_0907 and 1K23VH8-9_ 0907.

VH of 2K4. Positive VH clones from the 2K4 hybridoma were sequenced and found to encode 4 functional VH chains. These 4 clones were designated 2K4VH3-8, 2K4VH2-8, 2K4VH1-1 and 2K4VH 1-4.

VH of 1C9. Positive VH clones from the 1C9 hybridoma were sequenced and found to encode 8 functional VH chains. These 8 clones included 4 unique sequences designated 1C9VH2-404-8_1024, 1C9VH2-405-12_1024, 1C9VH2-411-1_1024, and 1C9VH2-406-4_ 1024.

VH of 1J20. Positive VH clones from 1J20 hybridoma were sequenced and found to encode 2 functional VH chains. These 2 clones were designated 1J20VH1-7_0910 and 1J20VH1-1-6_ 0829.

VH of 1L20. Sequencing of Positive VH clones from 1L20 hybridoma3 were found to encode functional VH chains. These 3 clones were named 1L20VH2-3_0903, 1L20VH2-1_0907 and 1L20VH2-3_ 0910.

VH of 1K3. Positive VH clones from 1K3 hybridoma were sequenced and 5 encoded functional VH chains were found. These 5 clones were designated 1K3VH6-7, 1K3VH6-8_0816, 1K3VH3-4, 1K3VH3-4 and 1K3VH3-3_ 0816.

VH of 1L5. Positive VH clones from the 1L5 hybridoma were sequenced and 9 clones were found to encode functional VH chains. These 9 clones were designated 1L5VH003-5-8_0907, 1L5VH003-6-3_0907, 1L5VH001-7-6_0907, 1L5VH001-6-5_0907, 1L5VH001-6-11_0907, 1L5VH003-6-2_0910, 1L5VH001-6-12_0907, 1L5VH003-3-4_0907 and 1L5VH003-3-8_ 0907.

Additional VH. VH sequences of 4 additional hybridoma antibodies called CTA5_ VH, CTB11_ VH, CTC2_ VH, CTD2_ VH were obtained.

VH sequence alignment. An alignment of all VH sequences described above is shown in figure 10. The figure indicates the approximate positions of the three CDR regions (bold, underlined) and the SEQ ID NOs corresponding to each sequence.

Unique VH CDR sequences. An alignment of the unique CDR sequences of VH in fig. 10 is shown in fig. 12. Of the 43 VH sequences, there were only 8 unique CDR1 sequences, 9 unique CDR2 sequences, and 10 unique CDR3 sequences as shown in fig. 12.

VH CDR consensus sequences. Based on the sequences disclosed in fig. 12, as well as the structural/functional characteristics of the naturally occurring amino acids, consensus sequences for VH CDRs can be determined.

For VH CDR1, one consensus sequence is VH CDR1 motif 1:

X₁X₂X₃X₄X₅X₆X₇X₈(SEQ ID NO:142)

wherein X₁Is G or A; x₂Is Y, F, W, D or E; x₃Is T, S, C orM；X₄Is F, Y, W, V, L, I, M or A; x₅Is T, S, C, N or Q; x₆Is S, T, C, A or G; x₇Is Y, F, W, N, Q, G or A; and X₈Is W, A, G, Y or F. In some embodiments, X₁Limited to G; and/or X₂Limited to Y, F or D; and/or X₃Limited to T or S; and/or X₄Limited to F or V; and/or X₅Limited to T, S or N; and/or X₆Limited to S, T or A; and/or X₇Limited to Y, F, N or G; and X₈Limited to W, A or Y. In some embodiments, subsequence X₁X₂X₃Limited to G Y T; and in some embodiments, subsequence X₁X₂X₃Limited to gft. Furthermore, in some embodiments, subsequence X₁X₇X₈Limited to sy W.

