HK1234068A - Novel modulators and methods of use - Google Patents

Description

Novel modulators and methods of use

The application is a divisional application of an invention patent application with the application date of 2011, 12 and 7, the application number of 201180065609.1 and the name of 'a novel regulator and a use method'.

Cross reference to the application

The present application claims priority from united states provisional application serial No. 61/421,157, filed 12/8/2010, and priority from Patent Cooperation Treaty (PCT) No. PCT/US2011/050451, filed 9/2/2011, each of which is incorporated herein by reference in its entirety.

Sequence listing

This application contains a sequence listing that has been submitted through the EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on day 22 of 11/2011 was named 11200pct.txt and was 80102 bytes in size.

Technical Field

The present application relates generally to novel compositions and methods for their use in preventing, treating or ameliorating hyperproliferative disorders and any expansion, recurrence or metastasis thereof. In a broad aspect, the invention relates to the use of ephrin-a ligand (EFNA) modulators, including anti-EFNA antibodies and fusion constructs, for the treatment or prevention of neoplastic disorders. A particularly preferred embodiment of the invention provides for the use of such EFNA modulators for immunotherapy of malignancies, including the reduction of tumor initiating cell frequency.

Background

Stem and progenitor cell differentiation and cell proliferation are normally ongoing processes that act synergistically to support tissue growth during organogenesis and cellular replacement and repair of most tissues throughout the life of all living organisms. Differentiation and proliferation decisions are usually governed by a multitude of factors and signals that are balanced to maintain cell fate decisions and tissue architecture. Normal tissue architecture is maintained primarily by cells in response to microenvironment cues that regulate cell division and tissue maturation. Thus, cell proliferation and differentiation typically occurs only for the purpose of replacing damaged or dying cells or for growth needs. Unfortunately, disruption of cell proliferation and/or differentiation can result from a number of factors, including, for example, deficiencies and overabundance of various signaling chemicals, the presence of altered microenvironments, genetic mutations, or some combination thereof. When normal cell proliferation and/or differentiation is disturbed or somehow disrupted, it can lead to various diseases or disorders, including hyperproliferative disorders such as cancer.

Conventional cancer treatments include chemotherapy, radiation therapy, surgery, immunotherapy (e.g., biological response modifiers, vaccines or targeted therapeutics) or combinations thereof. Unfortunately, too many cancers respond to such conventional treatments in no or minimal ways, leaving the patient with few options. For example, in certain patients, certain cancers exhibit genetic mutations that render them unresponsive, although the selected therapy is generally effective. Furthermore, depending on the type of cancer, some available treatments (e.g., surgery) may not be a viable alternative. Limitations inherent to current standard of care treatments are particularly evident when attempting to care for patients who have undergone prior treatment and subsequently relapsed. In this situation, a failed treatment regimen and the resulting patient exacerbation may contribute to refractory tumors, which often manifest themselves as more aggressive diseases that ultimately prove incurable. Although there have been great improvements in the diagnosis and treatment of cancer over the years, the overall survival rate of many solid tumors remains essentially unchanged due to the inability of existing therapies to prevent relapse, tumor recurrence and metastasis. Therefore, the development of more targeted and effective therapies remains a challenge.

One promising area of research involves the use of targeted therapeutics to chase tumor-initiating "seed" cells that appear to underlie many cancers. To this end, many solid tissues are now known to contain adult, tissue-resident stem cell populations that produce differentiated cell types that comprise the majority of the tissue. Tumors arising in these tissues are similarly composed of heterogeneous cell populations that also arise from stem cells, but differ significantly in their overall proliferation and structure. While it is increasingly recognized that most tumor cells have limited proliferative capacity, a small population of cancer cells (commonly known as cancer stem cells or CSCs) have the unique ability to self-renew in large numbers, thereby conferring an inherent tumor reinitiation capability. More specifically, the cancer stem cell hypothesis proposes a distinct subset of cells (i.e., CSCs) within each tumor (approximately 0.1-10%) that are capable of self-renewal indefinitely and producing tumor cells with progressively limited replication capacity as a result of differentiation into tumor progenitor cells and subsequently terminally differentiated tumor cells.

In recent years, it has become more apparent that these CSCs (also known as tumor perpetuating cells or TPC) may be more resistant to traditional chemotherapeutic agents or radiation and therefore persist after standard of care clinical treatment to later provoke the growth of refractory tumors, secondary tumors, and promote metastasis. Furthermore, there is increasing evidence that pathways regulating organogenesis and/or normal tissue-resident stem cell self-renewal are deregulated or altered in CSCs, leading to continued expansion of self-renewing cancer cells and tumor formation. See Al-Hajj et Al, 2004, PMID: 15378087, respectively; and Dalerba et al, 2007, PMID: 17548814, respectively; each of which is incorporated herein by reference in its entirety. Thus, the effectiveness of traditional, as well as recent, targeted therapies is apparently limited by the presence and/or emergence of resistant cancer cells that are capable of perpetuating cancer even in the face of these various therapies. Huff et al, European Journal of Cancer 42: 1293 1297(2006) and Zhou et al, Nature Reviews Drug Discovery 8: 806-823(2009), each of which is incorporated herein by reference in its entirety. These observations were confirmed by: traditional tumor-reducing agents consistently fail to substantially increase patient survival when having solid tumors, and by creating an increasingly detailed understanding of how tumors grow, recur, and metastasize. Thus, recent strategies for treating neoplastic disorders recognize the importance of eliminating, depleting, silencing, or promoting differentiation of tumor perpetuating cells to reduce the likelihood of tumor recurrence, metastasis, or patient relapse.

Efforts to develop this strategy have incorporated recent work involving non-traditional xenograft (NTX) models, in which primary human solid tumor samples were exclusively implanted and passaged in immunocompromised mice. This technique demonstrates the existence of a subpopulation of cells with the unique ability to produce heterogeneous tumors and provoke their unlimited growth in a number of cancers. As previously hypothesized, work in NTX models confirmed that the identified CSC subpopulations of tumor cells appear to be more resistant to tumor-reduction protocols (e.g., chemotherapy and radiation), potentially accounting for the difference between clinical response rate and overall survival. Furthermore, the use of NTX models in CSC studies has triggered fundamental changes in drug discovery and preclinical evaluation of drug candidates, which may lead to CSC-targeted therapies with major effects on tumor recurrence and metastasis, thereby increasing patient survival. Despite advances, the inherent technical difficulties associated with the treatment of primary and/or xenograft tumor tissue, as well as the lack of an experimental platform to characterize CSC identification and differentiation potential, form major challenges. Thus, there remains a great need for selectively targeting cancer stem cells and developing diagnostic, prophylactic or therapeutic compounds or methods that can be used to treat, prevent and/or manage hyperproliferative disorders.

Summary of The Invention

These and other objects provided by the present invention relate broadly to methods, compounds, compositions and articles of manufacture useful for treating EFNA associated disorders (e.g., hyperproliferative disorders or neoplastic disorders). To this end, the present invention provides novel EFNA (or ephrin-a ligand) modulators that effectively target tumor cells or cancer stem cells and can be used to treat patients with various malignancies. As will be discussed in greater detail herein, there are currently six known ephrin-a ligands (i.e., EFNA1-6) and the disclosed modulators may comprise or bind to any one, or more than one, ephrin-a ligand. Furthermore, in certain embodiments, the disclosed EFNA modulators may comprise any of the following compounds: that recognizes, agonizes, antagonizes, or competes with, interacts with, binds to, or associates with an EFNA polypeptide, its receptor, or its gene; and modulating, altering or modifying the effect of the EFNA protein on one or more physiological pathways. Thus, in broad terms the present invention relates to isolated EFNA modulators. In preferred embodiments, the invention more specifically relates to an isolated EFNA1 modulator or an isolated EFNA4 modulator (i.e., a modulator comprising or associated with at least EFNA1 or EFNA 4). Furthermore, such modulators may be used to provide pharmaceutical compositions, as discussed in detail below.

In selected embodiments of the invention, the EFNA modulator may comprise ephrin-a ligand itself or a fragment thereof, in isolated form or fused or otherwise associated with other moieties (e.g., Fc-EFNA, PEG-EFNA or EFNA associated with a targeting moiety). In other selected embodiments the EFNA modulator may comprise an EFNA antagonist, which for purposes of this application shall be taken to mean any of the following constructs or compounds: which recognizes or competes with, interacts with, binds to or associates with EFNA and neutralizes, eliminates, reduces, sensitizes, reprograms, inhibits or controls the growth of neoplastic cells, including tumor initiating cells. In preferred embodiments, EFNA modulators of the invention include anti-EFNA antibodies or fragments or derivatives thereof that have unexpectedly been found to silence, neutralize, reduce, deplete, modulate, reduce, reprogram, eliminate, or otherwise inhibit the ability of tumor initiating cells to multiply, maintain, expand, proliferate, or otherwise promote the survival, recurrence, regeneration, and/or metastasis of neoplastic cells. In particularly preferred embodiments, the antibody or immunoreactive fragment may be conjugated or conjugated to one or more anti-cancer agents.

In one embodiment, the EFNA modulator may comprise a humanized antibody, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 149, SEQ ID NO: 153, SEQ ID NO: 157 and SEQ ID NO: 161 and a heavy chain variable region amino acid sequence selected from SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 163, or a light chain variable region amino acid sequence. In other preferred embodiments the invention will be in the form of a composition comprising a humanized antibody selected from the group consisting of hsc4.5, hsc4.15, hsc4.22 and hsc4.47 and a pharmaceutically acceptable carrier. In another preferred embodiment, the EFNA modulator may comprise an antibody comprising one or more CDRs from FIG. 7A (SEQ ID NOS: 8-59 and 70-95). Preferably, an antibody comprising at least one CDR from fig. 7A will comprise a humanized antibody.

In certain other embodiments, the invention will include EFNA modulators that reduce the frequency of tumor initiating cells upon administration to a subject. Preferably, the reduction in frequency will be determined using in vitro or in vivo limiting dilution analysis. In particularly preferred embodiments, such assays can be performed using in vivo limiting dilution assays that include transplantation of live human tumor cells into immunocompromised mice. Alternatively, the limiting dilution assay may be performed using an in vitro limiting dilution assay comprising limiting dilution deposition of live human tumor cells into in vitro colony supporting conditions. In either case, the reduction in analysis, calculation, or quantification frequency will preferably include the use of poisson distribution statistics to provide accurate calculations. It will be understood that while such quantitative methods are preferred, other less labor intensive methods (e.g., flow cytometry or immunohistochemistry) may also be used to provide the desired values, and thus such methods are expressly contemplated as being within the scope of the present invention. In these cases, the reduction in frequency can be determined by flow cytometry analysis or immunohistochemical detection of tumor cell surface markers known to enrich for tumor initiating cells.

Thus, another preferred embodiment of the present invention includes a method of treating an EFNA associated disorder comprising administering to a subject in need thereof a therapeutically effective amount of an EFNA modulator thereby reducing the frequency of tumor initiating cells. Again, the reduction in tumor initiating cell frequency will preferably be determined using in vitro or in vivo limiting dilution analysis.

In this regard, it will be appreciated that the present invention is based, at least in part, on the following findings: EFNA polypeptides (particularly EFNA4, as discussed below) are associated with tumor perpetuating cells (i.e., cancer stem cells) involved in the pathogenesis of various tumors. More specifically, the present application surprisingly shows that administration of various exemplary EFNA modulators is capable of mediating, reducing, inhibiting, or eliminating tumorigenic signaling (i.e., reducing the frequency of tumor initiating cells) of tumor initiating cells. This reduced signaling, whether by reducing, eliminating, reprogramming or silencing tumor initiating cells or by modifying tumor cell morphology (e.g., induced differentiation, niche disruption), in turn allows for more effective treatment of EFNA-associated disorders by inhibiting tumorigenesis, tumor maintenance, expansion and/or metastasis, and recurrence. In other embodiments, the disclosed modulators may promote, support, or otherwise enhance EFNA-mediated signaling that may limit or inhibit tumor growth. In other embodiments, the disclosed modulators may interfere with, inhibit, or otherwise slow EFNA-mediated signaling that may provoke tumor growth. In addition, as will be discussed in more detail below, EFNA polypeptides are involved in generating adhesion and repulsion forces between cells through integrin and cytoskeletal rearrangements. Intervention in such cell-cell interactions using the novel EFNA modulators described herein may thus improve the condition by more than one mechanism (i.e., tumor-initiated cytoreduction and disruption of cell adhesion) to provide additive or synergistic effects. Other preferred embodiments may utilize endocytosis of ephrin-a ligands to deliver modulator-mediated anti-cancer agents. In this regard, it will be understood that the invention is not limited to any particular mechanism of action but encompasses the broad use of the disclosed modulators to treat EFNA associated disorders, including various tumors.

Accordingly, another preferred embodiment of the present invention includes a method of treating an EFNA associated disorder in a subject in need thereof, said method comprising the step of administering to said subject an EFNA modulator. In particularly preferred embodiments, the EFNA modulator will bind to (e.g., conjugate with) an anti-cancer agent. In addition, beneficial aspects of the invention include any cell adhesion disruption and related benefits that can be achieved whether the subject tumor tissue exhibits elevated or reduced levels of EFNA as compared to normal adjacent tissue.

As described above and discussed in more detail below, there are currently six known ephrin-A ligands (i.e., EFNA 1-6). It will be understood in accordance with the present invention that the disclosed modulators may be generated, manufactured and/or selected to react with a single ephrin-a ligand (e.g., EFNA4), a subset of ephrin-a ligands (e.g., EFNA4 and EFNA1) or all six ephrin-a ligands. More particularly, as described herein and shown in the examples below, preferred modulators (e.g., antibodies) may be generated and selected such that they react or bind to a domain or epitope expressed on a single ephrin-a ligand, or to a domain or epitope that is conserved (at least to some extent) and presented in multiple or all EFNA polypeptides (e.g., EFNA1 and 4 or EFNA3 and 4). This is significant with respect to the present invention in the following respects: as shown in example 18 below, it was discovered that certain ephrin-a ligands are preferentially expressed on TICs, and when combined they can serve as particularly effective therapeutic targets that provide for the selective reduction of the frequency of tumorigenic cells and/or the depletion of cancer stem cell populations.

Thus, in selected embodiments, the invention encompasses pan-EFNA modulators that immunospecifically bind to two or more ephrin-a ligands. In such embodiments, the selected modulator may be generated by immunization with a particular ligand (e.g., EFNA4) and it binds or cross-reacts more or less with the various test ligands. Thus, in other embodiments, the invention encompasses methods of treating a subject in need thereof comprising administering a therapeutically effective amount of a pan-EFNA modulator. Other embodiments include methods of treating a subject in need thereof comprising administering a therapeutically effective amount of an EFNA modulator that immunospecifically binds to one or more ephrin-a ligands.

Thus, in other embodiments, the present invention will comprise pan-EFNA modulators. In other embodiments, the invention will comprise a method of treating an EFNA associated disorder in a subject in need thereof, said method comprising the step of administering to said subject a pan-EFNA modulator.

Of course, it will be understood that the disclosed EFNA modulators may be generated, manufactured and/or selected to preferentially react or bind with a single ephrin-a ligand (e.g., EFNA4) while exhibiting minimal or no binding to any other ephrin-a ligand. Accordingly, other embodiments of the present invention relate to the following EFNA modulators: it immunospecifically binds to the selected ephrin-a ligand and shows little or no binding to any other ephrin-a ligand. In this regard, preferred embodiments disclosed herein will include methods of treating an EFNA associated disorder in a subject in need thereof, the methods comprising the step of administering an EFNA modulator, wherein the EFNA modulator immunospecifically binds to a selected ephrin-a ligand and is substantially non-reactive with any other ephrin-a ligand. Furthermore, methods of producing, manufacturing, and selecting such modulators are within the scope of the present invention.

Other aspects of the invention exploit the ability of the disclosed modulators to potentially disrupt cell adhesion interactions while simultaneously silencing tumor initiating cells. Such multi-active EFNA modulators (e.g., EFNA antagonists) may prove particularly effective when used in combination with standard of care anti-cancer agents or tumor-reducing agents. Furthermore, two or more EFNA antagonists (e.g., antibodies that specifically bind to two separate epitopes on ephrin-a ligands or bind to separate ligands) may be used in combination according to the present teachings. Furthermore, as discussed in detail below, the EFNA modulators of the present invention may be used in a conjugated or unconjugated state and optionally in combination with various chemical or biological anti-cancer agents as sensitizers.

Accordingly, another preferred embodiment of the present invention includes a method of sensitizing a tumor in a subject for treatment with an anti-cancer agent comprising the step of administering to the subject an EFNA modulator. In a particularly preferred aspect of the invention, the EFNA modulator will specifically cause a reduction in the frequency of tumor initiating cells (as determined using in vitro or in vivo limiting dilution assays) to sensitize the tumor for a concomitant or subsequent reduction in volume.

Similarly, since the compounds of the invention may exert therapeutic benefits through a variety of physiological mechanisms, the invention also relates to selected effectors or modulators that are specifically made to exploit certain cellular processes. For example, in certain embodiments, preferred modulators may be engineered to bind to EFNA on or near the surface of tumor initiating cells and stimulate an immune response in a subject. In other embodiments, the modulator may comprise antibodies directed to epitopes wherein the ephrin-a ligand activity and interaction with the ephrin receptor may affect the attachment and repulsion forces between cells through integrin and cytoskeleton rearrangement. In other embodiments, the disclosed modulators may function by depleting or eliminating EFNA-binding cells. Thus, it is important to understand that the present invention is not limited to any particular mode of action but encompasses any method or EFNA modulator that achieves the desired result.

Within such framework, preferred embodiments of the disclosed embodiments relate to methods of treating a subject having a neoplastic disorder, the method comprising the step of administering a therapeutically effective amount of at least one neutralizing EFNA modulator.

Other embodiments relate to methods of treating a subject having an EFNA associated disorder comprising the step of administering a therapeutically effective amount of at least one depleting EFNA modulator. A related method involves depleting EFNA binding cells in a subject in need thereof, the method comprising the step of administering an EFNA modulator.

In another embodiment, the invention provides a method of maintenance therapy wherein the disclosed effector or modulator is administered for a period of time after an initial procedure (e.g., chemotherapy, radiation, or surgery) designed to remove at least a portion of the tumor mass. This treatment regimen may be administered over a period of weeks, months, or even years, wherein the EFNA modulator may act prophylactically to inhibit metastasis and/or tumor recurrence. In other embodiments, the disclosed modulators may be administered in conjunction with known tumor reduction regimens to prevent or slow metastasis.

In addition to the therapeutic uses discussed above, it will also be appreciated that modulators of the invention may be used to diagnose EFNA associated disorders and, in particular, hyperproliferative disorders. In certain embodiments, the modulators may be administered to a subject and detected or monitored in vivo. One skilled in the art will appreciate that the modulators may be labeled with a label or reporter as disclosed below or conjugated thereto and detected using any of several standard techniques (e.g., MRI or CAT scans). In other cases, the modulators may be used in vitro diagnostic settings using art-recognized procedures. Thus, a preferred embodiment includes a method of diagnosing a hyperproliferative disorder in a subject in need thereof, the method comprising the steps of:

a. obtaining a tissue sample from the subject;

b. contacting the tissue sample with at least one EFNA modulator; and

c. detecting or quantifying the EFNA modulator bound to the sample.

The methods are readily discernible in conjunction with the present application and can be readily implemented using generally commercially available techniques (automated plate readers, dedicated reporting subsystems, etc.). In selected embodiments, the EFNA modulator will bind to tumor perpetuating cells present in the sample. In other preferred embodiments, the detecting or quantifying step will comprise reducing the frequency of tumor initiating cells and their detection. Furthermore, limiting dilution analysis may be performed as previously described above and will preferably employ the use of poisson distribution statistics to provide accurate calculations regarding frequency reduction.

Similarly, the invention also provides kits useful for diagnosing and monitoring EFNA associated disorders (e.g., cancer). To this end, the present invention preferably provides an article of manufacture useful for diagnosing or treating an EFNA associated disorder comprising a container containing an EFNA modulator and instructional material for using said EFNA modulator to treat or diagnose an EFNA associated disorder.

Other preferred embodiments of the invention also utilize the properties of the disclosed modulators as a means that can be used to identify, isolate, grade, or enrich a population or sub-population of tumor initiating cells by methods such as Fluorescence Activated Cell Sorting (FACS) or laser-mediated grading.

Thus, another preferred embodiment of the present invention relates to a method of identifying, isolating, fractionating or enriching a population of tumor initiating cells, said method comprising the step of contacting said tumor initiating cells with an EFNA modulator.

The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features, and advantages of the methods, compositions, and/or apparatus described herein and/or other subject matter will become apparent in the teachings presented herein. A summary is provided to provide an introduction to selected concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

Drawings

FIGS. 1A-C depict an alignment of the nucleic acid sequence encoding human EFNA4 (SEQ ID NO: 1), the corresponding amino acid sequence of human EFNA4 isoform a (SEQ ID NO: 2) and the human EFNA4a, b and C isoform sequences showing amino acid differences (SEQ ID NOS: 2-4), respectively, while FIGS. 1D-F depict an alignment of the nucleic acid sequence encoding human EFNA1 (SEQ ID NO: 5), the corresponding amino acid sequence of human EFNA1 isoform a (SEQ ID NO: 6) and the human EFNA1A and b isoform sequences showing amino acid differences (SEQ ID NOS: 6 and 7), respectively;

fig. 2A and 2B are diagrams depicting the following: gene expression levels of selected human ephrin-a ligands and ephrin-a receptors in untreated (fig. 2A) and irinotecan-treated (fig. 2B) mice as measured using whole transcriptome sequencing of highly enriched populations of tumor progenitor cells (TProg) and Tumor Perpetuating Cells (TPC) and non-tumorigenic cells (NTG) obtained from a subset of whole colorectal tumor specimens;

figures 3A and 3B are graphs depicting the gene expression levels of human ephrin-a 4 ligand in colorectal (fig. 3A) and pancreatic (fig. 3B) tumor samples, as measured using whole transcriptome sequencing of highly enriched tumor progenitor cells (TProg) and Tumor Perpetuating Cells (TPC), as well as populations of non-tumorigenic cells (NTG) or populations of Tumorigenic (TG) and non-tumorigenic cells (NTG);

FIG. 4 is a diagram showing the following: relative gene expression levels of human EFNA4 in highly enriched tumor progenitor (TProg) and Tumor Perpetuating Cells (TPC) populations obtained from mice harboring one of four different non-traditional xenograft (NTX) colorectal or pancreatic tumor cell lines normalized to a non-tumorigenic (NTG) enriched cell population as measured using quantitative RT-PCR;

fig. 5A and 5B are diagrams showing the following: relative gene expression levels of human EFNA4 in whole colorectal tumor samples (fig. 5A) or matched normal adjacent tissues (fig. 5B) from patients with stage I-IV disease, normalized to the mean expression in normal colon and rectal tissues as measured using RT-PCR;

FIGS. 6A-6E represent gene expression levels of the human EFNA gene, measured by RT-PCR for EFNA4 in whole tumor samples (gray dots) or matched NAT (white dots) from patients with one of eighteen different solid tumor types in FIGS. 6A and 6B, by RT-PCR for EFNA4 and EFNA1 in selected NTX tumor cell lines in FIGS. 6C and 6D, and by Western blot analysis for EFNA4 in normal tissues and selected NTX tumor cell lines in FIG. 6E;

FIGS. 7A-7R depict the sequences of several EFNA regulators, wherein FIG. 7A is a tabular display showing the genetic arrangement and heavy and light chain CDR sequences (derived from VBASE2 analysis) of discrete EFNA regulators isolated and cloned as described herein, FIGS. 7B-7N provide murine heavy and light chain variable region nucleic acid and amino acid sequences for the same regulators shown in FIG. 7A, and FIGS. 7O-7R provide heavy and light chain variable region nucleic acid and amino acid sequences of exemplary humanized versions of the disclosed EFNA regulators;

FIGS. 8A-8D show biochemical and immunological properties of exemplary modulators, as represented in tabular form in FIG. 8A; in fig. 8B and 8C are comparisons of the respective affinities of murine SC4.47 and humanized SC4.47, as determined by label-free interaction analysis using serial dilutions of fixed amounts of antibody and antigen; FIG. 8D is a tabular comparison of the properties of selected humanized and murine modulators;

FIG. 9 shows the cell surface binding properties of fifty exemplary ephrin-A ligand modulators of the invention with respect to Jurkat E6 cells and Z138 cells, respectively;

FIGS. 10A and 10B depict the binding of ephrin-A ligand to ephrin-A receptor expressing cells in a dose-dependent manner (FIG. 10A) and the inhibition of ephrin-A ligand cell surface binding by exposure to exemplary disclosed modulators (FIG. 10B);

11A-11D are diagrams showing the following: the ability of the disclosed modulators to inhibit cell surface binding of human and murine ephrin-a ligands, wherein fig. 11A shows a positive control curve and fig. 11B-11D show the ability of three exemplary EFNA modulators to reduce ligand binding;

FIGS. 12A-12E are diagrams showing the following: the ability of modulators of the invention to inhibit cell surface binding of soluble ephrin-a receptors, wherein figure 12A provides a standard curve for receptor binding, figure 12B shows that the characteristics of exemplary modulators are varied as the concentration of soluble receptors, figure 12C shows the results of varying the concentration of modulators while keeping the amount of receptor stable, figures 12D and 12E show the ability of the modulators to inhibit ephrin-a receptors from binding ephrin-a 4 and ephrin-a 1 ligands, respectively;

FIGS. 13A-13C show the ability of selected modulators of the invention to cross-react with the mouse ortholog of ephrin-A4 ligand, where FIG. 13A shows a non-reactive modulator, while FIGS. 13B and 13C show cross-reactive murine and humanized modulators, respectively;

FIGS. 14A-14D show that the expression of ephrin-A ligand is up-regulated in whole colorectal tumor samples (FIG. 14A) and tumorigenic subsets of colorectal NTX tumor cells (FIG. 14B) and tumorigenic subsets of lung NTX cell lines (FIG. 14D), but not on normal peripheral blood mononuclear cells (FIG. 14C);

FIGS. 15A-15D show the ability of selected modulators of the present invention to internalize upon binding to an ephrin-A ligand, wherein FIG. 15A shows the fluorescence migration associated with three exemplary modulators, FIG. 15B shows nineteen of the disclosed modulators show mean fluorescence intensity indicative of internalization, FIG. 15C shows relatively small internalization in low EFNA expressing cells, and FIG. 15D shows substantial internalization in cells expressing high levels of EFNA;

FIGS. 16A-16F provide the following evidence: the disclosed modulators can be effectively used as targeting moieties to direct cytotoxic payloads to cells expressing ephrin-a ligands, where downward slope curves indicate cell killing by internalization, where fig. 16A shows the killing effect of modulator SC4.5, fig. 16B shows the ability of the selected modulator to internalize and kill lung and skin NTX tumor cell lines, fig. 16C and 16D show that the modulator carries the bound cytotoxin into HEK293 fn 293T cells (fig. 16C) and HEK-. hea 4 cells (fig. 16D), fig. 16E shows that the humanized modulator reacts similarly, and fig. 16F shows that the target cells expressing mouse or human ephrin-a ligands are killed (note that the modulator can be referred to as E instead of SC4 throughout fig. 16);

FIGS. 17A-17E are graphical representations of various aspects of biochemical assays demonstrating the ability of the disclosed modulators to detect secreted ephrin-A ligand, where FIG. 17A provides a standard curve, FIG. 17B quantifies the level of secreted EFNA from selected hematologic tumors, FIG. 17C presents a correlation between tumor volume and secreted EFNA, FIG. 17D establishes the range of circulating ephrin-A ligand in healthy adults, and FIG. 17E shows that patients with selected solid tumors have significantly higher levels of circulating ephrin-A ligand;

figures 18A-18C are pictorial illustrations showing that various ephrin-a ligand modulators can be used as targeting moieties to bind cytotoxic payloads to selected cells, wherein downward slope curves indicate cell killing by internalized toxins, and wherein figures 18A-18C particularly show the ability of the modulators sc4.2.1 (or E2.1) and SC9.65 (or 9M065) to mediate killing of HEK293T cells overexpressing ephrin-a 4 ligand (figure 18A), ephrin-A3 ligand (figure 18B), and ephrin-a 1 ligand (figure 18C) in the presence of bound saponin;

figures 19A and 19B show the ability of ephrin-a ligand to interact selectively with numerous EPHA receptors, with HEK293T cells binding to the EPHA-ECD-Fc receptor construct to a limited extent only by endogenously expressed ephrin-a ligand (figure 19A), while HEK293t.hefna4 cells bind to all tested EPHA receptor constructs to various extents, except for EPHA1 that did not bind (figure 19B); and

FIGS. 20A and 20B show the ability of ephrin-A ligands to selectively interact with the EPHB receptors, where HEK293T cells interacted to a limited extent only with the EPHB-ECD-Fc receptor construct by endogenously expressed ephrin-A ligands (FIG. 20A), while HEK293T. hENFAA 4 cells bound to the EphB2 receptor but not to the EphB3 and EphB4 receptors (FIG. 20B).

Detailed Description

I. Introduction to the design reside in

While this invention is susceptible of embodiment in many different forms, there is disclosed herein certain illustrative embodiments of the invention which illustrate the principles of the invention. It should be emphasized that the invention is not limited to the particular embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As previously described, it has surprisingly been found that expression of ephrin-a ligand (or EFNA) is associated with neoplastic growth and hyperproliferative disorders, and that the ligand provides a useful tumor marker that can be employed in the treatment of related diseases. More specifically, it has been found that such EFNA modulators (such as those described herein) can be advantageously used for the diagnosis, theranostic, treatment, or prevention of a neoplastic disorder in a subject in need thereof. Thus, while preferred embodiments of the present invention will be discussed in detail below, particularly in the context of cancer stem cells and their interaction with the disclosed modulators, those skilled in the art will appreciate that the scope of the present invention is not limited by the exemplary embodiments. Rather, the present invention and the appended claims relate broadly and specifically to EFNA modulators and their use in the diagnosis, theranosis, treatment, or prevention of various EFNA associated or mediated disorders, including neoplastic or hyperproliferative disorders, regardless of any particular mechanism of action or specifically targeted tumor components.

It will also be appreciated that, contrary to the disclosure of much of the prior art, the present invention is primarily directed to ephrin ligand modulators (i.e., EFNs) rather than ephrin receptor (i.e., EPH) modulators. That is, while the ephrin receptor is widely implicated in several disorders and is generally targeted for therapeutic intervention, ephrin ligands have attracted much less attention to date. This is in part probably due to the cluttered behavior attributed to the ligands and the erroneous belief that these various interactions make them unreliable therapeutic targets, as pathway redundancy would likely compensate for any ligand antagonism. However, as shown herein, the disclosed ephrin-a ligand modulators can be effectively used to target and eliminate, or otherwise incapacitate, tumorigenic cells. Furthermore, in selected embodiments, the invention encompasses pan-EFNA modulators that bind to or react with more than one ephrin-a ligand to provide unexpected additive or synergistic effects that may allow silencing of more than one ephrin ligand-mediated pathway.

In addition to the general associations discussed above, the inventors have also discovered a hitherto unknown phenotypic association between selected "tumor initiating cells" (TICs) and ephrin-a ligands. In this regard, it was found that selected TICs express elevated levels of ephrin-a ligand when compared to normal tissues and non-tumorigenic cells (NTGs), which together comprise a substantial portion of a solid tumor. Thus, the ephrin-a ligand includes a tumor binding marker (or antigen) and has been found to provide effective reagents for detecting and inhibiting TIC and related neoplasia due to elevated protein levels on the cell surface or in the tumor microenvironment. More specifically, EFNA modulators (including immunoreactive antagonists and antibodies that bind to or react with the protein) have been found to effectively reduce the frequency of tumor initiating cells and thus may be used to eliminate, disable, reduce, promote differentiation of, or otherwise eliminate or limit the ability of these tumor initiating cells to latently and/or continue to provoke tumor growth, metastasis or recurrence in a patient. As discussed in more detail below, the TIC tumour cell subpopulation is composed of Tumour Perpetuating Cells (TPC) and highly proliferative tumour progenitor cells (TProg).

In view of these findings, those skilled in the art will appreciate that the present invention also provides EFNA modulators and their use in reducing the frequency of tumor initiating cells. As will be discussed in greater detail below, EFNA modulators of the present invention broadly encompass any compound that recognizes, responds, competes, antagonizes, interacts, binds, agonizes, or associates with an ephrin-a ligand or gene thereof. Through these interactions, EFNA modulators thereby reduce or mitigate the frequency of tumor initiating cells. Exemplary modulators disclosed herein include nucleotides, oligonucleotides, polynucleotides, peptides, or polypeptides. In certain preferred embodiments, the modulator selected will comprise an antibody to EFNA or an immunoreactive fragment or derivative thereof. The antibody may be antagonistic or agonistic in nature and may optionally be conjugated or conjugated to a cytotoxic agent. In other embodiments, modulators of the invention will include EFNA constructs comprising ephrin-a ligands or reactive fragments thereof. It will be appreciated that such constructs may comprise fusion proteins and may comprise reactive domains from other polypeptides (e.g. immunoglobulins or biological response modifiers). In other aspects, an EFNA modulator will comprise a nucleic acid assembly that exerts a desired effect at the genomic level. Other modulators compatible with the present teachings will be discussed in detail below.

Whatever form of modulator is ultimately selected, it will preferably be in an isolated and purified state prior to introduction into a subject. In this regard, the term "isolated EFNA modulator" should be interpreted in a broad sense and in accordance with standard pharmaceutical practice shall mean any preparation or composition comprising the modulator in a state substantially free of undesirable contaminants (biological or otherwise). As will be discussed in detail below, these preparations can be purified and formulated as desired using various art-recognized techniques. Of course, it will be understood that such "isolated" preparations may be intentionally formulated or combined with inert or active ingredients as necessary to improve the commercial, manufacturing or therapeutic aspects of the finished product and to provide pharmaceutical compositions.

EFNA physiology

Ephrin receptor tyrosine kinases (EPHs) (type I transmembrane proteins) comprise the largest family of receptor tyrosine kinases in the genome of animals and interact with ephrin ligands (EFNs), which are also cell surface bound. Receptors in the EPH subfamily typically have a single kinase domain and an extracellular region containing a Cys-rich domain and 2 fibronectin type III repeats. It is routinely believed that ephrin receptors are divided into two groups based on the similarity of their extracellular domain sequences and their affinity for binding ephrin-a and ephrin-B ligands. Previous studies have shown that EPH-mediated signaling events control multiple aspects of embryonic development (particularly in the nervous system) and are important mediators of cell-cell communication that regulate cell attachment, shape, and mobility. In addition, many members of the ephrin receptor family have been identified as important markers and/or regulators of cancer development and progression relative to ephrin ligands. To date, nine ephrin-a receptors and six ephrin-B receptors are known.

For the purposes of this application, the terms "ephrin receptor," "ephrin-a receptor," "ephrin-B receptor," "EPHA," or "EPHB" (or EPHA or EPHB) are used interchangeably and are considered to refer to the family of receptors, subfamily of receptors, or single receptor (i.e., EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6) as indicated above and below.

Based on sequence analysis, ephrin ligands can be divided into two groups: six ephrin-a ligands (or EFNAs), typically anchored to the cell surface through glycosylphosphatidylinositol linkages (although some non-GPI-anchored proteins are produced by alternative splicing of ephrin mRNA, such as EFNA 4); and three ephrin-B ligands (or EFNBs) containing a transmembrane domain and a short cytoplasmic region with conserved tyrosine residues and PDZ-binding motifs. EFNA ligands preferentially interact with any of nine different EPHA receptors and EFNB ligands preferentially interact with any of six different EPHB receptors, although certain specific EFNA-EPHB and EFNB-EPHA cross-interactions have been reported.

For the purposes of this application, the terms "ephrin ligand," "ephrin-a ligand," "ephrin-B ligand," "EFNA," or "EFNB" are used interchangeably and refer to a receptor family, receptor subfamily, or single receptor (i.e., EFNA1, EFNA2, EFNA3, EFNA4, EFNA5, EFNA6, EFNB1, EFNB2, EFNB3), as the context dictates. For example, the terms "ephrin-a 4", "ephrin-a 4 ligand" or "EFNA 4" should be considered to designate the same family of protein isoforms (e.g., as shown in fig. 1C), while the terms "ephrin-a ligand" and "ENFA" should be considered to refer to the subfamily of ephrin (i.e., a rather than B) that contains all six type a ligands and any isoforms thereof. In this regard, "ephrin-a modulator," "ephrin-a ligand modulator," or "EFNA modulator" refers to any modulator (as defined herein) that associates, binds, or reacts with one or more type a ligands or isoforms, or fragments or derivatives thereof.

A more detailed summary of ephrin receptor and ligand nomenclature can be found in table 1 below.

TABLE 1

Eph nomenclature Committee, cell.1997; 90(3) 403-4, which is hereby incorporated by reference in its entirety.

As with all cell surface receptor-ligand interactions, engagement of the ephrin receptor by an ephrin ligand ultimately leads to activation of intracellular signaling cascades. Although receptor-ligand interactions can occur between molecules on the same cell surface (cis interactions), it is generally believed that cis interactions do not result in triggering a signaling cascade, or that cis interactions may actually antagonize a signaling cascade initiated by trans interactions (e.g., between a receptor and a ligand on separate cells). One unique aspect of EPH-EFN trans-interaction is the ability to trigger two signaling cascades following receptor-ligand engagement-a forward signaling cascade in cells expressing the ephrin receptor, and a reverse signaling cascade in cells expressing the ephrin ligand. Activation of two separate signaling cascades can reflect the cell sorting and cell localization processes evolved by EPH and EFN to coordinate in animal embryonic development.

EPH-EFN signaling often activates cell signaling pathways that regulate cytoskeletal dynamics and cause modulation of adhesion and repulsion interactions between different cell types. In general, the levels of EPH and EFN proteins during embryogenesis were found to be much higher than those observed in adult tissues, although continued low level expression in adults with well-defined structures resulting from migration of differentiating cells from their source at tissue stem cells in the crypts to their final location at the villus surface towards the intestinal lumen may reflect the role of these molecules in normal function of the tissues (e.g., adult intestine). Since the ephrin receptor was first identified in hepatocellular carcinoma and EPH and EFN expression is generally restricted to adults, reactivation of the ephrin ligand and/or the expression of the ephrin receptor in human cancers can be linked to: dedifferentiation of cancer cells and/or the ability of these cancer cells to invade surrounding normal tissues and migrate from the site of the primary tumor to a distant location. Other studies have shown that EPH-EFN interactions also play a role in angiogenesis.

Consistent with the finding that the interaction of EPH-EFN in non-lymphoid tissues regulates cellular interactions by generating intercellular adhesion or repulsion through integrins and cytoskeletal rearrangement, it is shown that the EPH and EFN molecules found on lymphocytes mediate cell adhesion, chemotaxis and cell migration with extracellular matrix components. For example, EFNA1 (which binds to EphA2 receptor and comprises an amino acid sequence as in Genbank accession NM _ 004428) was found to engage primary CD4 and CD8T cells to stimulate cell migration and enhance chemotaxis. Similar to EFNA1, EFNA4 is expressed on primary CD4T cells, and it is unclear whether EFNA4 engagement has a similar effect on these cells due to the promiscuity of EPH-EFN interactions. However, it was shown that mature human B-lymphocytes express EFNA4 and secrete it upon activation. Furthermore, unlike any other EFN or EPH molecule, EFNA4 is also consistently expressed on or by B cells of Chronic Lymphocytic Leukemia (CLL) patients. Interestingly, expression of EFNA4 isoform as measured by Q-PCR can be correlated with clinical manifestations of disease. Furthermore, B cells from CLL patients known to have increased expression of EFNA4 showed impairment of transendothelial migration potential compared to B cells from healthy individuals. Apparently, conjugation of EFNA4 reduced the ability of CLL cells to attach extracellular matrix molecules and reduced their chemotactic response to CCL 1. Taken together, these reports suggest a role for EFNA4 in B and T cell trafficking, when viewed in combination with the intracellular signaling data discussed above, makes ephrin-a ligands (particularly EFNA4) a very attractive target for the development of anti-cancer therapeutics.

In addition to the foregoing features, the present disclosure shows that expression of EFNA4 is elevated in various cancer stem cell populations. Together with the concomitant upregulation of several EPHA receptors in large tumors, this raises the following possibilities: EFNA 4-mediated ligand receptor interactions can trigger cellular signaling cascades linked to tumor proliferation, angiogenesis, and/or tumor metastasis. While not wishing to be bound by any particular theory, it is believed that EFNA4 modulators of the present invention (particularly antagonistic or neutralizing embodiments) function (at least in part) by: reducing or eliminating tumor initiating cell frequency to interfere with tumor proliferation or survival in a manner different from traditional standard of care treatment regimens (e.g., irinotecan), or by immunotherapeutic signaling or delivering a payload capable of killing EFNA 4-expressing cells. For example, elimination of TPC by antagonizing EFNA4 may involve simply promoting cell proliferation in the face of chemotherapeutic regimens that eliminate proliferating cells, or promoting differentiation of TPC so that their self-renewal (i.e., unlimited proliferation and maintenance of multipotency) capability is lost. Alternatively, in preferred embodiments, recruitment of cytotoxic T cells to attack EFNA 4-expressing cells, or delivery of a potent toxin conjugated to an anti-EFNA 4 antibody capable of internalization, may selectively kill or otherwise disable TPC.

As used herein, the term EFNA4 (also known as a ligand for eph-related kinase 4, LERK 4; or eph-related receptor tyrosine kinase ligand 4, EFL-4) refers to naturally occurring human EFNA4, unless the context indicates otherwise. Representative orthologs of EFNA4 protein include, but are not limited to, human (i.e., hEFNA4, NP _005218, NP _872631 or NP _872632), mouse (NP _031936), chimpanzee (XP _001153095, XP _001152971, XP _524893 and XP _001152916) and rat (NP _ 001101162). The transcribed human EFNA4 gene comprises at least 5817bp from chromosome 1. Three mRNA transcript variants are described, each of which results from alternative splicing of transcribed RNA: (1) a 1276bp variant (NM-005227; EFNA4 transcript variant 1; SEQ ID NO: 1) which encodes a 201 amino acid proprotein (NP-005218; EFNA4 variant a; SEQ ID NO: 2); (2) a 1110bp variant (NM-182689; EFNA4 transcript variant 2) which encodes a 207 amino acid proprotein (NM-872631; EFNA4 variant b; SEQ ID NO: 3); and (3) a 1111bp variant (NM-182690; EFNA4 transcript variant 3) which encodes a 193 amino acid proprotein (NP-872632; EFNA4 variant c; SEQ ID NO: 4). It will be understood that each of the EFNA4 proteins includes a polypeptide comprising SEQ ID NO: 2 (i.e. 168-182 aa) which is spliced out to provide the mature form of the protein. This signal peptide targets the polypeptide to the cell surface/secretory pathway. Due to alternative splicing of mRNA (and the consequences for the protein coding sequence), the cell processes the protein isoforms differently-isoform a is membrane-localized and anchored to the cell surface by a Glycosylphosphatidylinositol (GPI) linkage, whereas isoforms b and c lack the GPI-anchoring signal sequence and are therefore expected to be secreted by the cell. An alignment of three protein isoforms of human EFNA4 is shown in fig. 1C. As indicated previously, unless directly referred to or the necessity of context otherwise indicates, the term EFNA4 shall relate to isoforms and immunoreactive equivalents of human EFNA 4. It will also be understood that this term may also refer to derivatives or fragments of EFNA4 in native or variant form (which contain an epitope to which an antibody or immunoreactive fragment may specifically bind).

Tumor perpetuating cells

Contrary to the teachings of the prior art, the present invention provides EFNA modulators that are particularly useful for targeting tumor initiating cells (particularly tumor perpetuating cells), thereby facilitating the treatment, management, or prevention of neoplastic disorders. More specifically, as shown previously, it was surprisingly found that specific tumor cell subpopulations express EFNA and are likely to modify the local coordination of morphogen signaling important for cancer stem cell self-renewal and cell survival. Thus, in preferred embodiments, modulators of EFNA according to the present teachings may be used to reduce tumor initiating cell frequency and thereby facilitate treatment or management of hyperproliferative diseases.

As used herein, the term Tumor Initiating Cells (TICs) encompasses tumor perpetuating cells (TPC; i.e. cancer stem cells or CSCs) and highly proliferative tumor progenitor cells (known as tprogs), which together typically constitute a distinct subpopulation (i.e. 0.1-40%) of a large tumor or mass. For the purposes of this disclosure, the terms tumor perpetuating cells and cancer stem cells are equivalent and are used interchangeably herein. Conversely, TPC differs from TProg in that TPC can completely reproduce the composition of tumor cells present within the tumor and has unlimited self-renewal capacity, as shown by serial transplantation of low numbers of isolated cells (by two or more passages in mice). As will be discussed in more detail below, Fluorescence Activated Cell Sorting (FACS) using suitable cell surface markers is a reliable method for isolating highly enriched cell subsets (> 99.5% purity) at least in part because of its ability to distinguish single cells from cell masses (i.e. pairings, etc.). Using such techniques, it has been shown that when low cell numbers of highly purified TProg cells are transplanted into immunocompromised mice, they can provoke tumor growth in primary transplants. However, unlike the purified TPC subpopulations, TProg-generated tumors do not fully reflect the parental tumor in terms of phenotypic cell heterogeneity and are significantly less efficient in terms of reinitiation of serial tumorigenesis in subsequent transplants. In contrast, the TPC subpopulation completely reconstitutes the cellular heterogeneity of the parent tumor and when serially isolated and transplanted can effectively initiate the tumor. Thus, one skilled in the art will recognize that, while both may be tumorigenic in primary transplants, a decisive difference between TPC and TProg is the ability of TPC to permanently provoke heterogeneous tumor growth after serial transplantation at low cell numbers. Other common means of characterizing TPC involve morphology as well as detection of cell surface markers, transcriptional profiles, and drug responses (although marker expression may vary in vitro with culture conditions and with cell line passage).

Thus, for the purposes of the present invention, tumor perpetuating cells (like normal stem cells that support the cellular hierarchy in normal tissue) are preferably defined by the ability to: they self-renew indefinitely while retaining the capacity for multisystem differentiation. Tumor perpetuating cells are thus capable of producing both tumorigenic (i.e., tumor initiating cells: TPC and TProg) and non-tumorigenic (NTG) progeny. As used herein, non-tumorigenic cells (NTGs) refer to tumor cells that are produced by tumor initiating cells but do not themselves self-renew or produce a heterogeneous tumor cell line that constitutes a tumor. Experimentally, NTG cells cannot reproducibly form tumors in mice, even when transplanted with excessive cell numbers.

As shown, TProg is also classified as a tumor initiating cell (or TIC) due to its limited ability to generate tumors in mice. TProg are progeny of TPC and are generally capable of a limited number of non-self-renewing cell divisions. In addition, TProg cells can be further divided into early tumor progenitor cells (ETP) and late tumor progenitor cells (LTP), each of which can be distinguished by phenotype (e.g., cell surface markers) and the ability to replicate the differential architecture of tumor cells. Despite this technological difference, both ETP and LTP differ functionally from TPC in that they are generally less able to serially reconstitute tumors when transplanted at low cell numbers and do not generally reflect the heterogeneity of the parental tumors. Despite the aforementioned differences, it has also been shown that various TProg populations are able to (in occasional cases) acquire self-renewal capacity that is normally attributed to stem cells and that themselves become TPC (or CSC). In any case, it is possible that both types of tumor-initiating cells are presented in the typical tumor mass of a single patient and are subjected to treatment with the modulators disclosed herein. That is, the disclosed compositions are generally effective in reducing the frequency of or altering the chemosensitivity of such EFNA-positive tumor initiating cells, regardless of the particular embodiment or whether present in admixture in the tumor.

In the context of the present invention, TPC are more tumorigenic, relatively more silent and generally more chemoresistant than TProg (both ETP and LTP), NTG cells and tumor-infiltrating non-TPC-derived cells (e.g. fibroblasts/stroma, endothelial & hematopoietic cells) that make up the bulk of the tumor. Given that conventional therapies and protocols are largely designed to reduce tumors and attack rapidly proliferating cells, TPC is likely to be more resistant to conventional therapies and protocols than faster proliferating TProg and other large tumor cell populations. In addition, TPC often express other features that make it relatively chemoresistant to conventional therapies, such as increased expression of multi-drug resistance transporters, enhanced DNA repair mechanisms, and anti-apoptotic proteins. These properties, each of which contributes to the drug tolerance of TPC, constitute a key reason why standard oncological treatment regimens cannot ensure long-term benefit in most patients with advanced neoplasia; i.e., those cells that provoke sustained tumor growth and recurrence (i.e., TPC or CSC) are not adequately targeted and cleared.