Of particular note are SEQ ID NOs: 109-110 and 112 the VH CDR1 sequence is distinct from the other sequences in FIG. 12, and an alternative consensus sequence is the VH CDR1 motif 2:

X₁X₂X₃X₄X₅X₆X₇X₈(SEQ ID NO:143)

wherein X₁Is G or A; x₂Is Y, F or W; x₃Is T, S, C or M; x₄Is F, Y or W; x₅Is T, S or C; x₆Is S, T or C; x₇Is Y, F or W; and X₈Is W. In some embodiments, X₁Limited to G; and/or X₂Limited to Y or F; and/or X₃Limited to T or S; and/or X₄Limited to F; and/or X₅Limited to T or S; and/or X₆Limited to S or T; and/or X₇Limited to Y or F; and X₈Limited to W. In some embodiments, subsequence X₁X₂X₃Limited to G Y T; and in some embodiments, subsequence X₁X₂X₃Limited to gft. Furthermore, in some embodiments, subsequence X₁X₇X₈Limited to sy W.

For VH CDR2, one consensus sequence is VH CDR2 motif 1:

Y₁Y₂Y₃Y₄Y₅Y₆Y₇Y₈Y₉Y₁₀(SEQ ID NO:144)

wherein Y is₁Is I, L, V, M or A; y is₂Is Y, F, H, R, K, S or T; y is₃Is P, S, T, Y, F, R, K or H; y is₄Is G, A, S, T, K, R, H, D or E; y is₅T, S or absent; y is₆R, K, H or absent; y is₇N, Q, D, E, G, A or absent; y is₈Is G, A, S, T, Y or F; y is₉Is D, E, A, G, N or Q; and Y is₁₀Is T, S, I, L, V, M, A, K, R or H. In some embodiments, Y is₁Limited to I; and/or Y₂Limited to Y, H, R, K or S; and/or Y₃Limited to P, S, Y or R; and/or Y₄Limited to G, S, K or D; and/or Y₅Limited to T or absent; and/or Y₆Limited to R or absent; and/or Y₇Limited to N, D, G or absent; and/or Y₈Limited to G, A, S or Y; and/or Y₉Limited to D, E, A or N; and/or Y₁₀Limited to T, I or K.

Of particular note are SEQ ID NOs: the VH CDR2 sequence of 120-121 is distinct from the other sequences in FIG. 12, and an alternative consensus sequence is VH CDR2 motif 2:

Y₁Y₂Y₃Y₄Y₅Y₆Y₇Y₈Y₉Y₁₀(SEQ ID NO:145)

wherein Y is₁Is I, L, V, M or A; y is₂Is Y, F, H, R, K, S or T; y is₃Is P, S, T, Y or F; y is₄Is G, A, S, T, K, R or H; y is₅T, S or absent; y is₆R, K, H or absent; y is₇N, Q, D, E or absent; y is₈Is G, A, S, T, Y or F; y is₉D, E, A, G,N or Q; and Y is₁₀Is T, S, I, L, V, M or A. In some embodiments, Y is₁Limited to I; and/or Y₂Limited to Y, H, R or S; and/or Y₃Limited to P, S or Y; and/or Y₄Limited to G, S or K; and/or Y₅Limited to T or absent; and/or Y₆Limited to R or absent; and/or Y₇Limited to N, D or absent; and/or Y₈Limited to G, A, S or Y; and/or Y₉Limited to D, E, A or N; and/or Y₁₀Limited to either T or I.

For VH CDR3, one consensus sequence is VH CDR3 motif 1:

Z₁Z₂Z₃Z₄Z₅Z₆Z₇Z₈Z₉Z₁₀Z₁₁Z₁₂Z₁₃Z₁₄Z₁₅(SEQ ID NO:146)

wherein Z₁Is A, G, V, L, I or M; z₂Is R, K, H, C or M; z₃G, A, R, K, H, S, T, Y, F, W, D, E or absent; z₄Y, F, W, N, Q, G, A, R, K, H or absent; z₅S, T, N, Q, E, D or absent; z₆D, E or absent; z₇L, I, V, M, A, S, T or absent; z₈L, I, V, M, A or absent; z₉G, A, R, K, H or absent; z₁₀I, L, V, M, A, N, Q, R, K, H or absent; z₁₁A, M, F, Y, W, S, T, G or absent; z₁₂W, Y, F, A, G or absent; z₁₃Is F, Y, W, G, A, M or C; z₁₄Is A, G, M, D, E, W, Y or F; and Z₁₅Is Y, F, W, G, A or V. In some embodiments, Z₁Limited to A or V; and/or Z₂Limited to R, K or C; and/or Z₃Limited to G, R, S, Y, D or absent; and/or Z₄Limited to Y, N, G, R or absent; and/or Z₅Limited to S, N, E or absent; and/or Z₆Limited to D or absent; and/or Z₇Limited to L, S or absent; and/or Z₈Limited to L or absent; and/or Z₉Limited to G, R or absent; and/or Z₁₀Limited to I, N, R, L or absent; and/or Z₁₁Limited to A, F, S, G or absent; and/or Z₁₂Limited to W, Y, A or absent; and/or Z₁₃Limited to F, Y, G or M; and/or Z₁₄Limited to A, D, W or Y; and/or Z₁₅Limited to Y, F, W or G.