Unlike many of the aforementioned prior art treatments, the novel compositions of the present invention preferably reduce the frequency of tumor initiating cells upon administration to a subject, regardless of the form of the selected modulator or the particular target (e.g., genetic material, EFNA antibody, or ligand fusion construct). As described above, the reduction in the frequency of tumor initiating cells can be produced as a result of: a) clearing, depleting, sensitizing, silencing or inhibiting tumor initiating cells; b) controlling the growth, expansion or recurrence of tumor initiating cells; c) interrupting the initiation, propagation, maintenance or proliferation of tumor initiating cells; or d) otherwise interfere with the survival, regeneration and/or metastasis of the tumorigenic cells. In certain embodiments, the reduction in tumor initiating cell frequency may occur as a result of a change in one or more physiological pathways. Alteration of the pathway (whether by reducing or eliminating tumor initiating cells or by modifying their potency (e.g., induced differentiation, niche disruption), or otherwise interfering with their ability to exert an effect on the tumor environment or other cells), in turn allows for more effective treatment of EFNA-related disorders by inhibiting tumorigenesis, tumor maintenance and/or metastasis and recurrence.

Among the methods that can be used to assess this reduction in tumor initiating cell frequency are in vitro or in vivo limiting dilution assays, preferably followed by counting using poisson distribution statistics; or assessing the frequency of predetermined defined events, such as the ability to produce or not produce a tumor in vivo. While such limiting dilution analysis is the preferred method of calculating the reduction in tumor-initiating cell frequency, other less demanding methods may also be used to effectively determine the desired value (albeit with somewhat less accuracy) and are fully compatible with the teachings herein. Thus, as will be appreciated by those skilled in the art, it is also possible to determine the reduction in frequency values by well-known flow cytometric analysis or immunohistochemical means. For all the foregoing methods, see, e.g., Dylla et al 2008, PMCID: PMC2413402 and Hoey et al 2009, PMID: 19664991, respectively; each of which is incorporated herein by reference in its entirety.

With respect to limiting dilution analysis, in vitro counting of tumor initiating cell frequency can be achieved by placing fractionated or unfractionated human tumor cells (e.g., from treated and untreated tumors, respectively) in vitro growth conditions that promote colony formation. In this way, colony forming cells can be counted by simple counting and colony characterization, or by an assay consisting of: for example, human tumor cells are placed in plates in serial dilutions and each well is scored as positive or negative for colony formation at least 10 days after plating. In vivo limiting dilution experiments or assays (which are often more accurate in their ability to determine tumor initiating cell frequency) include: human tumor cells are transplanted from untreated controls or treated conditions in serial dilutions, for example, into immunocompromised mice and each mouse is then scored as positive or negative for tumor formation at least 60 days after transplantation. The cell frequency values obtained by in vitro or in vivo limiting dilution analysis are preferably performed by applying poisson distribution statistics to the known frequencies of positive and negative events, thereby providing a frequency of events that satisfies the definition of positive events (in this case, colony or tumor formation, respectively).

As to other methods compatible with the present invention that can be used to calculate the frequency of tumor initiating cells, the most common include quantitative flow cytometry techniques and immunohistochemical staining procedures. While less accurate than the limiting dilution analysis techniques described above, these procedures are much less labor intensive and provide reasonable values in a relatively short time frame. Thus, it will be appreciated that one skilled in the art can use flow cytometry cell surface marker profiling to determine (using one or more antibodies or reagents that bind to cell surface proteins enriched for tumor initiating cells recognized in the art (e.g., potentially compatible markers as shown in example 1 below)) and thereby measure TIC levels from various samples. In another compatible method, one skilled in the art can count TIC frequencies in situ (e.g., in tissue sections) by immunohistochemistry using one or more antibodies or reagents capable of binding to cell surface proteins thought to distinguish these cells.

Then, using any of the above methods, it is possible to quantify the reduction in TIC (or TPC therein) frequency provided by the disclosed EFNA modulators, including those conjugated to cytotoxic agents, in accordance with the teachings herein. In certain instances, the compounds of the invention may reduce TIC frequency by 10%, 15%, 20%, 25%, 30% or even 35% (by various mechanisms described above, including clearance, induced differentiation, niche disruption, silencing, etc.). In other embodiments, the reduction in TIC frequency may be on the order of 40%, 45%, 50%, 55%, 60% or 65%. In certain embodiments, the disclosed compounds can reduce the frequency of TIC by 70%, 75%, 80%, 85%, 90% or even 95%. Of course, it will be appreciated that any reduction in TIC frequency is likely to result in a corresponding reduction in tumorigenesis, persistence, recurrence and aggressiveness of the neoplasia.

EFNA modulators

In any case, the present invention relates to the use of EFNA modulators (including EFNA antagonists) for the diagnosis, treatment and/or prevention of any of several EFNA-associated malignancies. The disclosed modulators may be used alone or in combination with various anti-cancer compounds (e.g., chemotherapeutic or immunotherapeutic agents or biological response modifiers). In other selected embodiments, two or more discrete EFNA modulators may be used in combination to provide enhanced anti-tumor effects or may be used to make multispecific constructs.

In certain embodiments, EFNA modulators of the invention will comprise a nucleotide, oligonucleotide, polynucleotide, peptide or polypeptide. Even more preferably, the modulator will comprise soluble EFNA (sffna) or a form, variant, derivative or fragment thereof, including, for example, EFNA fusion constructs (e.g., EFNA-Fc, EFNA-targeting moiety, etc.) or EFNA conjugates (e.g., EFNA-PEG, EFNA-cytotoxic agent, EFNA-brm, etc.). It will also be understood that in other embodiments, the EFNA modulator comprises an antibody (e.g., anti-EFNA 1 or anti-EFNA 4mAb) or an immunoreactive fragment or derivative thereof. In particularly preferred embodiments, modulators of the invention will comprise neutralizing antibodies or derivatives or fragments thereof. In other embodiments, the EFNA modulator may comprise an internalizing antibody or fragment thereof. In other embodiments, the EFNA modulator may comprise a depleting antibody or fragment thereof. Furthermore, as with the previously described fusion constructs, these antibody modulators may be conjugated, linked or otherwise associated with selected cytotoxic agents, polymers, Biological Response Modifiers (BRMs), etc., to provide targeted immunotherapy with various (optionally multiple) mechanisms of action. As described above, such antibodies may be pan-EFNA antibodies and bind to two or more ephrin-a ligands, or are immunospecific antibodies that selectively react with one of six ephrin-a ligands. In other embodiments, the modulator may function at the genetic level and may include compounds that are antisense constructs, sirnas, micro RNAs, and the like.

Based on the teachings herein one skilled in the art will appreciate that particularly preferred embodiments of the present invention may include sfna 4 or sfna 1 or an antibody modulator that binds to either or both of EFNA4 and EFNA 1.

It will also be appreciated that the disclosed EFNA modulators may deplete, silence, neutralize, eliminate or inhibit the growth, proliferation or survival of tumor cells (particularly TPC) and/or associated neoplasias by various mechanisms, including agonizing or antagonizing selected pathways or eliminating specific cells, depending, for example, on the form of the EFNA modulator, any associated payload or delivered dose and method. Thus, while the preferred embodiments disclosed herein relate to the depletion, inhibition, or silencing of a particular subpopulation of tumor cells (e.g., tumor perpetuating cells), it must be emphasized that such embodiments are illustrative only and not limiting in any sense. Rather, as shown in the appended claims, the present invention relates broadly to EFNA modulators and their use in treating, managing or preventing a variety of EFNA-associated hyperproliferative disorders, regardless of any particular mechanism or target tumor cell population.

To the same extent, the disclosed embodiments of the invention can include one or more EFNA antagonists. To this end, it will be understood that EFNA antagonists of the present invention may include any ligand, polypeptide, peptide, fusion protein, antibody or immunologically active fragment or derivative thereof that recognizes, reacts with, binds to, combines with, competes with, associates with or otherwise interacts with an EFNA protein or fragment thereof and eliminates, silences, reduces, inhibits, impedes, limits or controls the growth of tumor initiating cells or other neoplastic cells, including bulk tumors or NTG cells. In selected embodiments, the EFNA modulator comprises an EFNA antagonist.

As used herein, an antagonist refers to a molecule that is capable of neutralizing, blocking, inhibiting, abrogating, reducing, or interfering with the activity of a particular or designated protein, including binding of a receptor to a ligand or interaction of an enzyme with a substrate. More generally, the antagonists of the invention may include antibodies and antigen-binding fragments or derivatives thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, antisense constructs, siRNA, miRNA, bio-organic molecules, peptidomimetics, pharmacological agents and metabolites thereof, transcriptional and translational control sequences, and the like. Antagonists may also include small molecule inhibitors, fusion proteins, receptor molecules and derivatives that specifically bind to the protein thereby masking its binding to its substrate target, antagonist variants of the protein, antisense molecules directed against the protein, RNA aptamers, and ribozymes against the protein.

As used herein and applied to two or more molecules or compounds, the term recognition or binding shall refer to covalent or non-covalent reactions, associations, specific associations, combinations, interactions, associations, linkages, associations, coalesces, merges, or links of molecules in which one molecule exerts an effect on another.

Furthermore, as shown in the examples herein, modulators of certain human EFNAs may in some cases cross-react with EFNAs from species other than human (e.g., murine). In other cases, exemplary modulators may be specific for one or more isoforms of human EFNA and will not show cross-reactivity with EFNA orthologs from other species. Of course, in conjunction with the teachings herein, such embodiments may include pan-EFNA antibodies that bind to two or more ephrin-a ligands from a single species or antibodies that specifically bind to a single ephrin-a ligand.

In any event, and as will be discussed in further detail below, one skilled in the art will appreciate that the disclosed modulators can be used in conjugated or unconjugated form. That is, the modulator may be bound or conjugated (e.g., covalently or non-covalently) to a pharmaceutically active compound, a biological response modulator, an anti-cancer agent, a cytotoxic or cytostatic agent, a diagnostic moiety, or a biocompatible modulator. In this regard, it will be understood that such conjugates may comprise peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, organic molecules, and radioisotopes. Furthermore, as shown herein, the selected conjugate can be covalently or non-covalently linked to the EFNA modulator in various molar ratios, depending at least in part on the method used to achieve conjugation.

V. antibody

a. Overview

As previously mentioned, particularly preferred embodiments of the invention include EFNA modulators in the form of antibodies. The term antibody is used in the broadest sense and specifically covers synthetic antibodies, monoclonal antibodies, oligoclonal or polyclonal antibodies, polyclonal (polyclonal) antibodies, recombinantly produced antibodies, endosomes, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, human antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies, primatized antibodies, Fab fragments, F (ab') fragments, single chain fvfcs (scfvffc), single chain fvs (scfv), anti-idiotypic (anti-Id) antibodies, and any other immunologically active antibody fragment, so long as they exhibit the desired biological activity (i.e., EFNA association or binding). In a broader sense, the antibodies of the invention include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site, wherein these fragments may or may not be fused to another immunoglobulin domain, including but not limited to an Fc region or a fragment thereof. Furthermore, as described in more detail herein, the terms antibody (antibody) and antibody (antibodies) specifically include Fc variants as described below, including full-length antibodies fused to a variant Fc comprising an Fc region or fragment thereof, optionally comprising at least one amino acid residue modification and fused to an immunologically active fragment of an immunoglobulin.

As discussed in more detail below, the generic term antibody or immunoglobulin comprises five distinct antibody classes that can be biochemically distinguished, and depending on the amino acid sequence of their heavy chain constant domains, can be readily assigned to the appropriate class. For historical reasons, the main class of intact antibodies is known as IgA, IgD, IgE, IgG and IgM. In humans, classes IgG and IgA can be further divided into recognized subclasses (isotypes), i.e., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, depending on structure and certain biochemical properties. It will be understood that in humans the IgG isotypes are named in the order of their abundance in serum, with IgG1 being the most abundant.

Although all five antibodies (i.e., IgA, IgD, IgE, IgG and IgM) and all isotypes (i.e., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) and variants thereof are within the scope of the invention, preferred embodiments including the immunoglobulin IgG class will be discussed in detail hereinafter for illustrative purposes only. It is to be understood, however, that this disclosure is merely illustrative of exemplary compositions and methods for practicing the invention and does not limit the scope of the invention or the appended claims in any way.

In this regard, a human IgG immunoglobulin comprises two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chains having a molecular weight of 53,000-70,000 (depending on isotype). Heavy chain constant domains corresponding to different antibody classes are indicated by the corresponding lower case greek letters α, γ and μ, respectively. The light chain of an antibody from any vertebrate species can be assigned to one of two clearly distinct classes (called κ and λ) based on the amino acid sequence of its constant domain. Those skilled in the art will appreciate that the subunit structures and three-dimensional configurations of different immunoglobulin classes are well known.

The four chains are disulfide-linked in a Y configuration, with the light chain supporting the heavy chain, which starts at the mouth of the Y and continues through the variable region to both ends of the Y. Each light chain is linked to a heavy chain by one covalent disulfide bond and two disulfide linkages in the hinge region are linked to the heavy chain. Each of the heavy and light chains also has regularly spaced intrachain disulfide bridges, the number of which can vary based on the isotype of the IgG.

Each heavy chain has a variable domain at one end (V)_H) Followed by several constant domains. Each light chain has a variable domain at one end(V_L) And has a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the heavy chain variable domain. In this regard, it will be understood that the light chain (V)_L) And heavy chain (V)_H) The variable domain portion of (a) determines antigen recognition and specificity. In contrast, light chain (C)_L) And heavy chain (C)_H1，C_H2 or C_H3) The constant domains of (a) confer and modulate important biological properties, such as secretion, transplacental mobility, circulating half-life, complement fixation, etc. By convention, the numbering of the constant region domains increases as they are farther from the antigen binding site or amino terminus of the antibody. Thus, the amino or N-terminus of an antibody comprises the variable region and the carboxy or C-terminus comprises the constant region. Thus, C_H3 and C_LThe domains actually comprise the carboxy-termini of the heavy and light chains, respectively.

The term variable refers to the fact that: the sequences of certain portions of variable domains vary widely between immunoglobulins and these hot spots largely define the binding and specificity characteristics of a particular antibody. These hypervariable sites manifest themselves in three segments, known as Complementarity Determining Regions (CDRs), in the light and heavy chain variable domains, respectively. The more highly conserved portions of the variable domains flanking the CDRs are called Framework Regions (FRs). More specifically, in naturally occurring monomeric IgG antibodies, the six CDRs present on each arm of the antibody are short, non-contiguous amino acid sequences that are specifically positioned such that they form an antigen binding site when the antibody assumes its three-dimensional configuration in an aqueous environment.

The framework regions comprising the remainder of the heavy and light chain variable domains show less intermolecular variation in amino acid sequence. In contrast, the framework regions largely adopt a β -sheet conformation and the CDRs form connected loops, and in some cases form part of the β -sheet structure. Thus, these framework regions act to form a scaffold that provides for placing the six CDRs in the correct orientation through inter-chain, non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface complementary to an epitope on the immunoreactive antigen (i.e., EFNA 4). This complementary surface facilitates non-covalent binding of the antibody to the immunoreactive epitope. It will be appreciated that the location of the CDRs can be readily identified by those skilled in the art.

All or a portion of the heavy and light chain variable regions can be recombined or engineered using standard recombination and expression techniques to provide effective antibodies, as discussed in more detail below and shown in the accompanying examples. That is, the variable region of the heavy or light chain from the first antibody (or any portion thereof) may be mixed and matched with any selected portion of the variable region of the heavy or light chain from the second antibody. For example, in one embodiment, the entire light chain variable region comprising the three light chain CDRs of the first antibody can be paired with the entire heavy chain variable region comprising the three heavy chain CDRs of the second antibody to provide an operable antibody. Furthermore, in other embodiments, individual heavy and light chain CDRs derived from various antibodies can be mixed and matched to provide a desired antibody with optimized characteristics. Thus, an exemplary antibody can comprise three light chain CDRs from a first antibody, two heavy chain CDRs from a second antibody, and a third heavy chain CDR from a third antibody.

More specifically, in the context of the present invention, it will be understood that any of the disclosed heavy and light chain CDRs in fig. 7A may be rearranged in such a manner to provide optimized anti-EFNA (e.g., anti-hEFNA 4) antibodies in accordance with the present teachings. That is, one or more of the CDRs disclosed in figure 7A may be incorporated into an EFNA modulator, and in particularly preferred embodiments into a CDR-grafted or humanized antibody that immunospecifically binds to one or more ephrin-a ligands.

In any case, the number of complementarity determining region residues can be defined as those in Kabat et al (1991, NIH Publication91-3242, National Technical Information Service, Springfield, Va.), particularly residues 24-34(CDR1), 50-56(CDR2) and 89-97(CDR3) in the light chain variable domain and 31-35(CDR1), 50-65(CDR2) and 95-102(CDR3) in the heavy chain variable domain. It should be noted that the CDRs vary appreciably from antibody to antibody (and by definition they will not show homology to the Kabat consensus sequence). Maximum alignment of framework residues often requires the insertion of spacer residues in the numbering system to be used in the Fv region. Furthermore, the identity of certain individual residues at any given Kabat position numbering may vary from antibody chain to antibody chain due to inter-species or allelic differences. See also Chothia et al, J.mol.biol.196:901-917 (1987); chothia et al, Nature 342, pp.877-883(1989) and MacCallum et al, J.mol.biol.262:732-745(1996), wherein the definitions include overlaps or subsets of amino acid residues when compared to each other. Each of the foregoing references is hereby incorporated by reference in its entirety and encompasses the amino acid residues of the CDRs as defined in each of the references cited above are shown for comparison.

CDR definition

	Kabat1	Chothia2	MacCallum3
				VH CDR1	31-35	26-32	30-35
VH CDR2	50-65	53-55	47-58
				VH CDR3	95-102	96-101	93-101
VL CDR1	24-34	26-32	30-36
				VL CDR2	50-56	50-52	46-55
VL CDR3	89-97	91-96	89-96

¹Residue numbering is according to Kabat et al nomenclature, see above

²Residue numbering is according to the name of Chothia et al, supra

³Residue numbering is according to MacCallum et al nomenclature, supra

For convenience, the CDRs shown in FIG. 7A (SEQ ID NOS: 8-59 and 70-95) were derived from VBASE2 analysis, although those skilled in the art, in view of the present disclosure, will readily identify and count the CDRs as defined by Kabat et al or MacCallum et al for each of the corresponding heavy and light chain sequences. In this regard, the CDRs as defined by Kabat et al were used in the humanization analysis shown in example 7(b) and underlined in FIGS. 7O-7R (SEQ ID NO: 148-. Accordingly, antibodies comprising CDRs defined by all such designations are expressly included within the scope of the present invention. More broadly, the term variable region CDR amino acid residues includes amino acids in CDRs as identified using any of the sequence or structure based methods set forth above.

As used herein, the term variable region Framework (FR) amino acid residues refers to those amino acids in the framework regions of Ig chains. The term framework region or FR region as used herein includes amino acid residues that are part of the variable region but are not part of the CDRs (e.g. using the Kabat definition of CDRs). Thus, the variable region framework is a non-contiguous sequence of about 100-120 amino acids in length but including only those amino acids outside the CDRs.

For specific examples of heavy chain variable regions and for the CDRs defined by Kabat et al, framework region 1 corresponds to a domain encompassing the variable region of amino acids 1-30; framework region 2 corresponds to a domain encompassing the variable region of amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acid 103 to the end of the variable region. The framework regions of the light chain are similarly separated by each light chain variable region CDR. Similarly, using the definition of CDRs by Chothia et al or McCallum et al, the framework region boundaries are separated by the respective CDR ends as described above.

In view of the foregoing structural considerations, those skilled in the art will appreciate that the antibodies of the present invention may comprise any of a number of functional embodiments. In this regard, compatible antibodies can include any immunoreactive antibody (as that term is defined herein) that provides a desired physiological response in a subject. While any of the disclosed antibodies may be used in conjunction with the present teachings, certain embodiments of the present invention will comprise chimeric, humanized or human monoclonal antibodies or immunoreactive fragments thereof. Yet other embodiments may include, for example, homogeneous or heterogeneous multimeric constructs, Fc variants, and conjugated or glycosylation altered antibodies. Furthermore, it will be understood that such configurations are not mutually exclusive and that compatible individual antibodies may include one or more of the functional aspects disclosed herein. For example, compatible antibodies may include single chain diabodies with humanized variable regions, or fully human full length IgG3 antibodies with Fc modifications that alter glycosylation patterns to modulate serum half-life. Other exemplary embodiments will be apparent to those skilled in the art and can be readily discerned within the scope of the present invention.

b. Antibody production

As is well known, various host animals (including rabbits, mice, rats, etc.) can be vaccinated and used to provide antibodies according to the teachings herein. Adjuvants known in the art that can be used to increase the immune response depend on the species being vaccinated and include, but are not limited to, freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, Pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille calmette guerin) and corynebacterium parvum. Such adjuvants may protect the antigen from rapid dispersion by sequestering it in a local deposit, or they may contain the following: it stimulates the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is administered, the immunization schedule will include two or more administrations of the polypeptide, spread over several weeks.

After immunization of an animal with an EFNA immunogen (e.g., soluble EFNA4 or EFNA1), which may include selected isoforms and/or peptides, or live cells or cell preparations expressing a desired protein, antibodies and/or antibody-producing cells may be obtained from the animal using techniques recognized in the art. In some embodiments, serum containing polyclonal anti-EFNA antibodies is obtained by bleeding or sacrificing the animal. The serum may be used for research purposes in a form obtained from the animal or, alternatively, the anti-EFNA antibody may be partially or fully purified to provide an immunoglobulin fraction or a homogeneous antibody preparation.

c. Monoclonal antibodies

Although polyclonal antibodies may be used in conjunction with certain aspects of the invention, preferred embodiments include the use of EFNA reactive monoclonal antibodies. As used herein, the term monoclonal antibody or mAb refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations that may be present in minor amounts (e.g., naturally occurring mutations). Thus, the modifier monoclonal indicates that the antibody is not characterized as a mixture of discrete antibodies and can be used in conjunction with any type of antibody. In certain embodiments, such monoclonal antibodies include antibodies comprising a polypeptide sequence that binds or associates with EFNA, wherein the polypeptide sequence that binds EFNA is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences.

In a preferred embodiment, the antibody-producing cell line is prepared from cells isolated from an immunized animal. After immunization, the animals were sacrificed and lymph nodes and/or splenic B cells were immortalized by means well known in the art as shown in the appended examples). Methods of immortalizing cells include, but are not limited to, transfecting them with an oncogene, infecting them with an oncogenic virus and culturing them under conditions that select for immortalized cells, subjecting them to an oncogenic or mutagenic compound, fusing them with immortalized cells (e.g., myeloma cells) and inactivating the tumor suppressor gene. If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Immortalized cells are screened using ephrin-a ligands (including selected isoforms) or immunoreactive portions thereof. In a preferred embodiment, the initial screening is performed using enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay.

More generally, discrete monoclonal antibodies consistent with the present invention may be prepared using a variety of techniques known in the art, including hybridomas, recombinant techniques, phage display techniques, yeast cultureLibraries, transgenic animals (e.g. for example)Or HuMAb) Or some combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology as generally described above and as taught in more detail in the following: harlow et al, Antibodies: a Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); hammerling et al: monoconal Antibodies and T-Cell hybrids 563-681(Elsevier, N.Y., 1981), each of which is incorporated herein. Using the disclosed protocol, antibodies are preferably produced in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen and adjuvant. As discussed previously, such immunization typically elicits an immune response, which includes the production of antigen-reactive antibodies from activated spleen cells or lymphocytes (which may be fully human if the animal being immunized is transgenic). While the antibodies produced can be harvested from the serum of an animal to provide a polyclonal preparation, it is generally more desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide a homogeneous preparation of monoclonal antibodies. Most typically, lymphocytes are obtained from the spleen and immortalized to provide hybridomas.

For example, as described above, the selection process can be to select a unique clone from a plurality of clones (e.g., a collection of hybridoma clones, phage clones, or recombinant DNA clones). It will be appreciated that the EFNA binding sequence selected may be further altered, for example, to improve affinity for the target, to humanise the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce multispecific antibodies, etc., and that antibodies comprising the altered target binding sequence are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations, which typically include separate antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins which may be cross-reactive.

d. Chimeric antibodies

In another embodiment, an antibody of the invention may comprise a chimeric antibody derived from covalently linked protein segments from at least two different species or antibody types. It will be understood that, as used herein, the term chimeric antibody relates to a construct that: wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one exemplary embodiment, a chimeric antibody according to the teachings herein can comprise murine V_HAnd V_LAmino acid sequence and constant regions derived from human sources. In other compatible embodiments, the chimeric antibodies of the invention may comprise CDR grafted or humanized antibodies as described below.

Generally, the objective of making chimeric antibodies is to create chimeras in which the number of amino acids from the desired subject species is maximized. One example is a CDR-grafted antibody, wherein the antibody comprises one or more Complementarity Determining Regions (CDRs) from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain is identical or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass. For use in humans, the variable regions or selected CDRs from rodent antibodies are typically grafted into human antibodies, replacing the naturally occurring variable regions or CDRs of human antibodies. These constructs generally have the following advantages: providing full strength modulator functions (e.g., CDC, ADCC, etc.) while reducing an undesired immune response of the subject to the antibody.

e. Humanized antibodies

Similar to the CDR grafted antibody is a humanized antibody. Generally, humanized antibodies are produced from monoclonal antibodies that were originally produced in non-human animals. As used herein, a humanized form of a non-human (e.g., murine) antibody is a chimeric antibody containing minimal sequences derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient or recipient antibody) in which residues from a CDR of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity.

Generally, humanization of antibodies involves analysis of sequence homology and conventional structure of the donor and recipient antibodies. In selected embodiments, the acceptor antibody may comprise a consensus sequence. To create a consensus human framework, frameworks from several human heavy or light chain amino acid sequences can be aligned to identify a consensus amino acid sequence. Furthermore, in many cases, one or more framework residues in a human immunoglobulin variable domain are replaced by corresponding non-human residues from a donor antibody. These framework substitutions are identified by methods well known in the art, for example by modeling the interaction between the CDRs and framework residues to identify framework residues important for antigen binding, and performing sequence comparisons to identify unusual framework residues at specific positions. Such substitutions help maintain the proper three-dimensional configuration of the grafted CDRs and often improve affinity relative to similar constructs without framework substitutions. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications can be made using well known techniques to further improve antibody performance.

CDR grafted and humanized antibodies are described, for example, in U.S. Pat. nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101. Typically, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-E329 (1988); and Presta, curr, Op, Structure, biol.2:593-596 (1992). See also, e.g., Vaswani and Hamilton, ann. 105-115 (1998); harris, biochem. Soc. transactions 23: 1035-; hurle and Gross, curr. Op. Biotech.5: 428-; and U.S. patent nos. 6,982,321 and 7,087,409. Another method is known as manual engineering (humaneering) and is described, for example, in u.s.2005/0008625. For the purposes of this application, the term humanized antibody will be considered to expressly include CDR-grafted antibodies (i.e., human antibodies comprising one or more grafted non-human CDRs) with no or minimal framework substitutions.

Furthermore, non-human anti-EFNA antibodies may also be modified by specific deletion of human T cell epitopes or by de-immunization by the methods disclosed in WO98/52976 and WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody can be analyzed for peptides that bind MHC class II; these peptides represent potential T cell epitopes (as defined in WO98/52976 and WO 00/34317). To detect potential T cell epitopes, a computer modeling method known as peptide threading (threading) can be applied, and in addition, can be present in V_HAnd V_LMotifs in the sequences search for human MHC class II binding peptide libraries as described in WO98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by replacing a few amino acid residues in the variable region or by single amino acid substitutions. Conservative substitutions are made as far as possible. Typically, but not exclusively, amino acids common to positions in human germline antibody sequences may be used. After identifying the immunological changes of the patient, the patient can be treatedConstruction of encoded V by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, etc.)_HAnd V_LThe nucleic acid of (1). The mutagenized variable sequences may optionally be fused to human constant regions.

In selected embodiments, at least 60%, 65%, 70%, 75% or 80% of the humanized antibody variable region residues will correspond to those of the parent Framework Region (FR) and CDR sequences. In other embodiments, at least 85% or 90% of the humanized antibody residues will correspond to those of the parent Framework Region (FR) and CDR sequences. In other preferred embodiments, greater than 95% of the humanized antibody residues will correspond to those of the parent Framework Region (FR) and CDR sequences.

Humanized antibodies can be made using common molecular biology and biomolecular engineering techniques as described herein. These methods comprise isolating, manipulating and expressing a nucleic acid sequence encoding all or part of an immunoglobulin Fv variable region from at least one heavy or light chain. The source of such nucleic acids is well known to those skilled in the art and may be obtained, for example, from hybridomas, eukaryotic cells, or phages (as described above) that produce antibodies or immunoreactive fragments against the intended target, from germline immunoglobulin genes, or from synthetic constructs. The recombinant DNA encoding the humanized antibody can then be cloned into an appropriate expression vector.

Human germline sequences disclosed, for example, in Tomlinson, I.A. et al (1992) J.mol.biol.227: 776-; cook, g.p. et al (1995) immunol.today 16: 237-242; chothia, D.et al (1992) J.mol.Bio.227: 799-; and Tomlinson et al (1995) EMBO J14: 4628-one 4638. The V BASE catalog provides a comprehensive catalog of human immunoglobulin variable region sequences (see Retter et al, (2005) Nuc Acid Res 33: 671-674). These sequences can be used as a source of human sequences, for example for framework regions and CDRs. As shown herein, consensus human framework regions may also be used, for example as described in U.S. patent No. 6,300,064.

f. Human antibodies

In addition to the foregoing antibodies, those skilled in the art will appreciate that the antibodies of the invention may include fully human antibodies. For the purposes of this application, the term human antibody includes antibodies having the following amino acid sequences: which corresponds to the amino acid sequence of an antibody produced by a human and/or made using any of the techniques for making human antibodies as disclosed herein. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

Human antibodies can be produced using various techniques known in the art. As described above, phage display technology can be used to provide immunologically active binding regions according to the invention. Accordingly, certain embodiments of the present invention provide methods for producing an anti-EFNA antibody, or antigen-binding portion thereof, comprising the steps of: synthesizing a library of (preferably human) antibodies on phage, screening said library with selected EFNA or antibody-binding portion thereof, isolating phage that bind EFNA, and obtaining immunoreactive fragments from said phage. As an example, one method for preparing an antibody library for use in phage display technology includes the following steps: immunizing a non-human animal comprising a human or non-human immunoglobulin locus with selected EFNA or antigenic portion thereof to generate an immune response, extracting antibody-producing cells from the immunized animal; isolating RNA encoding the heavy and light chains of the antibody of the present invention from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector such that the antibody is expressed on a phage. More specifically, the code V is encoded by PCR_HAnd V_LThe DNA of the domain is recombined with the scFv linker and cloned into a phagemid vector (e.g. p CANTAB 6 or pComb 3 HSS). The vector can then be electroporated into E.coli and the E.coli subsequently infected with helper phage. The phage used in these methods are typically filamentous phage comprising fd and M13, and V_HAnd V_LThe domain is usually with phage gene III or gene VIII recombinant fusion.

The recombinant panels prepared as above can be screenedAntibody libraries are pooled to isolate recombinant human anti-EFNA antibodies of the invention. In a preferred embodiment, the library is a scFv phage display library, which uses human V prepared from mRNA isolated from B cells_LAnd V_HGeneration of cDNA. Methods for preparing and screening such libraries are well known in the art and kits for generating phage display libraries are commercially available (e.g., Pharmacia recombinant phage antibody System, catalog No. 27-9400-01; and Stratagene SurfZAP^TMPhage display kit, catalog No. 240612). There are also other methods and reagents that can be used to generate and screen antibody display libraries (see, for example, U.S. Pat. No.5,223,409; PCT publication No. WO92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al, Bio/Technology 9:1370-, Natl.Acad.Sci.USA 88: 7978-.

Antibodies generated from naive libraries (natural or synthetic) may be of intermediate affinity (K)_aIs about 10⁶To 10⁷M^-1) But affinity maturation can also be simulated in vitro by construction and re-selection from secondary libraries as described in the art. For example, the present invention can be found in Hawkins et al, j.mol.biol., 226: 889-: error-prone polymerases (reported by Leung et al, Technique, 1: 11-15 (1989)) were used in the methods of 3576-3580(1992) to randomly introduce mutations in vitro. In addition, affinity can be performed by randomly mutating one or more CDRs in a selected single Fv clone (e.g., by PCR using primers that carry random sequences spanning the CDR of interest) and screening for higher affinity clonesAnd sexual maturation. WO 9607754 describes a method for inducing mutations in the complementarity determining regions of an immunoglobulin light chain to produce a light chain gene library. Another effective way is to select V by phage display_HOr V_LThe domains are recombined with a pool of naturally occurring V domain variants obtained from unimmunized donors and screened for higher affinity in rounds of chain shuffling, as described by Marks et al, biotechnol, 10: 779 it is described in 783 (1992). This technique allows the dissociation constant K to be generated_d(k_off/k_on) Is about 10^-9M or less.

It will also be appreciated that a similar procedure may be employed, wherein a library comprising eukaryotic cells (e.g. yeast) expressing binding pairs on their surface is used. As with phage display technology, eukaryotic cell libraries are screened for the antigen of interest (i.e., EFNA) and cells expressing candidate binding pairs are isolated and cloned. Steps can be taken to optimize library content and affinity maturation for reactive binding pairs. See, for example, U.S. patent nos. 7,700,302 and u.s.s.n.12/404,059. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses a human antibody (Vaughan et al Nature Biotechnology 14: 309-; hoogenboom and Winter, J.mol.biol, 227:381 (1991); marks et al, J.MoI.biol, 222:581 (1991)). In other embodiments, human binding pairs can be isolated from combinatorial antibody libraries produced in eukaryotic cells (e.g., yeast). See, for example, U.S. patent No. 7,700,302. Such techniques advantageously allow screening of a large number of candidate modulators and provide relatively easy manipulation of candidate sequences (e.g., by affinity maturation or recombinant shuffling).

Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals (e.g., mice) in which endogenous immunoglobulin genes are partially or completely inactivated. After challenge (challenge), human antibody production was observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. The method is describedAs described inU.S. patent numbers 5,545,807 for technology; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,661,016 and U.S. Pat. Nos. 6,075,181 and 6,150,584, and in the following scientific publications: marks et al, Bio/Technology 10: 779 783 (1992); lonberg et al, Nature 368: 856-859 (1994); morrison, Nature 368:812-13 (1994); fisherworld et al, nature biotechnology 14: 845-51 (1996); neuberger, Nature Biotechnology 14: 826 (1996); lonberg and Huszar, Intern.Rev.Immunol.13:65-93 (1995). Alternatively, human antibodies can be made by immortalizing human B-lymphocytes that produce antibodies to the target antigen (such B-lymphocytes can be recovered from individuals with neoplastic disorders or can be immunized in vitro). See, e.g., Cole et al, Monoclonal Antibodies and cancer therapy, Alan R.Liss, p.77 (1985); boerner et al, J.Immunol,147(l):86-95 (1991); and U.S. Pat. No.5,750,373

Antibody characterization

Regardless of how obtained, or which of the aforementioned forms the antibody modulator takes (e.g., humanized, human, etc.), preferred embodiments of the disclosed modulators may exhibit a variety of characteristics. In this regard, cells producing anti-EFNA antibodies (e.g., hybridomas or yeast colonies) can be selected, cloned, and further screened for desired characteristics, including, for example, robust growth, high antibody production, and (as discussed in more detail below) desired antibody characteristics. Hybridomas can be expanded in vivo in syngeneic animals, in animals lacking the immune system (e.g., nude mice), or in vitro in cell culture. Methods of selecting, cloning and expanding hybridomas and/or colonies, each of which produces a separate antibody species, are well known to those of ordinary skill in the art.

a. Neutralizing antibodies

In particularly preferred embodiments, modulators of the invention will comprise neutralizing antibodies or derivatives or fragments thereof. The term neutralizing antibody or neutralizing antagonist refers to the following antibodies or antagonists: which binds to or interacts with ephrin-a ligand and prevents binding or association of the ligand with its binding partner (e.g., EPHA receptor) thereby disrupting the biological response that would otherwise result from the interaction of the molecules. In assessing the binding and specificity of an antibody or immunologically functional fragment or derivative thereof, an antibody or fragment will substantially inhibit the binding of a ligand to its binding partner or substrate when excess antibody reduces the amount of binding partner bound to the target molecule by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more, as measured, for example, in an in vitro competitive binding assay (see, e.g., examples 9-12 herein). In the case of antibodies to EFNA4, for example, a neutralizing antibody or antagonist will preferably reduce the ability of EFNA4 to bind EphA4 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more. It will be appreciated that such reduced activity may be measured directly by using art-recognized techniques or may be measured by the effect such reduction would have on the activity of the EPH (e.g., EPHA4) receptor.

b. Internalizing antibodies

While evidence suggests that selected ephrin-a ligands or their isoforms may exist in soluble form, at least some EFNAs (e.g., EFNA1 and EFNA4) are likely to remain bound to the cell surface to allow internalization of the disclosed modulators. Thus, the anti-EFNA antibodies of the invention may be internalized, at least to some extent, by cells expressing ephrin-a ligands. For example, anti-EFNA 4 antibodies that bind to EFNA4 on the surface of tumor initiating cells can be internalized by the tumor initiating cells. In particularly preferred embodiments, such anti-EFNA antibodies may be conjugated or conjugated to an anti-cancer agent (e.g., a cytotoxic moiety that kills cells upon internalization).

As used herein, an internalized anti-EFNA antibody is an antibody that: which is aspirated by the cells after binding to EFNA associated with mammalian cells. The internalizing antibodies include antibody fragments, human or humanized antibodies, and antibody conjugates. Internalization can occur in vitro or in vivo. For therapeutic applications, internalization can occur in vivo. The number of antibody molecules internalized may be sufficient or sufficient to kill EFNA expressing cells, particularly EFNA expressing tumor initiating cells. Depending on the potency of the antibody or antibody conjugate, in some cases, uptake of a single antibody molecule into the cell may be sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly effective in killing such that internalization of one molecule of the toxin conjugated to an antibody is sufficient to kill tumor cells. Whether an anti-EFNA antibody is internalized upon binding to EFNA on a mammalian cell can be determined by various assays, including those described in the examples below (e.g., examples 15 and 16). Methods of detecting whether an antibody is internalized into a cell are also described in U.S. patent No. 7,619,068, which is incorporated herein by reference in its entirety.

c. Depleting antibodies

In other preferred embodiments, modulators of the invention will comprise depleting antibodies or derivatives or fragments thereof. The term depleting antibody refers to an antibody or fragment that binds or associates with EFNA on or near the surface of a cell and induces, promotes or causes death or clearance of the cell (e.g., by complement-dependent cytotoxicity or antibody-dependent cytotoxicity). In certain embodiments discussed more fully below, the selected depleting antibody will be conjugated or conjugated to a cytotoxic agent. Preferably, the depleting antibody will be capable of removing, eliminating or killing at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97% or 99% of tumor perpetuating cells in a defined cell population. In certain embodiments, the cell population can comprise enriched, fractionated, purified, or isolated tumor perpetuating cells. In other embodiments, the cell population can include a whole tumor sample or a heterogeneous tumor extract comprising tumor perpetuating cells. One skilled in the art will appreciate that the depletion of tumor-initiating cells or tumor perpetuating cells according to the teachings herein can be monitored and quantified using standard biochemical techniques as described in the examples (e.g., example 16) below.

d. Epitope binding

It will also be understood that the disclosed anti-EFNA antibodies will associate or bind to discrete epitopes or determinants presented by the selected target. As used herein, the term epitope refers to that portion of the target antigen that is capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide (e.g., EFNA), the epitope can be formed of contiguous amino acids as well as non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by contiguous amino acids are typically retained after protein denaturation, while epitopes formed by tertiary folding are typically lost after protein denaturation. Epitopes typically comprise at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. More specifically, the skilled person will understand that the term epitope includes any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active molecular surface groups (e.g., amino acids or carbohydrates or sugar side chains) and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. Furthermore, epitopes can be linear or conformational. In a linear epitope, all the interaction points between the protein and the interacting molecule (e.g., antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the interaction points are present in amino acid residues on the protein that are linearly separated from each other.

Once the desired epitope on the antigen has been determined, it is possible to generate antibodies against this epitope, for example by immunization with a peptide comprising the epitope using the techniques described in the present invention. Alternatively, in the discovery process, the production and characterization of antibodies may elucidate information about the desired epitope. From this information, it is then possible to competitively screen for antibodies that bind to the same epitope. One way to achieve this is to conduct competition studies to find antibodies that compete for binding to each other, i.e., antibodies that compete for binding to the antigen. High throughput procedures for binning antibodies based on their cross-competition are described in WO 03/48731.

As used herein, the term binning refers to a method of grouping antibodies based on their antigen binding properties. The distribution of the bins is somewhat arbitrary, depending on how different the binding pattern of the tested antibodies is observed. Thus, while the technique is a useful tool for the classification of antibodies of the invention, the bins are not always directly related to an epitope and this initial determination should be further confirmed by other art-recognized methods.

With this alert, one can determine whether a selected first antibody (or fragment thereof) binds to the same epitope as a second antibody or cross-competes for binding by using methods known in the art and methods shown in the examples herein. In one embodiment, one allows a first antibody of the invention to bind to EFNA under saturating conditions, and then measures the ability of a second antibody to bind to EFNA. The second antibody binds a different epitope than the first antibody if the test antibody is capable of binding to EFNA simultaneously with the first anti-EFNA antibody. However, if the second antibody is not capable of binding to EFNA at the same time, the second antibody binds to the same epitope, an overlapping epitope, or an epitope in close proximity to the epitope bound by the first antibody. As known in the art and as detailed in the examples below, solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays, Biacore may be used^TMThe system (i.e. surface plasmon resonance-GEHealthcare),an analyzer (i.e., bio-layer interferometry-ForteBio, Inc.) or flow cytometry method to obtain the desired data. As used herein, the term surface plasmon resonance refers to an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration within the biosensor matrix. In a particularly preferred embodimentIn embodiments, as shown in the examples below, the analysis is performed by using Biacore or ForteBio means.

The term competition, when used in the context of antibodies that compete for the same epitope, refers to competition between antibodies determined by an assay in which the antibody or immunologically functional fragment tested prevents or inhibits specific binding of a reference antibody to a common antigen. Typically, such assays involve the use of purified antigen bound to a solid surface or cell carrying one of an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Antibodies identified by competition assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to a nearby epitope close enough to the epitope bound by the reference antibody to be sterically hindered. Further details of methods for determining competitive binding are provided in the examples herein. Typically, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In certain instances, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

In addition to epitope specificity, the disclosed antibodies can be characterized using several different physical characteristics, including, for example, binding affinity, melting temperature (Tm), and isoelectric point.

e. Binding affinity

In this regard, the invention also encompasses the use of antibodies with high binding affinity for the selected EFNA (or in the case of pan-antibodies, for more than one type of ephrin-a ligand). When dissociation constant K_d(k_off/k_on)≤10^-8M, an antibody of the invention is said to specifically bind its target antigen. When K is_d≤5x10^-9M, the antibody specifically binds to the antigen with high affinity, when K_d≤5x10^-10M specifically binds to antigen with very high affinity. In one embodiment of the invention, the antibody has a K_dIs less than or equal to 10^-9M and an off-rate (off-rate) of about 1x10^-4And/sec. In one embodiment of the invention, the dissociation rate is<1x10^-5And/sec. In other embodiments of the invention, the antibody will be present at about 10^-8M to 10^-10K of M_dIn combination with EFNA, in another embodiment, it will be ≦ 2 × 10^-10K of M_dAnd (4) combining. Other selected embodiments of the invention include the following antibodies: having a dissociation constant or K_d(k_off/k_on) Is less than 10^-2M, less than 5x10^-2M, less than 10^-3M, less than 5x10^-3M, less than 10^-4M, less than 5x10^-4M, less than 10^-5M, less than 5x10^-5M, less than 10^-6M, less than 5x10^{- 6}M, less than 10^-7M, less than 5x10^-7M, less than 10^-8M, less than 5x10^-8M, less than 10^-9M, less than 5x10^-9M, less than 10^-10M, less than 5x10^-10M, less than 10^-11M, less than 5x10^-11M, less than 10^-12M, less than 5x10^-12M, less than 10^-13M, less than 5x10^-13M, less than 10^-14M, less than 5x10^-14M, less than 10^-15M or less than 5x10^-15M。

In particular embodiments, an antibody of the invention that immunospecifically binds EFNA has an association rate constant or k_onRate (EFNA (Ab) + antigen (Ag)^k _onRadix Seu caulis Opuntiae Dillenii (Ab-Ag) of at least 10⁵M^-1s^-1At least 2x10⁵M^-1s^-1At least 5x10⁵M^-1s^-1At least 10⁶M^-1s^-1At least 5x10⁶M^-1s^-1At least 10⁷M^-1s^-1At least 5x10⁷M^-1s^-1Or at least 10⁸M^-1s^-1。

In another embodiment, an antibody of the invention that immunospecifically binds EFNA has a k_offRate (EFNA (Ab) + antigen (Ag)^k _offRadix Seu caulis Opuntiae Dillenii No. Ab-Ag) of less than 10^-1s^-1Less than 5x10^-1s^-1Less than 10^-2s^-1Less than 5x10^-2s^-1Less than 10^-3s^-1Less than 5x10^-3s^-1Less than 10^-4s^-1Less than 5x10^-4s^-1Less than 10^-5s^-1Less than 5x10^-5s^-1Less than 10^{- 6}s^-1Less than 5x10^-6s^-1Less than 10^-7s^-1Less than 5x10^-7s^-1Less than 10^-8s^-1Less than 5x10^-8s^-1Less than 10^-9s^-1Less than 5x10^-9s^-1Or less than 10^-10s^-1。

In other selected embodiments of the invention, the anti-EFNA antibody will have an affinity constant or K_a(k_on/k_off) Is at least 10²M^-1At least 5x10²M^-1At least 10³M^-1At least 5x10³M^-1At least 10⁴M^-1At least 5x10⁴M^-1At least 10⁵M^-1At least 5x10⁵M^-1At least 10⁶M^-1At least 5x10⁶M^-1At least 10⁷M^-1At least 5x10⁷M^-1At least 10⁸M^-1At least 5x10⁸M^-1At least 10⁹M^-1At least 5x10⁹M^-1At least 10¹⁰M^-1At least 5x10¹⁰M^-1At least 10¹¹M^-1At least 5x10¹¹M^-1At least 10¹²M^-1At least 5x10¹²M^-1At least 10¹³M^-1At least 5x10¹³M^-1At least 10¹⁴M^-1At least 5x10¹⁴M^-1At least 10¹⁵M^-1Or at least 5x10¹⁵M^-1。

f. Isoelectric point

In addition to the foregoing binding properties, like all polypeptides, anti-EFNA antibodies and fragments thereof have an isoelectric point (pI), which is generally defined as the pH at which the polypeptide carries no net charge. It is known in the art that the solubility of proteins is usually the lowest when the pH of the solution is equal to the isoelectric point (pI) of the protein. Thus, it is possible to optimize solubility by altering the number and position of ionizable residues in the antibody to adjust pI. For example, the pI of a polypeptide can be manipulated by making appropriate amino acid substitutions (e.g., by substituting an uncharged residue, such as alanine, for a charged amino acid, such as lysine). Without wishing to be bound by any particular theory, amino acid substitutions of an antibody that cause a change in the pI of the antibody may improve the solubility and/or stability of the antibody. Those skilled in the art will understand which amino acid substitutions will be most appropriate for a particular antibody to achieve the desired pI.