While the disclosed subject matter has been described and illustrated in the foregoing exemplary embodiments, it is understood that the present disclosure has been made only by way of example, and that numerous changes in the details of implementation of the disclosed subject matter may be made without departing from the spirit and scope of the disclosed subject matter, which is limited only by the following claims.

Claims (17)

1. An antibody that specifically binds to a human VASA protein, comprising:

an immunoglobulin heavy chain and an immunoglobulin light chain,

wherein the variable region of the light chain comprises:

(i) a CDR1 region comprising an amino acid sequence selected from SEQ ID NOs: 83-88;

(ii) a CDR2 region comprising an amino acid sequence selected from SEQ ID NOs 89-95; and/or

(iii) A CDR3 region comprising an amino acid sequence selected from SEQ ID NOs 96-104.

2. An antibody that specifically binds to a human VASA protein, comprising:

an immunoglobulin heavy chain and an immunoglobulin light chain,

wherein the variable region of the heavy chain comprises:

(i) a CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NO 105-112;

(ii) a CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NO 113-121; and/or

(iii) A CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 122-131.

3. An antibody that specifically binds to a human VASA protein, comprising:

an immunoglobulin heavy chain and an immunoglobulin light chain,

wherein the variable region of the light chain comprises:

(i) a CDR1 region comprising an amino acid sequence selected from VL CDR1 motifs 1-2;

(ii) a CDR2 region comprising an amino acid sequence selected from VL CDR2 motifs 1-3; and/or

(iii) A CDR3 region comprising an amino acid sequence selected from VL CDR3 motifs 1-5.

4. An antibody that specifically binds to a human VASA protein, comprising:

an immunoglobulin heavy chain and an immunoglobulin light chain,

wherein the variable region of the heavy chain comprises:

(i) a CDR1 region comprising an amino acid sequence selected from VH CDR1 motifs 1-2;

(ii) a CDR2 region comprising an amino acid sequence selected from VL CDR2 motifs 1-2; and/or

(iii) A CDR3 region comprising an amino acid sequence selected from VL CDR3 motif 1.

5. An antibody preparation comprising the antibody of any one of claims 1-4.

6. The antibody preparation of claim 5, wherein the preparation is a monoclonal antibody preparation.

7. The antibody preparation of claim 5, wherein the preparation is a mixture of at least two monoclonal antibody preparations.

8. An isolated nucleic acid molecule encoding the heavy or light chain of any one of claims 1-4.

9. The isolated nucleic acid molecule of claim 8, wherein the nucleic acid is selected from the group consisting of a cloning vector, an expression vector, a heterologous recombinant vector, and a viral integration vector.

10. A cell transformed with a nucleic acid according to any one of claims 8 to 9.

11. The cell of claim 10, wherein the cell is a mammalian cell.

12. The cell of claim 11, wherein the cell is a rodent cell.

13. The cell of claim 11, wherein the cell is a CHO cell.

14. The cell of claim 11, wherein the cell is a human cell.

15. A method of isolating a cell expressing a VASA protein, comprising:

(a) obtaining a population of cells;

(b) contacting the population of cells with a plurality of antibodies of any one of claims 1-4; and

(c) separating cells in the population that specifically bind to the antibody from cells in the population that do not specifically bind to the antibody.

16. The method of claim 15, wherein the cells are separated by fluorescence activated cell sorting.

17. The method of claim 15, wherein the cells are separated by fluorescence activated cell sorting using an immobilized secondary antibody.