The pI of a protein can be determined by a variety of methods including, but not limited to, isoelectric focusing and various computer algorithms (see, e.g., Bjellqvist et al, 1993, electrophoresinis 14: 1023). In one embodiment, the pI of the anti-EFNA antibody of the invention is greater than about 6.5, about 7.0, about 7.5, about 8.0, about 8.5 or about 9.0. In another embodiment, the pI of the anti-EFNA antibody of the invention is higher than 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0. In another embodiment, substitutions that cause a change in the pI of an antibody of the invention will not significantly reduce their binding affinity for EFNA. As discussed in more detail below, it is specifically contemplated that substitutions in the Fc region that cause altered binding to Fc γ R may also cause changes in pI. In a preferred embodiment, the substitutions in the Fc region are specifically selected to achieve the desired alteration of Fc γ R binding and any desired change in pI. As used herein, the pI value is defined as the pI of the predominantly charged form.

g. Thermal stability

It will also be appreciated that the Tm of the Fab domain of an antibody may be a good indicator of the thermostability of the antibody and may further provide an indication of shelf life. The Tm is simply the temperature at which a given domain or sequence unfolds 50%. Lower Tm indicates more aggregation/less stability, while higher Tm indicates less aggregation/more stability. Thus, antibodies or fragments or derivatives with higher Tm are preferred. Furthermore, it is possible to alter the composition of the anti-EFNA antibody or domain thereof to increase or optimize molecular stability using techniques recognized in the art. See, for example, U.S. patent No. 7,960,142. Thus, in one embodiment, the Fab domain of the selected antibody has a Tm value that is higher than at least 50 ℃, 55 ℃,60 ℃, 65 ℃,70 ℃,75 ℃,80 ℃,85 ℃, 90 ℃,95 ℃, 100 ℃, 105 ℃, 110 ℃, 115 ℃ or 120 ℃. In another embodiment, the Fab domain of the antibody has a Tm value that is greater than at least about 50 ℃, about 55 ℃, about 60 ℃, about 65 ℃, about 70 ℃, about 75 ℃, about 80 ℃, about 85 ℃, about 90 ℃, about 95 ℃, about 100 ℃, about 105 ℃, about 110 ℃, about 115 ℃ or about 120 ℃. The thermal melting temperature (Tm) of a protein domain (e.g., Fab domain) can be measured using any standard method known in the art, such as by differential scanning calorimetry (see, e.g., Vermeer et al, 2000, Biophys.J.78: 394: 404; Vermeer et al, 2000, Biophys.J.79: 2150-.

EFNA modulator fragments and derivatives

Whether the agents of the invention comprise intact fusion constructs, antibodies, fragments or derivatives, the selected modulator will react with, bind to, combine with, complex with, link to, attach to, link to, interact with or otherwise bind to EFNA and thereby provide the desired anti-tumor effect. One skilled in the art will appreciate that modulators comprising anti-EFNA antibodies interact or bind to EFNA through one or more binding sites expressed on the antibody. More specifically, as used herein, the term binding site includes a region of a polypeptide that is responsible for selectively binding a target molecule of interest (e.g., an enzyme, antigen, ligand, receptor, substrate, or inhibitor). The binding domain includes at least one binding site (e.g., an intact IgG antibody will have two binding domains and two binding sites). Exemplary binding domains include antibody variable domains, receptor binding domains of ligands, ligand binding domains of receptors, or enzymatic domains. For the purposes of the present invention, a typical EFNA active region (e.g., as part of an Fc-EFNA fusion construct) may include a binding site for a substrate (e.g., an Eph receptor).

a. Fragments

Regardless of the form of the modulator chosen (e.g., chimeric, humanized, etc.) to practice the present invention, it will be understood that immunoreactive fragments thereof may be used in accordance with the teachings herein. In the broadest sense, the term antibody fragment includes at least a portion of an intact antibody (e.g., a naturally occurring immunoglobulin). More particularly, the term fragment refers to a part or portion of an antibody or antibody chain (or EFNA molecule in the case of Fc fusion) that comprises fewer amino acid residues than the intact or complete antibody or antibody chain. The term antigen-binding fragment refers to an immunoglobulin or polypeptide fragment of an antibody that binds to an antigen or competes with (i.e., specifically binds to) an intact antibody (i.e., the intact antibody from which it was derived). As used herein, the term fragment of an antibody molecule includes antigen-binding fragments of an antibody, such as the antibody light chain (V)_L) Antibody heavy chain (V)_H) Single chain antibodies (scFv), F (ab')2 fragments, Fab fragments, Fd fragments, Fv fragments, single domain antibody fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Similarly, active fragments of EFNA contain portions of the EFNA molecule that retain their ability to interact with EFNA substrates or receptors and modify them in a manner similar to intact EFNA (although perhaps somewhat less efficiently).

It will be appreciated by those skilled in the art that such fragments may be obtained by chemical or enzymatic treatment of the whole or intact modulator (e.g. antibody or antibody chain) or by recombinant means. In this regard, while various antibody fragments are defined with respect to the digestion of intact antibodies, one skilled in the art will appreciate that the fragments can be synthesized de novo either chemically or by using recombinant DNA methods. Thus, as used herein, the term antibody specifically includes antibodies, or fragments or derivatives thereof, produced by modification of whole antibodies, or newly synthesized by using recombinant DNA methods.

More specifically, papain digestion of antibodies produces two identical antigen-binding fragments (called Fab fragments, each with a single antigen-binding site) and a residual Fc fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produces F (ab') which has two antigen binding sites and is still capable of cross-linking antigens₂And (3) fragment. The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (C)_H1). Fab' fragments by heavy chain C_H1 domain(s) differ from the Fab fragment by the addition of a few residues at the carboxy terminus (including one or more cysteines from the antibody hinge region). Fab' -SH is referred to herein for the following: fab' in which the cysteine residue of the constant domain bears at least one free thiol group. F (ab')₂Antibody fragments were originally produced as a Fab' fragment pair with a hinge cysteine between them. Other chemical couplings of antibody fragments are also known. For a more detailed description of other antibody fragments see, for example, Fundamental Immunology, w.e.paul, ed., Raven Press, n.y. (1999).

It will also be appreciated that Fv fragments are antibody fragments that contain an intact antigen recognition and binding site. This region is formed by a dimer of one heavy and one light chain variable domain that are tightly bound (may be covalent in nature, for example in an scFv). It is in this configuration that the three CDRs of each variable domain interact to define V_H-V_LAntigen binding sites on the surface of the dimer. In summary, six CDRs, or subsets thereof, confer antigen binding specificity to an antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the recognition sumThe ability to bind antigen, although usually with a lower affinity than the entire binding site.

In other embodiments, the antibody fragment is, for example, a fragment of: which comprises an Fc region and retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to an intact antibody. For example, such antibody fragments may comprise an antigen-binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

b. Derivatives of the same

In another embodiment, it will be further understood that modulators of the invention may be monovalent or multivalent (e.g., bivalent, trivalent, etc.). As used herein, the term valency refers to the number of binding sites of a potential target (i.e., EFNA) that bind to an antibody. Each target binding site specifically binds to one target molecule or a specific location or portion on a target molecule. When an antibody of the invention comprises more than one target binding site (multivalent), each target binding site may specifically bind to the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes or locations on the same antigen). For the purposes of the present invention, an antibody of the invention will preferably have at least one binding site specific for human EFNA. In one embodiment, the antibody of the invention will be monovalent, wherein each binding site of the molecule will specifically bind to a single EFNA location or epitope. In other embodiments, the antibodies will be multivalent, in that they comprise more than one binding site and different binding sites specifically bind to more than a single site or epitope. In such a case, the multiple epitopes may be present on the selected EFNA polypeptide or splice variant, or a single epitope may be present on the EFNA and a second, different epitope may be present on another molecule or surface. See, for example, U.S. patent No. 2009/0130105.

As described above, multivalent antibodies can immunospecifically bind to different epitopes of a desired target molecule or can immunospecifically bind to a target molecule and a heterologous epitope (e.g., a heterologous polypeptide or a solid support material). While preferred embodiments of anti-EFNA antibodies bind only two antigens (i.e., bispecific antibodies), antibodies with additional specificity (e.g., trispecific antibodies) are also encompassed by the invention. Examples of bispecific antibodies include, without limitation, those having one arm directed against EFNA and another arm directed against any other antigen (e.g., a modulator cell marker). Methods for making bispecific antibodies are known in the art. The traditional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, 1983, Nature, 305: 537-539). Other more complex compatible multispecific constructs and methods for their manufacture are shown in U.S. patent No. 2009/0155255.

In other embodiments, an antibody variable domain (antibody-antigen combining site) with the desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion is preferably with an immunoglobulin heavy chain constant domain comprising a hinge, C_H2 and/or C_H3 at least a portion of the area. In one example, a first heavy chain constant region (C) containing a site required for light chain binding_H1) Is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain (and, if desired, immunoglobulin light chain) fusions is inserted into a separate expression vector and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in an embodiment, when unequal proportions of the three polypeptide chains are used in the construction to provide optimal yields. However, when at least two polypeptide chains are expressed in equal ratios resulting in high yields or when the ratios are not particularly affected, it is possible to insert the coding sequences for two or all three polypeptide chains in one expression vector.

In one embodiment of the methodIn one embodiment, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain having a first binding specificity (e.g., EFNA4) in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from the undesired immunoglobulin chain combinations, since the presence of the immunoglobulin light chain in only half of the bispecific molecule provides a simple way of separation. Such a process is disclosed in WO 94/04690. For additional details on the generation of bispecific antibodies, see, e.g., Suresh et al, 1986, Methods in Enzymology, 121: 210. According to another approach described in WO96/27011, the antibody molecule pair can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces include C of the antibody constant domain_H3 domain. In this approach, one or more small amino acid side chains of the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities ranging from the same or smaller size to large side chains are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers relative to other undesired end products (e.g., homodimers).

Bispecific antibodies also include cross-linked or heteroconjugate (heteroconjugate) antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and are useful in the treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies can be made by using any convenient crosslinking method. Suitable crosslinking reagents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several crosslinking techniques.

Efna modulator-constant region modification

Fc region and Fc receptor

In addition to various modifications, substitutions, additions or deletions of the variable or binding regions of the disclosed modulators (e.g., Fc-EFNA or anti-EFNA antibodies) as described above, those skilled in the art will appreciate that selected embodiments of the invention may also include substitutions or modifications of the constant region (i.e., Fc region). More specifically, it is contemplated that the EFNA modulators of the present invention may specifically contain one or more other amino acid residue substitutions, mutations, and/or modifications that result in compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased production, altered Fc ligand binding, enhanced or reduced ADCC or CDC activity, altered glycosylation and/or disulfide bonding, and modified binding specificity. In this regard, it will be appreciated that these Fc variants may advantageously be used to improve the effective anti-tumor properties of the disclosed modulators.

The term Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinantly engineering a nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibodies may comprise a population of antibodies with all K447 residues removed, a population of antibodies without K447 residues removed, and a population of antibodies with a mixture of antibodies with and without K447 residues. A functional Fc region has the effector functions of a native sequence Fc region. Exemplary effector functions include C1q binding; CDC; fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays such as disclosed in the definitions herein.

Fc receptor FcR describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, an FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the Fc γ RI, Fc. Fc γ II receptors include Fc γ RIIA (activating receptor) and Fc γ RIIB (inhibitory receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor F γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron, Annu. Rev. Immunol.15:203-234 (1997)). Fcrs are reviewed, for example, in ravatch and Kinet, annu.rev.immunol 9:457-92 (1991); capel et al, immunolmethods 4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). Other fcrs (including those that will be identified in the future) are encompassed by the term FcR herein. The term Fc receptor or FcR also includes the neonatal receptor FcRn, which in some cases is responsible for the transfer of maternal IgG to the fetus (Guyer et al, j.immunol.117:587(1976) and Kim et al, j.immunol.24:249(1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol.today 18(12):592-598 (1997); Ghetie et al, Nature Biotechnology, 15(7):637-640 (1997); Hinton et al, J.biol.chem.279(8):6213-6216 (2004); WO 2004/92219(Hinton et al)).

Fc function

As used herein, complement-dependent cytotoxicity and CDC refer to the lysis of target cells in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule, such as an antibody complexed with a cognate antigen. To assess complement activation, CDC assays can be performed, for example as described in Gazzano-Santoro et al, 1996, J.Immunol.methods, 202: 163.

Furthermore, antibody-dependent cell-mediated cytotoxicity or ADCC refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (fcrs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Specific high affinity IgG antibodies directed against the target arm the cytotoxic cells and are necessary for such killing. Lysis of the target cells is extracellular, requires direct cell-to-cell contact and does not involve complement.

EFNA modulator variants with altered FcR binding affinity or ADCC activity are those with enhanced or reduced FcR binding activity and/or ADCC activity as compared to the parent or unmodified antibody or modulator comprising a native sequence Fc region. A modulator variant that exhibits enhanced binding to an FcR binds at least one FcR with better affinity than the parent or unmodified antibody or a modulator comprising a native sequence Fc region. Variants that exhibit reduced binding to an FcR bind at least one FcR with poorer affinity than the parent or unmodified antibody or a modulator comprising a native sequence Fc region. Such variants that exhibit reduced binding to an FcR may have little or no significant binding to an FcR, e.g., 0-20% binding to an FcR compared to a native sequence IgG Fc region, e.g., as determined by techniques well known in the art.

As to FcRn, the antibodies of the invention also include or encompass Fc variants having modifications to the constant region that provide a half-life (e.g., serum half-life) in a mammal (preferably a human) of greater than 5 days, greater than 10 days, greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-life of the antibody (or Fc-containing molecule) of the invention in a mammal (preferably a human) results in a higher serum titer of said antibody or antibody fragment in the mammal and thus reduces the frequency of administration of said antibody or antibody fragment and/or reduces the concentration of said antibody or antibody fragment to be administered. Antibodies with increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, antibodies with increased half-life in vivo can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International publication No. WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737,056, and U.S. Pat. No. 2003/0190311). The in vivo binding to human FcRn and the serum half-life of human FcRn high affinity binding polypeptides can be determined, for example, in: for example in transgenic mice or transfected human cell lines expressing human FcRn, or primates administered with polypeptides having variant Fc regions. WO 2000/42072 describes antibody variants with increased or decreased binding to FcRn. See also, for example, Shields et al J.biol.chem.9(2):6591-6604 (2001).

c. Glycosylation modification

In other embodiments, the glycosylation pattern or composition of the antibodies of the invention is modified. More specifically, preferred embodiments of the present invention may include engineered glycoforms, i.e., altered glycosylation patterns or altered carbohydrate compositions covalently attached to a molecule comprising an Fc region. Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function, increasing the affinity of an antibody for a target antigen, or promoting production of an antibody. Where reduced effector function is desired, it will be understood that the molecule may be engineered to be expressed in an unglycosylated form. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. That is, one or more amino acid substitutions that result in the removal of one or more variable region framework glycosylation sites can be made to eliminate glycosylation at that site (see, e.g., U.S. Pat. nos. 5,714,350 and 6,350,861). Conversely, enhanced effector function or improved binding may be imparted to the Fc-containing molecule by engineering in one or more other glycosylation sites.

In addition orAlternatively, Fc variants with altered glycosylation compositions can be made, such as low fucosylated antibodies with reduced amounts of fucose residues or antibodies with increased bisecting GlcNAc structures. These and similar altered glycosylation patterns are shown to increase the ADCC ability of an antibody. Engineered glycoforms can be produced by any method known to those skilled in the art, for example by using engineered or variant expressing strains, by co-expression with one or more enzymes, such as N-acetylglucosaminyltransferase III (GnTI11), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms or by modifying a carbohydrate after expression of a molecule comprising an Fc region. See, e.g., Shield, R.L. et al (2002) J.biol.chem.277: 26733-26740; umana et al (1999) nat. Biotech.17:176-1, and European patent No. EP 1,176,195; PCT publications WO 03/035835; WO 99/54342, Umana et al, 1999, nat. Biotechnol 17: 176-180; davies et al, 20017 Biotechnol Bioeng 74: 288-294; shield et al, 2002, J Biol Chem 277: 26733-26740; shinkawa et al, 2003, J Biol Chem 278: 3466-; U.S. serial No. 10/277,370; 10/113,929, respectively; PCT WO 00/61739a 1; PCT WO 01/292246a 1; PCT WO 02/311140a 1; PCT WO 02/30954a 1; potilllegent^TMTechnique (Biowa, Inc.); GlycoMAb^TMGlycosylation engineering technology (GLYCART biotechnology AG); WO 00061739; EA 01229125; U.S. patent nos. 2003/0115614; okazaki et al, 2004, JMB, 336: 1239-49.

IX. regulon expression

a. Overview

DNA encoding the desired EFNA modulator can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody). If the modulator is an antibody, isolated and subcloned hybridoma cells (or phage-or yeast-derived colonies) may be the preferred source of such DNA. If desired, the nucleic acids can be further manipulated as described herein to produce reagents including fusion proteins, or chimeric, humanized, or fully human antibodies. More specifically, isolated DNA (which can be modified) can be used to clone the constant and variable region sequences for antibody production, as described in U.S. patent No. 7,709,611.

This exemplary method entails extracting RNA from selected cells, converting to cDNA, and amplifying by PCR using antibody-specific primers. Suitable primers are well known in the art and, as exemplified herein, are readily available from a number of commercial sources. It will be understood that, for expression of recombinant human or non-human antibodies isolated by screening combinatorial libraries, the DNA encoding the antibodies is cloned into recombinant expression vectors and introduced into host cells, including mammalian cells, insect cells, plant cells, yeast, and bacteria. In other embodiments, the modulator is introduced into and expressed by a simian COS cell, NS0 cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell that does not otherwise produce the desired construct. As will be discussed in more detail below, transformed cells expressing the desired modulator can be cultured in relatively large quantities to provide a clinical and commercial supply of the fusion construct or immunoglobulin.

Whether the nucleic acid encoding the desired portion of the EFNA modulator is obtained or derived from phage display technology, yeast libraries, hybridoma-based technology, synthetically or from commercial sources, it is to be understood that the invention specifically encompasses nucleic acid molecules and sequences encoding EFNA modulators including fusion proteins and anti-EFNA antibodies or antigen-binding fragments or derivatives thereof. The invention also encompasses nucleic acids or nucleic acid molecules (e.g., polynucleotides) that hybridize under high stringency, or alternatively, moderate or low stringency hybridization conditions (e.g., as defined below) to polynucleotides that are complementary to: the nucleic acid has a polynucleotide sequence encoding a modulator of the invention or a fragment or variant thereof. As used herein, the term nucleic acid molecule or isolated nucleic acid molecule is intended to include at least DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. In addition, the invention includes any vehicle or construct incorporating a polynucleotide encoding the modulator, including without limitation a vector, plasmid, host cell, cosmid, or viral construct.

The term isolated nucleic acid means that the nucleic acid is (i) amplified in vitro, e.g., by Polymerase Chain Reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, e.g., fractionated by shearing and gel electrophoresis, or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid is a nucleic acid that is useful for manipulation by recombinant DNA techniques.

More specifically, nucleic acids encoding the modulators are also provided, including one or both strands of an antibody of the invention, or fragments, derivatives, muteins or variants thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding polypeptides, antisense nucleic acids for inhibiting expression of polynucleotides, and the complements of the foregoing. The nucleic acid can be of any length. They may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and/or they may comprise one or more additional sequences, such as regulatory sequences, and/or be part of a larger nucleic acid (e.g., a vector). These nucleic acids may be single-stranded or double-stranded and may include RNA and/or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids). The nucleic acid encoding the modulator of the invention (including antibodies or immunoreactive fragments or derivatives thereof) is preferably isolated as described above.

b. Hybridization and identity

As shown, the present invention also provides nucleic acids that hybridize to other nucleic acids under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For the purposes of this application, moderately stringent hybridization conditions use a precleaning solution containing 5x sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0mM EDTA (pH 8.0); about 50% formamide, 6XSSC in hybridization buffer and 55 ℃ hybridization temperature (or other similar hybridization conditions, e.g., containing about 50% formamide, hybridization temperature of 42 ℃), and wash conditions of 60 ℃ in 0.5XSSC, 0.1% SDS. Stringent hybridization conditions are hybridization in 6XSSC at 45 ℃ followed by one or more washes in 0.1XSSC, 0.2% SDS at 68 ℃. Furthermore, one skilled in the art can manipulate hybridization and/or wash conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% identical to each other typically remain hybridized to each other. More generally, for the purposes of this disclosure, the term substantially identical with respect to a nucleic acid sequence can be construed as a nucleotide sequence that exhibits at least about 85%, or 90%, or 95%, or 97% sequence identity to a reference nucleic acid sequence.

The basic parameters that influence the selection of hybridization conditions and guide the design of appropriate conditions are shown below: for example, Sambrook, Fritsch and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current protocols in Molecular Biology, 1995, Ausubel et al, eds., John Wiley & Sons, Inc., sections 2.10and 6.3-6.4), and can be readily determined by one of ordinary skill in the art based on, for example, the length and/or base composition of the nucleic acid.

It will also be understood that, according to the invention, the nucleic acid may be present alone or in combination with other nucleic acids (which may be homologous or heterologous). In a preferred embodiment, the nucleic acid is functionally linked to an expression control sequence which may be homologous or heterologous with respect to said nucleic acid. In this context, the term homologous means that the nucleic acid is also naturally functionally linked to said expression control sequence and the term heterologous means that the nucleic acid is not naturally functionally linked to the expression control sequence.

c. Expression of

Nucleic acids (e.g., nucleic acids expressing RNA and/or proteins or peptides) and expression control sequences are functionally linked to each other if they are covalently linked to each other in such a way that expression or transcription of the nucleic acid is under the control or influence of the expression control sequence. If the nucleic acid is to be translated into a functional protein, then with the expression control sequence functionally linked to the coding sequence, induction of the expression control sequence results in transcription of the nucleic acid without causing a shift in reading frame of the coding sequence or without causing the coding sequence to be unable to be translated into the desired protein or peptide.

The term expression control sequence includes according to the invention promoters, ribosome binding sites, enhancers and other control sequences which regulate the transcription of a gene or the translation of an mRNA. In a particular embodiment of the invention, the expression control sequence is capable of being modulated. The exact structure of the expression control sequence may vary with the species or cell type, but generally includes 5' -nontranscribed and 5' -and 3' -nontranslated sequences involved in initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. More specifically, the 5' -non-transcribed expression control sequence comprises a promoter region comprising a promoter sequence for transcriptional control of the functionally linked nucleic acid. The expression control sequence may also comprise an enhancer sequence or an upstream activator sequence.

According to the invention, the term promoter or promoter region relates to a nucleic acid sequence which is located upstream (5') of the nucleic acid sequence to be expressed and controls the expression of said sequence by providing recognition and binding sites for RNA polymerase. The promoter region may include other recognition and binding sites for other factors involved in regulating gene transcription. Promoters can control the transcription of prokaryotic or eukaryotic genes. In addition, a promoter may be inducible and may initiate transcription in response to an inducing agent, or may be constitutive if transcription is not under the control of an inducing agent. If no inducer is present, the gene under the control of the inducible promoter is not expressed or is expressed only to a small extent. In the presence of an inducer, the gene is opened or the transcription level is increased. This is generally mediated by the binding of specific transcription factors.

Preferred promoters according to the present invention include promoters for SP6, T3, and T7 polymerase, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (e.g., CMV), a portion of which is fused with a portion of a promoter of a gene for other cellular proteins (e.g., human GAPDH (glyceraldehyde-3-phosphate dehydrogenase)), and other introns are included or excluded.

According to the present invention, the term expression is used in its most general sense and comprises the production of RNA or RNA and proteins/peptides. It also includes partial expression of nucleic acids. Furthermore, expression can be performed transiently or stably.

In a preferred embodiment, the nucleic acid molecule according to the invention is present in a vector, where appropriate with a promoter, which controls the expression of the nucleic acid. The term vector is used herein in its most general sense and includes any intermediate vehicle for nucleic acids which enables the nucleic acids to be introduced, for example, into prokaryotic and/or eukaryotic cells and, where appropriate, integrated into the genome. Such vectors are preferably replicated and/or expressed in cells. Vectors may include plasmids, phagemids, bacteriophages or viral genomes. As used herein, the term plasmid generally relates to a construct of extrachromosomal genetic material, typically a circular DNA duplex, that can replicate independently of chromosomal DNA.

In practicing the present invention, it will be understood that many conventional techniques in molecular biology, microbiology and recombinant DNA techniques may optionally be used. Such conventional techniques involve vectors, host cells and recombinant methods as defined herein. These techniques are well known and are explained in, for example, the following: berger and Kimmel, Guide to Molecular cloning technologies, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif.; sambrook et al, Molecular Cloning-A Laboratory Manual (3rd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 and Current protocols in Molecular Biology, F.M. Ausubel et al, eds., supra. Other useful references, for example, for cell isolation and Culture (e.g., for subsequent nucleic acid or protein isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and references cited therein; payne et al (1992) Plant Cell and Tissue Culture in liquid systems John Wiley & Sons, Inc. New York, N.Y.; gamborg and Phillips (Eds.) (1995) plant cell, Tissue and Organ Culture; fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (Eds.) The handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. Methods of making nucleic acids (e.g., by in vitro amplification, purification from cells, or chemical synthesis), methods for manipulating nucleic acids (e.g., site-directed mutagenesis, by restriction enzyme digestion, ligation, etc.), and various vectors, cell lines, etc., useful for manipulating and making nucleic acids are described in the above references. In addition, essentially any polynucleotide (including, for example, labeled or biotinylated polynucleotides) can be custom made or ordered from any of a variety of commercial sources.

Thus, in one aspect, the invention provides a recombinant host cell that allows for recombinant expression of an antibody of the invention, or a portion thereof. Antibodies produced by expression in such recombinant host cells are referred to herein as recombinant antibodies. The invention also provides progeny cells of such host cells, and antibodies produced therefrom.

As used herein, the term recombinant host cell (or simply host cell) refers to a cell into which a recombinant expression vector is introduced. It is understood that recombinant host cells and host cells refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term host cell, as used herein. Such cells may comprise a vector according to the invention as defined above.

In another aspect, the invention provides methods for making the antibodies or portions thereof described herein. According to one embodiment, the method comprises culturing a cell transfected or transformed with the vector described above, and retrieving the antibody or portion thereof.

As indicated above, expression of an antibody (or fragment or variant thereof) of the invention preferably comprises expression of a vector comprising a polynucleotide encoding the desired anti-EFNA antibody. Methods well known to those skilled in the art can be used to construct expression vectors comprising antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, embodiments of the present invention provide replicable vectors comprising a nucleotide sequence encoding an anti-EFNA antibody of the present invention (e.g., a whole antibody, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody, or a portion thereof, or a heavy or light chain CDR, single chain Fv, or a fragment or variant thereof) operably linked to a promoter. In preferred embodiments, such vectors may comprise a nucleotide sequence encoding a heavy chain of an antibody molecule (or fragment thereof), a nucleotide sequence encoding a light chain of an antibody (or fragment thereof), or both a heavy chain and a light chain.

Once the nucleotides of the present invention are isolated and modified according to the teachings herein, they can be used to generate selected modulators including anti-EFNA antibodies or fragments thereof.

Modulator production and purification

Using molecular biology techniques recognized in the art and current methods of protein expression, a large number of desired modulators can be generated. More specifically, nucleic acid molecules encoding modulators (e.g., antibodies) obtained and engineered as described above can be incorporated into well-known and commercially available protein production systems (including various types of host cells) to provide preclinical, clinical, or commercial quantities of a desired pharmaceutical product. It will be appreciated that in a preferred embodiment, the nucleic acid molecule encoding the modulator is engineered into the following vector or expression vector: which provides efficient integration into the selected host cell and subsequent high expression levels of the desired EFNA modulator.

Preferably, nucleic acid molecules encoding EFNA modulators and vectors comprising these nucleic acid molecules may be used to transfect suitable mammalian, plant, bacterial or yeast host cells, although it will be appreciated that prokaryotic systems may be used for modulator production. Transfection may be by any known method for introducing a polynucleotide into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus. In addition, the nucleic acid molecule can be introduced into a mammalian cell by a viral vector. Methods for transforming mammalian cells are well known in the art. See, for example, U.S. Pat. nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455. In addition, methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are well known in the art.

In addition, a host cell may be co-transfected with two expression vectors of the invention, e.g., a first vector encoding a polypeptide derived from the heavy chain and a second vector encoding a polypeptide derived from the light chain. The two vectors may contain the same selectable marker that enables substantially equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes and is capable of expressing both the heavy and light chain polypeptides. In this case, the light chain is preferably placed before the heavy chain to avoid an excess of non-toxic heavy chains. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

a. Host expression system

Various host expression vector systems (many are commercially available) are compatible with the teachings herein and can be used to express the modulators of the invention. Such host expression systems represent the vehicle from which the coding sequence of interest can be expressed and subsequently purified, but also represent the cell (which can express the molecule of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequence). Such systems include, but are not limited to, microorganisms such as bacteria (e.g., E.coli, Bacillus subtilis, Streptomyces) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing regulator coding sequences; yeast (e.g., saccharomyces cerevisiae, pichia pastoris) transfected with recombinant yeast expression vectors containing regulator coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing regulator coding sequences; plant cell systems (e.g., tobacco, Arabidopsis, duckweed, maize, wheat, potato, etc.) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transfected with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a regulator coding sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) with recombinant expression constructs containing promoters derived from the genome of a mammalian cell (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, several expression vectors may be advantageously selected, depending on the intended use of the expressed molecule. For example, where large quantities of such proteins are to be produced, vectors directing the expression of high levels of fusion protein products that are readily purified may be required for pharmaceutical compositions that produce modulators. Such vectors include, but are not limited to, the E.coli expression vector pUR278(Ruther et al, EMBO 1.2:1791(1983)) in which the coding sequence may be ligated separately into the vector in frame with the lacZ coding region to produce a fusion protein; pIN vector (Inouye & Inouye, Nucleic Acids Res.13: 3101-; and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence may be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, several virus-based expression systems can be used to introduce the desired nucleotide sequence. In the case of using an adenovirus as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription/translation control complex (e.g., late promoter and tripartite leader sequence). This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g.region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the molecule in an infected host (see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 355-359 (1984)). Specific initiation signals may also be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, including natural and synthetic. The efficiency of expression can be increased by including appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., Bittner et al, Methods in enzymol.153:51-544 (1987)). Compatible mammalian cell lines that can be used as hosts for expression are therefore well known in the art and include many immortalized cells available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 free cells (Life Technologies), NIH-3T3 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, African green monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells and several other cell lines.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. Thus, cell lines stably expressing the selected modulator can be engineered using standard art-recognized techniques. Rather than using an expression vector containing a viral origin of replication, host cells can be transformed with DNA and a selectable marker under the control of appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). Following the introduction of the foreign DNA, the engineered cells may be allowed to grow in the enrichment medium for 1-2 days and then switched to the selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosome and grow to form foci, which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines expressing the molecule. Such engineered cell lines may be particularly useful in screening and evaluating compositions that interact directly or indirectly with the molecule.

Several selection systems are well known in the art and may be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al, Cell 11:223(1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202(1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell 22: 817 (1980)) genes may be employed in tk-, hgprt-or aprt-cells, respectively. Furthermore, antimetabolite resistance can be used as a basis for the selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, Natl. Acad. Sci. USA 77:357 (1980); O' Hare et al, Proc. Natl. Acad. Sci. USA78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, proc.Natl.Acad.Sci.USA78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12: 488-505; Wu and Wu, Biotherapy 3:87-95 (1991)); tolstoshiev, Ann.Rev.Pharmacol.Toxicol.32:573-596 (1993); mulligan, Science 260:926 and 932 (1993); and Morgan and Anderson, ann.rev.biochem.62: 191-217 (1993); TIB TECH 11(5) 155-; and hygro, which confers resistance to hygromycin (Santerre et al, Gene 30:147 (1984)). Methods of recombinant DNA technology generally known in the art can be routinely employed to select the desired recombinant clone and are described, for example, in Ausubel et al (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al (eds), Current Protocols in human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al, J.mol.biol.150:1 (1981). It will be appreciated that one particularly preferred method of establishing a stable, high-yield cell line includes the glutamyl ammonia synthase gene expression system (the GS system), which provides an efficient method for increasing expression under certain conditions. The GS system is discussed in whole or in part in connection with european patents 0216846, 0256055, 0323997 and 0338841, each of which is hereby incorporated by reference.

In addition, a host cell strain may be selected which modulates the expression of the inserted sequences or modifies and processes the gene product in a specific manner as desired. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function and/or purification of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. As known in the art, appropriate cell lines or host systems may be selected to ensure the desired modification and processing of the expressed polypeptide. For this reason, eukaryotic host cells with cellular mechanisms for proper processing of primary transcripts, glycosylation, and phosphorylation of gene products are particularly effective for use in the present invention. Thus, particularly preferred mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, NS0, MDCK, 293, 3T3, W138 and breast cancer cell lines, such as BT483, Hs578T, HTB2, BT2O and T47D, as well as normal breast cell lines, such as CRL7O3O and HsS78 Bst. Depending on the modulator and the production system chosen, the skilled person can easily select and optimize suitable host cells for efficient expression of the modulator.

b. Chemical synthesis

In addition to the aforementioned host cell systems, it will be understood that modulators of the invention may be chemically synthesized using techniques known in the art (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H.Freeman & Co., N.Y., and Hunkapiller, M. et al, 1984, Nature 310: 105-. For example, peptides corresponding to the polypeptide fragments of the present invention can be synthesized by using a peptide synthesizer. In addition, non-classical amino acids or chemical amino acid analogs can be introduced into the polypeptide sequence as substitutions or additions, if desired. Non-canonical amino acids include, but are not limited to, the D-isoforms of common amino acids, 2, 4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer (designer) amino acids such as b-methyl amino acid, Ca-methyl amino acid, Na-methyl amino acid, and amino acid analogs in general. Furthermore, the amino acid may be D (dextrorotatory) or L (levorotatory).

c. Transgenic system

The EFNA modulators of the invention may be produced transgenically by producing mammals or plants that are transgenic for the immunoglobulin heavy and light chain sequences of interest (or fragments or derivatives or variants thereof) and producing the desired compounds in recoverable form. In connection with transgenic production in mammals, anti-EFNA antibodies can be produced and recovered from, for example, emulsions in goats, cattle or other mammals. See, for example, U.S. Pat. nos. 5,827,690, 5,756,687, 5,750,172 and 5,741,957. In certain embodiments, a non-human transgenic animal comprising a human immunoglobulin locus is immunized with EFNA or an immunogenic portion thereof, as described above. Methods for making antibodies in plants are described, for example, in U.S. Pat. nos. 6,046,037 and 5,959,177.

Non-human transgenic animals or plants may be produced by introducing one or more nucleic acid molecules encoding the EFNA modulators of the invention into an animal or plant via standard transgenic techniques in accordance with the teachings herein. See Hogan and U.S. Pat. No. 6,417,429. The transgenic cell used to make the transgenic animal may be an embryonic stem cell or a somatic cell or a fertilized egg. The transgenic non-human organism may be chimeric, non-chimeric hybrid and non-chimeric homozygote. See, e.g., Hogan et al, Manipulating the Mouse Embryo: a Laboratory Manual 2nd ed., Cold Spring harborPress (1999); jackson et al, Mouse Genetics and Transgenics: a Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: arabidopsis Handbook, Academic Press (1999). In certain embodiments, the transgenic non-human animal is targeted for disruption and replacement by a targeting construct encoding, for example, a heavy and/or light chain of interest. In one embodiment, the transgenic animal comprises and expresses nucleic acid molecules encoding the heavy and light chains that specifically bind EFNA. Although anti-EFNA antibodies can be made in any transgenic animal, in particularly preferred embodiments, the non-human animal is a mouse, rat, sheep, pig, goat, cow, or horse. In other embodiments, the non-human transgenic animal expresses a desired pharmaceutical product in blood, milk, urine, saliva, tears, mucus, and other bodily fluids, from which the pharmaceutical product can be readily obtained using purification techniques recognized in the art.

Regulators (including antibodies) expressed by different cell lines or in transgenic animals will likely have different glycosylation patterns from each other. However, all regulators encoded by, or comprising, the nucleic acid molecules provided herein are of the inventionIn part, regardless of the glycosylation state of the molecule, and more generally, regardless of the presence or absence of post-translational modifications. Furthermore, the invention encompasses modulators that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protective/blocking groups, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, and the like. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄Specific chemical cleavage, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc. The invention also encompasses various post-translational modifications, including, for example, N-linked or O-linked carbohydrate chains, processing of the N-terminus or C-terminus, attachment of chemical moieties to the amino acid backbone, chemical modification of N-linked or O-linked carbohydrate chains, and addition or deletion of the N-terminal methionine residue as a result of expression in prokaryotic host cells. Furthermore, as shown in the text and examples below, the polypeptide may also be modified with a detectable label (e.g., an enzymatic, fluorescent, radioisotope, or affinity label) to allow detection and isolation of the modulator.

d. Purification of

Once the modulators of the invention are produced by recombinant expression or any of the other techniques disclosed herein, the modulators may be purified by any method known in the art for immunoglobulin purification, or more generally by any other standard technique for protein purification. In this regard, the modulator may be isolated. As used herein, an isolated EFNA modulator is one that is identified and separated and/or recovered from a component of its natural environment. The contaminant components of their natural environment are such materials: which would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. Isolated modulators include modulators that are in situ in recombinant cells, as at least one component of the polypeptide's natural environment will not be present.

When recombinant techniques are used, the EFNA modulator (e.g., anti-EFNA antibody or derivative or fragment thereof) may be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the desired molecule is produced intracellularly, as a first step, particulate debris (host cells or lysed fragments) can be removed, for example, by centrifugation or ultrafiltration. For example, Carter et al, Bio/Technology 10:163(1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) in about 30 minutes. Cell debris can be removed by centrifugation. When the antibody is secreted into the culture medium, the supernatant from the expression system is typically first concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors (e.g., PMSF) may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

The modulator (e.g., fc-EFNA or anti-EFNA antibody) composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (with affinity chromatography being the preferred purification technique). The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the chosen construct. Protein A can be used to purify antibodies based on human IgG1, IgG2, or IgG4 heavy chains (Lindmark et al, J Immunolmeth 62:1 (1983)). Protein G is recommended for all mouse isotypes as well as human IgG3(Guss et al, EMBO J5: 1567 (1986)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene allow faster flow rates and shorter processing times than can be achieved with agarose. When the antibody comprises C_H3 domain, Bakerbond ABX^TMResin (J.T.Baker; Phillipsburg, N.J..) Can be used for purification. Depending on the antibody to be recovered, other techniques for protein purification may also be used, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica gel, chromatography on heparin, agarose chromatography on anion or cation exchange resins (e.g. polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation. In a particularly preferred embodiment, the modulators of the invention will be purified, at least in part, using protein a or protein G affinity chromatography.

XI conjugation to EFNA modulators

Once the modulators of the invention have been purified according to the teachings herein, they may be linked, fused, conjugated (e.g., covalently or non-covalently) or otherwise associated with a pharmaceutically active or diagnostic moiety or biocompatible modification. As used herein, the term conjugate will be used broadly and is considered to refer to any molecule that binds to the disclosed modulator regardless of the method of binding. In this regard, it will be understood that such conjugates can include peptides, polypeptides, proteins, polymers, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, organic molecules, and radioisotopes. Furthermore, as indicated above, the selected conjugates can be covalently or non-covalently linked to the modulator and exhibit various molar ratios, depending at least in part on the method used to effect the conjugation.

In a preferred embodiment, the following will be apparent: modulators of the invention may be conjugated or conjugated to proteins, polypeptides or peptides that impart selected characteristics (e.g., biotoxins, biomarkers, purification tags, etc.). More generally, in selected embodiments, the invention encompasses the use of modulators or fragments thereof recombinantly fused or chemically conjugated (including covalent and non-covalent conjugations) to a heterologous protein or polypeptide, wherein the polypeptide comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. The constructs need not necessarily be directly linked, but may occur through linker sequences. For example, antibodies can be used to target heterologous polypeptides to specific cell types expressing EFNA (in vitro or in vivo) by fusing or conjugating modulators of the invention to antibodies specific to specific cell surface receptors. In addition, modulators fused or conjugated to heterologous polypeptides may also be used in vitro immunoassays and are compatible with purification methods known in the art. See, for example, international publication nos. WO 93/21232; european patent numbers EP 439,095; naramura et al, 1994, Immunol.Lett.39: 91-99; U.S. patent nos. 5,474,981; gillies et al, 1992, PNAS 89: 1428-; and Fell et al, 1991, J.Immunol.146: 2446-2452.

a. Biocompatible modulators

In preferred embodiments, modulators of the invention may be conjugated or otherwise associated with biocompatible modifications that may be used to adjust, alter, improve or modulate the characteristics of the modulator as desired. For example, antibodies or fusion constructs with increased in vivo half-life can be generated by attaching relatively high molecular weight polymer molecules, such as commercially available polyethylene glycol (PEG) or similar biocompatible polymers. One skilled in the art will appreciate that PEG can be obtained in many different molecular weights and molecular configurations, which can be selected to impart specific characteristics (e.g., customizable half-life) to the antibody. The attachment of PEG to the modulator or antibody fragment or derivative may be by site-specific conjugation of PEG to the N or C terminus of the antibody or antibody fragment or by an-amino group present on a lysine residue, with or without a multifunctional linker. Linear or branched polymers that cause minimal loss of biological activity may be used for derivatization. The extent of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure optimal conjugation of the PEG molecule to the antibody molecule. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography. In a similar manner, the disclosed modulators can be conjugated to albumin to make the antibodies or antibody fragments more stable in vivo or have a longer half-life in vivo. Such techniques are well known in the art, see, for example, international publication nos. WO 93/15199, WO 93/15200, and WO 01/77137; and european patent No. 0413,622. Other biocompatible conjugates will be apparent to those skilled in the art and can be readily identified in light of the teachings herein.

b. Diagnostic or detection reagents

In other preferred embodiments, modulators of the invention, or fragments or derivatives thereof, are conjugated to a diagnostic or detectable agent, label or reporter, which may be a biomolecule (e.g., peptide or nucleotide), small molecule, fluorophore or radioisotope. The labeled modulators may be used to monitor the development or progression of hyperproliferative disorders or as part of a clinical testing procedure to determine the efficacy of a particular therapy (i.e., theranosis) that includes the disclosed modulators. Such markers or reporters may also be used to purify selected modulators, to isolate or isolate TICs or in preclinical procedures or toxicology studies.

The diagnosis and detection may be accomplished by coupling the modulator to a detectable substance including, but not limited to, various enzymes including, for example, horseradish peroxidase, alkaline phosphatase, β -galactosidase or acetylcholinesterase, prosthetic groups such as, but not limited to, streptavidin biotin and avidin/biotin, fluorescent substances such as, but not limited to, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride or phycoerythrin, luminescent substances such as, but not limited to, luminol, bioluminescent substances such as, but not limited to, luciferase, fluorescein and aequorin, radioactive substances such as, but not limited to, iodine (I), (II), (III), (IV), (¹³¹I，¹²⁵I，¹²³I，¹²¹I), carbon (C: (¹⁴C) Sulfur (S) (S)³⁵S), tritium (³H) Indium (I)¹¹⁵In，¹¹³In，¹¹²In，¹¹¹In, and technetium (C) ()⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga，⁶⁷Ga), palladium (A)¹⁰³Pd), molybdenum (C)⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F)，¹⁵³Sm，¹⁷⁷Lu，¹⁵⁹Gd，¹⁴⁹Pm，¹⁴⁰La，¹⁷⁵Yb，¹⁶⁶Ho，⁹⁰Y，⁴⁷Sc，¹⁸⁶Re，¹⁸⁸Re，¹⁴²Pr，¹⁰⁵Rh，⁹⁷Ru，⁶⁸Ge，⁵⁷Co，⁶⁵Zn，⁸⁵Sr，³²P，¹⁵³Gd，¹⁶⁹Yb，⁵¹Cr，⁵⁴Mn，⁷⁵Se，¹¹³Sn and¹¹⁷tin; positron emitting metals using various positron emission tomography, nonradioactive paramagnetic metal ions, and molecules radiolabeled or conjugated with specific radioisotopes. In such embodiments, suitable detection methods are well known in the art and are readily available from a number of commercial sources.

As indicated above, in other embodiments, the modulator or fragment thereof may be fused to a marker sequence (e.g., a peptide or fluorophore) to facilitate purification or diagnostic procedures (e.g., immunohistochemistry or FAC). In a preferred embodiment, the tag amino acid sequence is a hexa-histidine (SEQ ID NO: 166) peptide, such as the tag provided in the pQE vector (Qiagen), many of which are commercially available. For example, hexahistidine (SEQ ID NO: 166) provides convenient purification of the fusion protein as described in Gentz et al, 1989, Proc.Natl.Acad.Sci.USA 86: 821-824. Other peptide tags that may be used for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, 1984, Cell 37:767), and the "flag" tag (U.S. Pat. No. 4,703,004).

c. Therapeutic moieties

As previously mentioned, the modulator, or fragment or derivative thereof, may also be conjugated, bound, fused or otherwise associated with a therapeutic moiety such as an anti-cancer agent, a cytotoxin or cytotoxic agent, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., an α or β emitterExamples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax, maytansinoids (maytansinoids) such as DM-1 and DM-4(Immunogen, Inc.), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs or homologs thereof(Pyrrolobinadiazepine) dimers (PBDs, Spirogen, Ltd.). Furthermore, in one embodiment, the EFNA modulators of the invention may be conjugated to anti-CD 3 binding molecules to recruit cytotoxic T cells and target them to tumor initiating cells (BiTE technology; see, e.g., Fuhrmann, s. et al, Annual Meeting of aac r Abstract No.5625(2010), which is incorporated herein by reference).

Other compatible therapeutic moieties include cytotoxic agents, including, but not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisplatin (DDP), amikacin (e.g., daunorubicin (previously referred to as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (previously referred to as actinomycin), bleomycin, mithramycin and Anthranilamycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). A more exhaustive list of therapeutic agents can be found in PCT publication WO 03/075957 and U.S. patent No. 2009/0155255, each of which is incorporated herein by reference.

The selected modulator may also be conjugated to the following therapeutic moiety: such as radioactive materials or macrocyclic chelators that may be used to conjugate radioactive metal ions (see the examples of radioactive materials above). In certain embodiments, the macrocyclic chelator is1, 4,7, 10-tetraazacyclododecane-N, N', N "-tetraacetic acid (DOTA), which can be attached to an antibody through a linker molecule. Such linker molecules are generally known in the art and are described in Denardo et al, 1998, ClinCancer res.4: 2483; peterson et al, 1999, bioconjugate. chem.10: 553; and Zimmerman et al, 1999, Nucl. Med. biol.26: 943.

Exemplary radioisotopes compatible with this aspect of the invention include, but are not limited to, iodine (I), (II), (III), (IV¹³¹I，¹²⁵I，¹²³I，¹²¹I), carbon (C: (¹⁴C) Copper (C)⁶²Cu，⁶⁴Cu，⁶⁷Cu, sulfur (S) ((S))³⁵S), tritium (³H) Indium (I)¹¹⁵In，¹¹³In，¹¹²In，¹¹¹In), bismuth (²¹²Bi，²¹³Bi), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga，⁶⁷Ga), palladium (A)¹⁰³Pd), molybdenum (C)⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F)，¹⁵³Sm，¹⁷⁷Lu，¹⁵⁹Gd，¹⁴⁹Pm，¹⁴⁰La，¹⁷⁵Yb，¹⁶⁶Ho，⁹⁰Y，⁴⁷Sc，¹⁸⁶Re，¹⁸⁸Re，¹⁴²Pr，¹⁰⁵Rh，⁹⁷Ru，⁶⁸Ge，⁵⁷Co，⁶⁵Zn，⁸⁵Sr，³²P，¹⁵³Gd，¹⁶⁹Yb，⁵¹Cr，⁵⁴Mn，⁷⁵Se，¹¹³Sn，¹¹⁷Tin，²²⁵Ac，⁷⁶Br and²¹¹at. Other radionuclides may also be useful as diagnostic and therapeutic agents, particularly those in the energy range of 60 to 4,000 keV. Depending on the condition to be treated and the desired therapeutic properties, one skilled in the art can readily select an appropriate radioisotope for use with the disclosed modulator.

The EFNA modulators of the invention may also be conjugated to a therapeutic moiety or drug that modifies a given biological response (e.g., a biological response modulator or BRM). That is, therapeutic agents or moieties compatible with the present invention should not be construed as limited to classical chemotherapeutic agents. For example, in a particularly preferred embodiment, the drug moiety may be a protein or polypeptide or fragment thereof having the desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin a, Onconase (or another cytotoxic rnase), pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, interferon-alpha, interferon-beta, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis agents, such as TNF-alpha, TNF-beta, AIM I (see International publication No. WO 97/33899), AIM II (see International publication No. WO 97/34911), Fas ligand (Takahashi et al, 1994, J.Immunol., 6:1567) and VEGI (see International publication No. WO 99/23105), thrombotic agents or anti-angiogenic agents, such as angiostatin or endostatin; or biological response modifiers, such as lymphokines (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), and granulocyte colony stimulating factor ("G-CSF")), or growth factors (e.g., growth hormone ("GH")). As indicated above, methods for fusing or conjugating modulators to polypeptide moieties are known in the art. In addition to the previously disclosed references, see, e.g., U.S. Pat. nos. 5,336,603; 5,622,929, respectively; 5,359,046, respectively; 5,349,053; 5,447,851 and 5,112,946; EP 307,434; EP 367,166; PCT publications WO96/04388 and WO 91/06570; ashkenazi et al, 1991, PNAS USA 88: 10535; ZHEN et al, 1995, J Immunol 154: 5590; and Vil et al, 1992, PNAS USA 89:11337, each of which is incorporated herein by reference. Binding of a modulator to a moiety need not necessarily be direct, but may occur through a linker sequence. Such linker molecules are generally known in the art and are described in Denardo et al, 1998, ClinCancer Res 4: 2483; peterson et al, 1999, bioconjugateg Chem 10: 553; zimmerman et al, 1999, Nucl Med Biol 26: 943; garnett, 2002, Adv Drug Deliv Rev 53:171, each of which is incorporated herein.

More generally, techniques for conjugating therapeutic moieties or cytotoxic agents to modulators are well known. The moiety may be conjugated to the modulator by any method recognized in the art, including but not limited to aldehyde/Schiff linkage, sulfhydryl linkage, acid labile linkage, cis aconitate linkage, hydrazone linkage, enzymatically degradable linkage (see generally Garnett, 2002, Adv Drug Deliv Rev 53: 171). See also, e.g., Amon et al, "Monoclonal Antibodies against immunological targeting Of Drugs In Cancer Therapy, In Monoclonal Antibodies and Cancer Therapy, Reisfeld et al (eds.), pp.243-56(Alan R.Liss, Inc.1985); hellstrom et al, "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al (eds.), pp.623-53(Marcel Dekker, Inc.1987); thorpe, "antibodies Of Cytoxic Agents In Cancer Therapy: a Review ", in monoclonal antibodies' 84: biologic and Clinical Applications, Pinchera et al (eds.), pp.475-506 (1985); "Analysis, Results, And d Future productive Of The Therapeutic Use Of radioactive amplification In Cancer Therapy," In Monoclonal Antibodies For Cancer Therapy And Therapy, Baldwin et al (eds.), pp.303-16(Academic Press 1985), And Thorpe et al, 1982, Immunol.Rev.62: 119. In preferred embodiments, EFNA modulators conjugated to a therapeutic moiety or cytotoxic agent may be internalized by a cell upon binding of an EFNA molecule bound to the cell surface, thereby delivering a therapeutic payload.

XII diagnosis and screening

a. Diagnosis of

As indicated, the present invention provides in vitro or in vivo methods for detecting, diagnosing or monitoring hyperproliferative disorders, as well as methods for screening cells from patients to identify tumorigenic cells, including TPC. Such methods comprise identifying an individual having cancer for the purpose of treating or monitoring the progression of cancer, comprising contacting the patient or a sample obtained from the patient with a selected EFNA modulator described herein, and detecting the presence or level of association of the modulator with bound or free ephrin-a ligand in the sample. When the modulator comprises an antibody or immunologically active fragment thereof, binding to a particular EFNA in a sample is likely to indicate that the sample may contain tumor perpetuating cells (e.g., cancer stem cells), indicating that the individual with cancer may be effectively treated by the EFNA modulators described herein. The method may further comprise the step of comparing the level in combination with a control. Conversely, where the modulator of choice is Fc-EFNA, the binding characteristics of the selected ephrin-a ligand when contacted with the sample can be utilized and monitored (directly or indirectly, in vivo or in vitro) to provide the desired information. Other diagnostic or theranostic methods compatible with the teachings herein are well known in the art and can be implemented using commercial materials (e.g., dedicated reporting systems).

In particularly preferred embodiments, modulators of the invention may be used to detect and quantify EFNA levels in patient samples (e.g., plasma or blood), which in turn may be used to detect, diagnose or monitor EFNA-associated disorders, including hyperproliferative disorders. One such embodiment is shown in example 17 below, which provides for the detection of EFNA in a plasma sample.

Exemplary compatible assay methods include radioimmunoassays, enzyme immunoassays, competitive binding assays, fluorescent immunoassays, immunoblot assays, western blot analysis, flow cytometry assays, and ELISA assays. More generally, detection of EFNA in a biological sample or measurement of EFNA enzymatic activity (or inhibition thereof) can be accomplished by using any assay known in the art. Compatible in vivo therapeutic diagnosis or diagnosis may include imaging or monitoring techniques recognized in the art such as Magnetic Resonance Imaging (MRI), computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like. Based on the etiology, pathological manifestation, or clinical progression of the condition, one of skill in the art will be readily able to identify and implement appropriate detection, monitoring, or imaging techniques (typically including commercially available sources).

In another embodiment, the invention provides methods for analyzing cancer progression and/or pathogenesis in vivo. In another embodiment, the analysis of cancer progression and/or pathogenesis in vivo comprises determining the extent of tumor progression. In another embodiment, the analysis comprises identification of a tumor. In another embodiment, the analysis of tumor progression is performed on a primary tumor. In another embodiment, the analysis is performed over time, depending on the type of cancer as known to those skilled in the art. In another embodiment, the secondary tumor derived from metastatic cells of the primary tumor is further analyzed in an in vivo assay. In another embodiment, the size and shape of the secondary tumor is analyzed. In some embodiments, additional ex vivo analyses are performed.

In another embodiment, the invention provides a method of analyzing cancer progression and/or pathogenesis in vivo comprising determining cell metastasis. In another embodiment, the analysis of cell metastasis includes determining the progressive growth of cells at sites that are not continuous with the primary tumor. In another embodiment, the site of the cell metastasis analysis comprises a pathway of tumor dissemination. In some embodiments, the cells may be disseminated through blood vessels, lymphatic vessels, within a body cavity, or a combination thereof. In another embodiment, the cell transfer assay is performed in view of cell migration, spreading, extravasation, proliferation, or a combination thereof.

In certain examples, tumorigenic cells in a subject or in a sample from a subject can be assessed or characterized using the disclosed modulators to establish a baseline prior to treatment or protocol. In other examples, the sample is derived from a treated subject. In some examples, the sample is taken from the subject at least about 1, 2,4, 6,7, 8, 10, 12, 14, 15, 16, 18, 20, 30, 60, 90 days, 6 months, 9 months, 12 months, or >12 months after the subject initiates or terminates treatment. In certain examples, the tumorigenic cells are evaluated or characterized after a number of doses (e.g., 2, 5, 10, 20, 30 or more therapeutic doses). In other examples, the tumorigenic cells are characterized or assessed after 1 week, 2 weeks, 1 month, 2 months, 1 year, 2 years, 3 years, 4 years after receiving one or more therapies.

In another aspect, the invention provides a kit for detecting, monitoring or diagnosing a hyperproliferative disorder, identifying an individual having such a disorder for possible treatment of said disorder or monitoring of the progression (or regression) of said disorder in a patient, wherein said kit comprises a modulator as described herein and reagents for detecting the effect of said modulator on a sample, as discussed in more detail below.

b. Screening

EFNA modulators, and cells, cultures, populations, and compositions comprising the same (including progeny thereof) may also be used to screen for or identify compounds or agents (e.g., drugs) that affect the function or activity of tumor initiating cells or progeny thereof by interacting with an ephrin-a ligand (e.g., a polypeptide or genetic component thereof). Thus, the present invention also provides systems and methods for evaluating or identifying compounds or agents that can affect the function or activity of a tumor initiating cell or progeny thereof by binding to EFNA or a substrate thereof. Such compounds and agents may be, for example, drug candidates screened for the treatment of hyperproliferative disorders. In one embodiment, a system or method includes a tumor initiating cell exhibiting EFNA and a compound or agent (e.g., a drug), wherein the cell and the compound or agent (e.g., a drug) are in contact with each other.

The invention also provides methods for screening and identifying EFNA modulators or agents and compounds for altering the activity or function of tumor initiating cells or progeny cells. In one embodiment, the method comprises contacting a tumor initiating cell or progeny thereof with a test agent or compound; and determining whether the test agent or compound modulates the activity or function of a tumor initiating cell that binds an ephrin-a ligand.

A test agent or compound in a population that modulates EFNA-associated activity or function of such tumor initiating cells or progeny thereof identifies the test agent or compound as an active agent. Exemplary activities or functions that may be modulated include alterations in cell morphology, expression of markers, differentiation or dedifferentiation, maturation, proliferation, survival, apoptosis or cell death of the neural progenitor cell or progeny thereof.

When used in reference to a cell or cell culture or method step or treatment, contacting refers to a direct or indirect interaction between a composition (e.g., an ephrin-a ligand-binding cell or cell culture) and another recited entity. A specific example of a direct interaction is a physical interaction. A specific example of an indirect interaction is where the composition acts on an intermediate molecule which in turn acts on the referenced entity (e.g. a cell or cell culture).

In this aspect of the invention, modulation means affecting the activity or function of a tumor initiating cell or progeny cell in a manner compatible with: the effect on cellular activity or function that has been determined to be associated with a particular aspect (e.g., metastasis or proliferation) of the tumor initiating cell or progeny cell of the invention is detected. Exemplary activities and functions include, but are not limited to, measuring morphology, developmental markers, differentiation, proliferation, survival, cellular respiration, mitochondrial activity, membrane integrity, or expression of markers associated with certain conditions. Thus, by contacting a cell or progeny cell with a compound or agent and measuring any modulation of the activity or function of the tumor initiating cell or progeny cell as disclosed herein or as would be known to one of skill in the art, the effect of the compound or agent (e.g., drug candidate) on the tumor initiating cell or progeny cell can be assessed.

Methods of screening and identifying agents and compounds include those suitable for high throughput screening, including cell arrays (e.g., microarrays) that are positioned or placed, optionally at predetermined locations or addresses. High throughput automated or manual processing methods can detect chemical interactions and determine the expression levels of many genes in a short time. The following techniques were developed: it utilizes molecular signals (e.g., fluorophores) and automated analysis to process information at very rapid rates (see, e.g., Pinhasov et al, comb. chem. high through screen.7:133 (2004)). For example, microarray technology is widely used to simultaneously probe the interaction of thousands of genes, while providing information about specific genes (see, e.g., Mocellin and Rossi, adv.exp.med.biol.593:19 (2007)).

Such screening methods (e.g., high throughput) can rapidly and efficiently identify active agents and compounds. For example, the cells may be positioned or placed (pre-seeded) in a culture dish, tube, shake flask, spinner flask or plate (e.g. a single multi-well plate or dish, e.g. 8, 16, 32, 64, 96, 384 and 1536 multi-well plates or dishes) (optionally in defined locations) for the identification of potential therapeutic molecules. Libraries that can be screened include, for example, small molecule libraries, phage display libraries, fully human antibody yeast display libraries (Adimab, LLC), siRNA libraries, and adenoviral transfection vectors.

Medicament preparation and therapeutic use

a. Formulations and routes of administration

Depending on the form of the modulator and any optional conjugates, the mode of delivery desired, the disease to be treated or monitored, and numerous other variables, the compositions of the invention can be formulated as desired using art-recognized techniques. That is, in various embodiments of The present invention, compositions comprising an EFNA modulator are formulated with various pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts andCoomporisons: drugs Plus, 20th ed. (2003); ansel et al, Pharmaceutical Dosageforms and Drug Delivery Systems, 7^thed., Lippencott Williams and Wilkins (2004); kibbe et al, Handbook of Pharmaceutical Excipients, 3^rded., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers (which include vehicles, adjuvants, and diluents) are readily available from a number of commercial sources. In addition, various pharmaceutically acceptable auxiliary substances are also available, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like. Certain non-limiting exemplary carriers include saline solution, buffered saline solution, dextrose, water, glycerol, ethanol, and combinations thereof.

More specifically, it will be understood that in some embodiments, the therapeutic compositions of the present invention may be administered neat or with a minimum of other components. Conversely, the EFNA modulators of the present invention may optionally be formulated with a suitable pharmaceutically acceptable carrier, which includes the following excipients and adjuvants: they are well known in the art and are relatively inert substances that facilitate the administration of the modulator or aid in the processing of the active compound into preparations that are pharmaceutically optimized for delivery to the site of action. For example, excipients may impart a form or consistency or act as diluents to improve the pharmacokinetics of the modulator. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for altering permeability, encapsulating agents, buffers and skin permeation enhancers.

The disclosed modulators for systemic administration can be formulated for enteral, parenteral, or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Excipients and formulations for parenteral and non-parenteral drug delivery are shown in the following: remington, The Science and Practice of pharmacy 20th ed. Mack Publishing (2000). Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, e.g. as water-soluble salts. Additionally, suspensions of the active compounds may be administered as appropriate for oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into cells.

Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants, and controlled release forms thereof.

In general, the compounds and compositions of the present invention comprising EFNA modulators may be administered in vivo to a subject in need thereof by a variety of routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or otherwise by implantation or inhalation. The compositions of the present invention may be formulated as a preparation in solid, semi-solid, liquid or gaseous form; this includes, but is not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected depending on the intended application and treatment regimen.

b. Dosage form

Similarly, the specific dosage regimen (i.e., dose, time and repetition) will depend on the particular individual and the medical history of that individual. Empirical considerations, such as pharmacokinetics (e.g., half-life, clearance rate, etc.), will contribute to the determination of dosage. The frequency of administration can be determined and adjusted over the course of treatment and is based on reducing the number of hyperproliferative or neoplastic cells, including tumor initiating cells, maintaining a reduction in such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastases. Alternatively, sustained continuous release formulations of the subject therapeutic compositions may be suitable. As noted above, various formulations and devices for achieving sustained release are known in the art.

From a therapeutic standpoint, the pharmaceutical composition is administered in an amount effective for treating or preventing the particular indication. The therapeutically effective amount will generally depend upon the weight of the subject being treated, his or her physical or health condition, the severity of the condition being treated, or the age of the subject being treated. Generally, the EFNA modulators of the present invention may be administered in an amount ranging from about 10 μ g/kg body weight to about 100mg/kg body weight/dose. In certain embodiments, the EFNA modulators of the present invention may be administered in an amount ranging from about 50 μ g/kg body weight to about 5mg/kg body weight/dose. In certain other embodiments, the EFNA modulators of the present invention may be administered in an amount ranging from about 100 μ g/kg body weight to about 10mg/kg body weight/dose. Optionally, the EFNA modulators of the present invention may be administered in an amount ranging from about 100 μ g/kg body weight to about 20mg/kg body weight/dose. Also optionally, the EFNA modulators of the present invention may be administered in an amount ranging from about 0.5mg/kg body weight to about 20mg/kg body weight/dose. In certain embodiments, compounds of the invention are provided that are administered at a dose of at least about 100 μ g/kg body weight, at least about 250 μ g/kg body weight, at least about 750 μ g/kg body weight, at least about 3mg/kg body weight, at least about 5mg/kg body weight, at least about 10mg/kg body weight.

Other dosage regimens may be predicted based on Body Surface Area (BSA) calculations, as disclosed in U.S. patent No. 7,744,877, which is incorporated herein by reference in its entirety. As is well known in the art, BSA is calculated using the height and weight of the patient and provides a measure of the size of the patient represented by the surface area of his or her body. In selected embodiments of the invention utilizing BSA, 10mg/m may be used²To 800mg/m²Administering the modulator. In other preferred embodiments, it will be at 50mg/m²To 500mg/m²Even more preferably at 100mg/m²，150mg/m²，200mg/m²，250mg/m²，300mg/m²，350mg/m²，400mg/m²Or 450mg/m²Administering the modulator.Of course, it will be understood that regardless of how the dose is calculated, multiple doses may be administered over a selected period of time to provide an absolute dose that is significantly higher than a single administration.

In any case, it is preferred to administer the EFNA modulator to a subject in need thereof as needed. The frequency of administration can be determined by one of skill in the art (e.g., an attending physician) based on considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like. Typically, an effective dose of the EFNA modulator is administered to the subject one or more times. More specifically, an effective dose of the modulator is administered to the subject once a month, more than once a month, or less than once a month. In certain embodiments, an effective dose of the EFNA modulator may be administered multiple times, including a period of at least one month, at least six months, or at least one year.

Dosages and schedules for the disclosed therapeutic compositions can also be determined empirically in individuals who have been administered one or more administrations. For example, an incremental dose of a therapeutic composition produced as described herein can be administered to an individual. To assess the efficacy of the selected composition, markers of a particular disease, disorder, or condition can be tracked as previously described. In embodiments where the individual has cancer, these include measuring the size of the tumor directly by palpation or visual observation, measuring the tumor size indirectly by X-ray or other imaging techniques; improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples; measuring a reduction in indirect tumor markers (e.g., PSA for prostate cancer) or antigens identified according to the methods described herein, pain or paralysis; improved language, vision, breathing or other disabilities associated with tumors; increased appetite; or an increase in quality of life, as measured by accepted tests or prolongation of survival. It will be apparent to those skilled in the art that the dosage will vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other locations in the individual, and the past and present treatments used.

c. Combination therapy

The combination therapies contemplated by the present invention may be particularly useful in: reducing or inhibiting unwanted proliferation of neoplastic cells (e.g., endothelial cells), reducing the appearance of cancer, reducing or preventing recurrence of cancer, or reducing or preventing dissemination or metastasis of cancer. In this case, the compounds of the present invention may act as sensitizers or chemosensitizers by removing TPC (e.g., NTG cells) that support and perpetuate the tumor mass and allow for more effective use of current standard of care tumor-reducing or anti-cancer agents. That is, combination therapy comprising an EFNA modulator and one or more anti-cancer agents may be used to reduce established cancer, for example, to reduce the number of cancer cells present and/or to reduce tumor burden, or to ameliorate at least one manifestation or side effect of cancer. Thus, combination therapy refers to the administration of EFNA modulators in combination with one or more anti-cancer agents, including but not limited to cytotoxic agents, cytostatic agents, chemotherapeutic agents, targeted anti-cancer agents, biological response modifiers, immunotherapeutic agents, cancer vaccines, anti-angiogenic agents, cytokines, hormonal therapy, radiation therapy, and anti-metastatic agents.

When each treatment (e.g. anti-EFNA antibody and anti-cancer agent) is performed separately, the combined result is not required to be an addition of the observed effects according to the methods of the invention. While it is generally desirable to have an additive effect, any increased anti-tumor effect over one of the monotherapies would be beneficial. Furthermore, the present invention does not require that the combined treatments show a synergistic effect. However, one skilled in the art will appreciate that synergy may be observed with certain selected combinations comprising the preferred embodiments.

To practice a combination therapy according to the present invention, EFNA modulators (e.g., anti-EFNA antibodies) may be administered to a subject in need thereof in combination with one or more anti-cancer agents in a manner effective to produce anti-cancer activity in the subject. The EFNA modulators and anti-cancer agents are provided as needed in an effective amount and for an effective period of time to cause their combined presence in the tumor environment and their combined action. To achieve this goal, the EFNA modulator and the anti-cancer agent may be administered to the subject simultaneously, either in a single composition or as two or more distinct compositions using the same or different routes of administration.

Alternatively, the modulator may be before or after the anti-cancer agent treatment, by intervals of, for example, minutes to weeks. In certain embodiments, wherein the anti-cancer agent and the antibody are separately administered to the subject, the time period between each delivery time is such that the anti-cancer agent and modulator are capable of exerting a combined effect on the tumor. In particular embodiments, the anti-cancer agent and the EFNA modulator are expected to be administered within about 5 minutes to about two weeks of each other.

In other embodiments, several days (2, 3, 4, 5, 6, or 7), several weeks (1, 2, 3, 4, 5, 6, 7, or 8), or several months (1, 2, 3, 4, 5, 6, 7, or 8) may elapse between administration of the modulator and the anti-cancer agent. EFNA modulators and one or more anti-cancer agents (combination therapy) may be administered once, twice or at least for a period of time until the condition is treated, alleviated or cured. Preferably, the combination therapy is administered multiple times. The combination therapy may be administered from three times per day to once every six months. The administration can be according to the following schedule: for example, three times daily, twice daily, once every two days, once every three days, once weekly, once every two weeks, once monthly, once every two months, once every three months, once every six months, or may be administered continuously by a mini-pump. As indicated previously, the combination therapy may be administered by oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral, intratumoral or topical routes. The combination therapy may be administered at a site remote from the tumor site. The combination therapy will generally be administered as long as a tumor is present, provided that the combination therapy causes the tumor or cancer to stop growing or to decrease in weight or volume.

In one embodiment, EFNA modulators are administered in combination with one or more anti-cancer agents for a short treatment period to a subject in need thereof. The duration of treatment with the antibody may vary depending on the particular anti-cancer agent used. The invention also contemplates discontinuous administration or daily doses divided into several partial administrations. One skilled in the art will appreciate the appropriate treatment times for a particular anti-cancer agent, and the present invention contemplates the continued evaluation of an optimal treatment plan for each anti-cancer agent.

The present invention contemplates administration of the combination therapy for at least one cycle, preferably more than one cycle. One skilled in the art will appreciate the appropriate time period for one cycle, as well as the total number of cycles and the intervals between cycles. The present invention contemplates the continuous evaluation of the optimal treatment plan for each modulator and anti-cancer agent. In addition, the present invention also provides more than one administration of the anti-EFNA antibody or the anticancer agent. The modulator and the anti-cancer agent may be administered alternately every other day or every other week; alternatively, a series of antibody treatments may be administered followed by one or more anti-cancer treatments. In any event, as will be understood by those skilled in the art, appropriate dosages of chemotherapeutic agents will generally encompass those already employed in clinical therapy, where chemotherapy is administered alone or in combination with other chemotherapy.

In another preferred embodiment, the EFNA modulators of the invention may be used in maintenance therapy to reduce or eliminate the chance of tumor recurrence following the initial presentation of the disease. Preferably, the condition will have been treated and the initial tumor mass eliminated, reduced or otherwise improved so that the patient is asymptomatic or in remission. In such a period, a pharmaceutically effective amount of the disclosed modulators can be administered to a subject one or more times, even if there is little or no indication of disease using standard diagnostic procedures. In some embodiments, the effector will be administered on a regular schedule over a period of time. For example, the EFNA modulator may be administered weekly, biweekly, monthly, hexaweekly, bimonthly, every three months, every six months, or annually. In view of the teachings herein, one of skill in the art can readily determine advantageous dosages and dosage regimens to reduce the potential for disease recurrence. Furthermore, such treatments may last for periods of weeks, months, years, or even indefinitely, depending on the patient's response and clinical and diagnostic parameters.

In another preferred embodiment, the effectors of the present invention may be used prophylactically to prevent or reduce the likelihood of tumor metastasis following a tumor reduction procedure. As used in this disclosure, a tumor reduction procedure is broadly defined and shall refer to any procedure, technique, or method that eliminates, reduces, treats, or ameliorates a tumor or tumor proliferation. Exemplary tumor reduction procedures include, but are not limited to, surgery, radiation therapy (i.e., beam radiation), chemotherapy, or resection. At an appropriate time, as readily determined by one of skill in the art in view of this disclosure, the EFNA modulator may be administered as suggested by clinical and diagnostic or theranostic procedures to reduce tumor metastasis. The modulator may be administered in one or more doses in a pharmaceutically effective amount (as determined using standard techniques). Preferably, the dosage regimen will be accompanied by appropriate diagnostic or monitoring techniques which allow it to be modified as necessary.

d. Anti-cancer agents

As used herein, the term anti-cancer agent refers to any agent that can be used to treat a cell proliferative disorder, such as cancer, including cytotoxic agents, cytostatic agents, anti-angiogenic agents, tumor reducing agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, biological response modifying agents, antibodies and immunotherapeutic agents. It will be appreciated that in selected embodiments as discussed above, the anti-cancer agent may comprise a conjugate, which may be combined with the modulator prior to administration.

The term cytotoxic agent refers to a substance that reduces or inhibits the function of a cell and/or causes destruction of a cell, i.e., the substance is toxic to a cell. Typically, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or bacterial enzymatically active toxins (e.g., diphtheria toxin, pseudomonas endotoxin and exotoxin, staphylococcal enterotoxin a), fungi (e.g., α -sarcin, restrictocin), plants (e.g., abrin, ricin, madecasin, quercitrin, pokeweed antiviral protein, saponin, gelonin, momoridin, trichosanthin, barley toxin, aleuritol, dianilin, american pokeweed protein (PAPI, PAPII and PAP-S), momordica charantia inhibitors, croton toxic protein, saponaria officinalis inhibitors, gelonin, mitegellin, restrictocin, phenomycin, enomycin and trichothecene) or animals, such as cytotoxic rnases, e.g., extracellular pancreatic rnases; dnase I, including fragments and/or variants thereof.

Chemotherapeutic agents refer to compounds that nonspecifically reduce or inhibit the growth, proliferation and/or survival of cancer cells (e.g., cytotoxic or cytostatic agents). Such chemical agents are typically involved in intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancer cells (which generally grow and divide rapidly). For example, vincristine depolymerizes microtubules and thus inhibits cells from entering mitosis. In general, a chemotherapeutic agent may include any chemical agent that inhibits, or is designed to inhibit, a cancerous cell or a cell that is likely to become cancerous or produce tumorigenic progeny (e.g., TIC). Such agent agents are typically administered in combination and are typically most effective when combined, for example in the formulation CHOP.

Examples of anti-cancer agents that may be used in combination with (or conjugated to) the modulators of the present invention include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimine and methylmelamine, polyacetyl, camptothecin, bryostatin, calalystatin, CC-1065, cryptophycin, urodolin, duocarmycin, orizanin, pancratistatin, sarcodictyin, halichondrin, nitrogen mustards, antibiotics, enediynes antibiotics, daptomycin, diphosphate, esperamycin, chromene diyne chromophore, aclacinomycin, actinomycin, auroramycin, azaserine, bleomycin, cactinomycin, carabicin, carmycin, carvacycin, carvacomycin, chromamycin, dactinomycin, daunomycin, 6-diazo-5-oxo-L-norleucine, or a pharmaceutically acceptable salt thereof,doxorubicin, epirubicin, esorubicin, idarubicin, sisomicin, mitomycin, mycophenolic acid, nogomycin, eleomycin, pelomycin, potfiromycin, puromycin, quelemycin, rodorubicin, streptonigrin, streptozotocin, tubercidin, ubenimex, setastatin, zorubicin; antimetabolites, folic acid analogs, purine analogs, androgens, anti-adrenals, folic acid supplements such as frolinicacid, acetoglucuronolactone, aldphosphoramide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestraucil, bison, edatraxate, defofamine, dimecorsin, mitoquinone, elfornithine, etiloamine, epothilone, etogluteny, gallium nitrate, hydroxyurea, lentinan, lonadinine, ligninoalkaloids, mitoguazone, mitoxantrone, mopidanmol, nitrerine, pentostatin, methionine, pirarubicin, losoxantrone, podophyllic acid, 2-ethyl hydrazide, procarbazine,polysaccharide complex (JHS Natural Products, Eugene, OR), ralozogene; rhizomycin; a texaphyrin; a germanium spiroamine; alternarionic acid; 2,2' -trichlorotriethylamine; trichothecene toxins (especially T-2 toxin, veracurin A, fisetin A and anguidine); urethane; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulcitol; pipobroman; a polycytidysine; arabinoside ("Ara-C"); cyclophosphamide, thiotepa; taxanes, chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16), ifosfamide, mitoxantrone, vincristine;vinorelbine;norxialine, teniposide, edatrexate, nordstycin, aminopterin, ceroda, ibandronate, irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS 2000, Diflurometlylornitine (DMFO), retinoids, capecitabine, compritin, Leucovorin (LV), oxaliplatin, inhibitors of cell proliferation-reducing PKC- α, Raf, H-Ras, EGFR, and VEGF-A, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing, also included in this definition are anti-hormonal agents that modulate or inhibit hormonal effects on tumors, such as anti-estrogens and Selective Estrogen Receptor Modulators (SERM), aromatase inhibitors that inhibit enzyme aromatase, which modulate the production of adrenal estrogens, and anti-androgens, and troxacitabine (1, 3-dioxolane nucleoside cytosine analogs), antisense oligonucleotides, ribosomal enzyme inhibitors such as expression inhibitors and HER2 vaccines,rIL-2；a topoisomerase 1 inhibitor;rmRH; vinorelbine and epothilones (Esperamicins), and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Other embodiments include the use of antibodies that have been approved for cancer therapy including, but not limited to, rituximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, tositumomab, bevacizumab, cetuximab, patitumumab, ofatumumab, ipilimumab, and brentuximab vedotin. One skilled in the art will be readily able to identify other anti-cancer agents that are compatible with the teachings herein.

e. Radiotherapy

The present invention also provides for the combination of EFNA modulators with radiation therapy (i.e., any mechanism that induces DNA damage locally within tumor cells, such as gamma radiation, X-rays, UV radiation, microwaves, electron emission, etc.). Combination therapies using targeted delivery of radioisotopes to tumor cells are also contemplated and may be used in conjunction with targeted anti-cancer agents or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 to about 2 weeks. The radiation therapy can be administered to a subject having a head and neck cancer for about 6 to 7 weeks. Optionally, the radiation therapy may be administered as a single dose or as multiple, sequential doses.

f. Neoplastic conditions

The EFNA modulators of the invention, whether administered alone or in combination with anti-cancer agents or radiotherapy, are particularly useful for treating neoplastic conditions in patients or subjects in general, which may include benign or malignant tumors (e.g., kidney, liver, kidney, bladder, breast, stomach, ovary, colorectal, prostate, pancreas, lung, thyroid, liver cancer; sarcomas; glioblastomas; and various head and neck tumors); leukemia and lymphoid malignancies; other disorders, such as neurological, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, interstitial and blastocoel disorders; as well as infectious, angiogenic, immunological disorders and disorders caused by pathogens. Particularly preferred targets for treatment with the therapeutic compositions and methods of the invention are neoplastic conditions including solid tumors. In other preferred embodiments, modulators of the invention may be used in the diagnosis, prevention or treatment of hematological malignancies. Preferably, the subject or patient to be treated will be a human, although as used herein, the term is expressly considered to include any mammalian species.

More specifically, the neoplastic condition undergoing treatment according to the present invention may be selected from the group including, but not limited to: tumors of the adrenal gland, AIDS-related cancers, alveolar soft tissue sarcoma, astrocytic tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), bone cancer (enamel tumor, aneurysmal bone cyst, osteochondrosis, osteosarcoma), brain and spinal column cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrosarcoma of the skin, profibrogenic small round cell tumor, ependymoma, Ewing's tumor, extraosseous mucus-like chondrosarcoma, bone fibrogenesis deficiency, bone fibroblastic dysplasia, gallbladder and biliary tract cancer, gestational trophoblastic cell disease, germ cell tumor, head and neck cancer, islet cell tumor, Kaposi's sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemia, lipoma/benign adipose tumor, liposarcoma/malignant adipose tumor, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphoma, lung cancer (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.), medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterious uniform melanoma, rare blood disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymus tumor, thyroid metastasis, uterine cancer (cervical cancer, endometrial cancer, uterine fibroids). In certain preferred embodiments, the cancerous cell is selected from the group consisting of a solid tumor, including but not limited to breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, colon cancer, prostate cancer, sarcoma, renal metastatic cancer, thyroid metastatic cancer, and clear cell carcinoma.

With respect to hematologic malignancies, it will also be understood that the compounds and methods of the present invention may be particularly effective in treating a variety of B cell lymphomas, including low grade/NHL follicular cell lymphoma (FCC), Mantle Cell Lymphoma (MCL), Diffuse Large Cell Lymphoma (DLCL), Small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high immunoblastic NHL, high lymphoblastic NHL, high small non-dividing cell NHL, large tumor disease NHL, Waldenstrom macroglobulinemia, lymphoplasmacytoid lymphoma (LPL), Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Diffuse Large Cell Lymphoma (DLCL), Burkitt Lymphoma (BL), AIDS related lymphoma, monocytic B cell lymphoma, angioimmunoblastic lymph node enlargement, small lymphocytes, follicular, diffuse large cells, diffuse small-dividing cells, large-cell immunoblastic lymphomas, small, non-dividing, burkitt and non-burkitt, follicular, mainly large cells; follicular, mainly small dividing cells; and follicular, mixed small and large cell lymphomas. See Gaidono et al, "Lymphomas", IN cancel: PRINCIPLES & PRACTIICE OF ONCOLOGY, Vol.2: 2131- "2145 (Devita et al, eds., 5.sup. th ed. 1997). It will be clear to the skilled person that these lymphomas will generally have different names due to changes in the classification system, and patients with lymphomas classified according to different names may also benefit from the combination treatment regimen of the present invention.

In other preferred embodiments, the EFNA modulators may be used to effectively treat certain bone marrow and hematologic malignancies, including leukemias, such as chronic lymphocytic leukemia (CLL or B-CLL). CLL is a disease mainly in the elderly, the incidence of which starts to increase after the age of fifty and peaks at nearly seventy years. Which typically involves proliferation of neoplastic peripheral blood lymphocytes. Clinical findings of CLL include lymphocytosis, lymphadenopathy, splenomegaly, anemia, and thrombocytopenia. CLL is characterized by the proliferation of monoclonal B cells and the accumulation of B-lymphocytes arrested in an intermediate state of differentiation, wherein said B cells express surface igm (smim) or both smim and sIgD, and a single light chain at a lower density than on normal B cells. However, as discussed above and shown in the accompanying examples, selected EFNA expression (e.g., EFNA) is upregulated on B-CLL cells to provide attractive targets for the disclosed modulators.

The invention also provides prophylactic or preventative treatment of a subject exhibiting a benign or precancerous tumor. It is not believed that any particular type of tumor or neoplastic disorder should be excluded from treatment using the present invention. However, the type of tumor cell may be relevant for the combined use of the present invention with a second therapeutic agent (particularly a chemotherapeutic agent and a targeted anti-cancer agent).

Other preferred embodiments of the invention include the use of EFNA modulators to treat subjects having solid tumors. In these subjects, many of these solid tumors comprise tissues that exhibit various genetic mutations that can make the tissues particularly susceptible to treatment with the disclosed effectors. For example, KRAS, APC and CTNNB1 and CDH1 mutations are relatively common in patients with colorectal cancer. Furthermore, patients with tumors with these mutations are often the most recalcitrant to current therapies; particularly those with KRAS mutations. KRAS activating mutations (which often result in single amino acid substitutions) are also implicated in other refractory malignancies, including lung adenocarcinoma, mucinous adenocarcinoma, and ductal carcinoma of the pancreas.

Currently, the most reliable prediction of whether a colorectal cancer patient will respond to EGFR or VEGF inhibitory drugs is, for example, testing for certain KRAS "activating" mutations. KRAS is mutated in 35-45% of colorectal cancers, and patients whose tumors express mutated KRAS do not respond well to these drugs. For example, KRAS mutations are predictive of a lack of response to panitumumab and cetuximab therapy in colorectal Cancer (Lievre et al Cancer Res66: 3992-5; Karapetis et al NEJM 359: 1757-. Approximately 85% of patients with colorectal cancer have mutations in the APC gene (Markowitz & Bertagnolli. NEJM 361:2449-60) and over 800 APC mutations are characterized in patients with familial adenomatous polyposis and colorectal cancer. Most of these mutations result in truncated APC proteins with reduced ability to mediate the disruption of β -catenin function. Mutations in the β -catenin gene CTNNB1 can also cause increased stability of the protein, which leads to nuclear import and subsequent activation of several oncogenic transcription programs, a mechanism of oncogenesis that is caused by the inability of mutated APCs to properly mediate β -catenin destruction, which is required to maintain normal cellular proliferation and differentiation programs under control.

Loss of CDH1 (E-cadherin) expression is another common event in colorectal cancer, which is often observed at more advanced stages of the disease. E-cadherin is the central member of the adhesive link to cells in the epithelial layer of a tissue. Generally, E-cadherin physically chelates beta catenin at the plasma membrane (CTNNB 1); loss of E-cadherin expression in colorectal cancer leads to nuclear localization of beta-catenin and transcriptional activation of the beta-catenin/WNT pathway. Aberrant β -catenin/WNT signaling is a central carcinogen and nuclear β -catenin is implicated in the sternness of cancer (stemness) (Schmalhofer et al, 2009 PMID 19153669). E-cadherin (Dodge Zantek et al, 1999PMID 10511313; Orsulic S and KemlerrR, 2000PMID 10769210) is required for expression and function of EphA2 (a known binding partner for EFNA ligands in epithelial cells). The oncogenic process can be modified, interrupted or reverted back to using modulators that bind to EFNA ligands and agonize or antagonize Eph receptor binding. Alternatively, EFNA modulators may preferentially bind to tumor cells with aberrant Eph/ephrin interactions based on their binding preference. Thus, patients with cancers that carry the above-described genetic traits may benefit from treatment with the aforementioned EFNA modulators.

XIV. product

Pharmaceutical packages and kits are provided that include one or more containers containing one or more doses of an EFNA modulator. In certain embodiments, unit doses are provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an anti-EFNA antibody, with or without one or more additional agents. For other embodiments, such unit doses are supplied in a single use prefilled syringe for injection. In other embodiments, the composition contained in the unit dose may contain saline, sucrose, or the like; buffers such as phosphates and the like; and/or formulated at a stable and effective pH range. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder, which may be reconstituted upon addition of a suitable liquid (e.g., sterile water). In certain preferred embodiments, the compositions comprise one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the contained composition is used to diagnose or treat the selected condition.

The invention also provides kits for producing single or multiple dose administration units of an EFNA modulator and, optionally, one or more anti-cancer agents. The kit comprises a container and a label or instructions on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container carries a composition effective to treat the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits will generally contain the pharmaceutically acceptable formulation of the EFNA modulator in a suitable container, and optionally one or more anti-cancer agents in the same or different container. The kit may also contain other pharmaceutically acceptable formulations for use in diagnosis or combination therapy. For example, the kit may contain, in addition to the EFNA modulators of the invention, any one or more of a range of anti-cancer agents (e.g., chemotherapeutic or radiotherapeutic drugs); an anti-angiogenic agent; an anti-transfer agent; a targeted anti-cancer agent; a cytotoxic agent; and/or other anti-cancer agents. Such kits may also provide suitable reagents for conjugating the EFNA modulator to an anti-cancer agent or a diagnostic agent (see, e.g., U.S. patent No. 7,422,739, which is incorporated herein by reference in its entirety).

More specifically, the kit may have a single container containing the EFNA modulator with or without other components, or the kit may have different containers for each desired reagent. When a combination therapy is provided for conjugation, the individual solutions may be pre-mixed, either in molar equivalent combinations or in one component over the other. Alternatively, the EFNA modulator and any optional anti-cancer agent of the kit may be stored separately in separate containers prior to administration to a patient. The kit may also comprise a second/third container means for containing sterile, pharmaceutically acceptable buffers or other diluents, such as bacteriostatic water for injection (BWFI), Phosphate Buffered Saline (PBS), Ringer's solution and dextrose solution.

When the components of the kit are provided in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, with sterile aqueous solutions being particularly preferred. However, the components of the kit may be provided as a dry powder. When the reagents or components are provided as a dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container.

As briefly indicated above, the kit may also contain means by which the antibody and any optional components are administered to the animal or patient, such as one or more needles or syringes, or even an eye dropper, pipette or other such device, from which the formulation may be injected or introduced into the animal or applied to the affected body area. Kits of the invention will also typically include means for containing the vials and the like, as well as other components, in a closed, confined space for commercial sale, such as injection or blow molded plastic containers in which the desired vials and other devices are placed and retained. Any label or instructions indicates that the EFNA modulator composition is for use in treating cancer, e.g., colorectal cancer.

XV. research reagent

Other preferred embodiments of the invention also utilize the properties of the disclosed modulators as a means that can be used to identify, isolate, grade, or enrich a population or subpopulation of tumor initiating cells by, for example, Fluorescence Activated Cell Sorting (FACS), Magnetic Activated Cell Sorting (MACS), or laser-mediated grading. One skilled in the art will appreciate that the modulators can be used in several compatible techniques for characterizing and manipulating TIC, including cancer stem cells (see, e.g., each of U.S. serial nos. 12/686,359, 12/669,136, and 12/757,649, which are hereby incorporated by reference in their entireties).

XVI. miscellaneous

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "cells" includes mixtures of cells and the like. Furthermore, the ranges provided in this specification and the appended claims include the endpoints and all points between the endpoints. Thus, the range of 2.0 to 3.0 includes 2.0, 3.0 and all points between 2.0 and 3.0.

Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the specification. See, e.g., Sambrook J. & Russell d. molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); ausubel et al, Short Protocols in Molecular Biology: a Complex of Methods from Current protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); harlow and Lane Using Antibody: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al, Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry laboratory procedures and techniques described herein is those well known and commonly employed in the art.

All references or documents disclosed or cited in this specification are herein incorporated by reference in their entirety without limitation. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Examples

Accordingly, the invention generally described hereinabove will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention. The examples are not intended to represent that the following experiments are all and only experiments performed. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

Example 1

Enrichment of tumor initiating cell populations

To characterize the cellular heterogeneity of solid tumors (as they exist in cancer patients), elucidation of the identity of tumor perpetuating cells (TPC; i.e., cancer stem cells: CSCs) using specific phenotypic markers, and identification of clinically relevant therapeutic targets, large non-traditional xenograft (NTX) tumor pools were developed and maintained using art-recognized techniques. The NTX tumor bank (containing many discrete tumor cell lines) is propagated in immunocompromised mice through multiple passages of heterogeneous tumor cells initially obtained from numerous cancer patients afflicted with various malignant solid tumors. The continuous availability of a number of discrete early passage NTX tumor cell lines with defined cell lineagesIdentification and isolation of TPC are greatly facilitated as they allow reproducible and reproducible characterization of cells purified from the cell line. More specifically, the isolated or purified TPC are most accurately defined retrospectively by their ability to produce phenotypically and morphologically heterogeneous tumors in mice that replicate patient tumor samples from which the cells were derived. Thus, the ability to use small isolated cell populations to generate fully heterogeneous tumors in mice strongly suggests the fact that the isolated cells contain TPC. In this work, the use of minimally passaged NTX cell lines greatly simplified the in vivo experiments and provided easily verifiable results. In addition, early passage of NTX tumors is also responsive to therapeutic agents such as irinotecan (i.e.) This provides clinically relevant insight into the underlying mechanisms that drive tumor growth, resistance to current therapies, and tumor recurrence.

When NTX tumor cell lines are established, the constituent tumor cell phenotypes are analyzed using flow cytometry to identify discrete markers that can be used to characterize, isolate, purify, or enrich for Tumor Initiating Cells (TICs) and to isolate or analyze TPC and TProg cells in such populations. In this regard, the inventors have adopted a proprietary platform based on proteomics (i.e., PhenoPrint)^TMArrays) that provide rapid cellular characterization based on protein expression and concomitant identification of potentially useful markers. The phonoprint array is a proprietary proteomics platform that contains hundreds of discrete binding molecules, many of which are commercially available, arranged in a 96-well plate, where each well contains a discriminating antibody in a phycoerythrin fluorescence channel and a plurality of other antibodies in different fluorescent dyes arranged in each well on the plate. This allows the determination of the expression level of the antigen of interest in a selected subpopulation of tumor cells by rapidly including relevant cells or eliminating irrelevant cells via non-phycoerythrin channels. When the PhenoPrint array is used in combination with tissue dissociation, transplantation and stem cell techniques well known in the art (Al-Hajj et Al,2004, Dalerba et al, 2007 and Dylla et al, 2008, supra, each of which is incorporated herein by reference in its entirety), it is possible to efficiently identify relevant markers and subsequently isolate and transplant specific subpopulations of human tumor cells with great efficiency.

Thus, after establishment of various NTX tumor cell lines in severely immunocompromised mice (as is commonly done for human tumors), up to 800-³The tumors were then excised from the mice and the cells dissociated into single cell suspensions using art-recognized enzymatic digestion techniques (see, e.g., U.S. patent No. 2007/0292414, incorporated herein). The data obtained from these suspensions using the PhenoPrint array provided absolute (per cell) and relative (relative to other cells in the population) surface protein expression on a cell-by-cell basis, leading to more complex characterization and stratification of cell populations. More specifically, the use of PhenoPrint arrays allows for the rapid identification of proteins or markers that would prospectively distinguish TIC or TPC from NTG bulk tumor cells and tumor stroma, and which, when isolated from NTX tumor models, provide relatively rapid characterization of tumor cell subpopulations expressing different levels of specific cell surface proteins. In particular, proteins that are heterogeneously expressed in a tumor cell population allow for the isolation and transplantation into immunocompromised mice of distinct and highly purified tumor cell subpopulations expressing high and low levels of specific proteins or markers, facilitating the assessment of whether TPC is enriched in one subpopulation or another.

The term enrichment is used synonymously with isolating cells and refers to the fraction of one type of cell that increases in yield (in part) over the other cell type as compared to the starting or initial population of cells. Preferably, enrichment refers to an increase in the percentage of one type of cell in the cell population as compared to the starting cell population of about 10%, about 20%, about 30%, about 40%, about 50% or greater than 50%.

As used herein, in the context of a cell or tissue, a marker refers to any characteristic of a form of a chemical or biological entity that can be differentially associated or specifically bound in or on a particular cell, group of cells, or tissue, including those identified in or on a tissue or group of cells affected by a disease or disorder. As will be apparent, the labels may be morphological, functional or biochemical in nature. In preferred embodiments, the marker is a cell surface antigen that is differentially or preferentially expressed by a particular cell type (e.g., TPC) or by cells under certain conditions (e.g., in particular points in the cell life cycle or in cells of a particular niche). Preferably, such a label is a protein, and more preferably, it has an epitope for an antibody, aptamer or other binding molecule known in the art. However, the marker may be composed of any molecule found on the cell surface or within the cell, including but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological marker features or characteristics include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional marker characteristics or properties include, but are not limited to, the ability to attach to a particular substrate, the ability to incorporate or exclude a particular dye (such as, but not limited to, excluding lipophilic dyes), the ability to migrate under particular conditions, and the ability to differentiate along a particular lineage. The marker may also be a protein expressed by a reporter gene, e.g., a reporter gene expressed by a cell, resulting from the introduction into the cell of a nucleic acid sequence encoding the reporter gene and its transcription that results in the production of a reporter protein that can be used as a marker. The reporter group that can be used as a marker is exemplified by, but not limited to, fluorescent protease, staphylokinin, resistance gene, and the like.

In a related sense, the term marker phenotype (e.g., a stable TPC phenotype) in the context of a tissue, cell, or cell population refers to any marker or combination of markers that can be used to characterize, identify, separate, isolate, or enrich for a particular cell or cell population (e.g., by FACS). In particular embodiments, the marker phenotype is a cell surface phenotype that can be determined by detecting or identifying expression of a combination of cell surface markers.

One skilled in the art will recognize that numerous markers (or the absence thereof) have been associated with various populations of cancer stem cells and used to isolate or characterize subpopulations of tumor cells. In this regard, exemplary cancer stem cell markers include OCT4, Nanog, STAT3, EPCAM, CD24, CD34, NB84, TrkA, GD2, CD133, CD20, CD56, CD29, B7H3, CD46, transferrin receptor, JAM3, carboxypeptidase M, ADAM9, oncostatin M, Lgr5, Lgr6, CD324, CD325, nestin, Sox 6, Bmi-1, 6, easyh 6, easyh 6, mf 6, yai 6, arca 6, smscharka 6, srcd 6, arccd 6, smard 6, smarcce 6, mllt 6, FZD6, fczd 6, phct 72, phct 6, phct 72, phct 6, phct 72, ph, GPR54, TGFBR3, SEMA4B, PCDHB2, ABCG2, CD166, AFP, BMP-4, β -catenin, CD2, CD3, CD9, CD14, CD31, CD38, CD44, CD45, CD74, CD90, CXCR4, decorin, EGFR, CD105, CD64, CD16, CD16a, CD16b, GLI1, GLI2, CD49b and CD49 f. See, e.g., Schulenburg et al, 2010, PMID: 20185329, U.S. patent No. 7,632,678 and U.S. patent nos. 2007/0292414, 2008/0175870, 2010/0275280, 2010/0162416, and 2011/0020221, each of which is incorporated herein by reference. It will be appreciated that many of these labels are included in the phonoprint arrays described above.

Similarly, non-limiting examples of cell surface phenotypes associated with cancer stem cells of a certain tumor type include CD44^hiCD24^low，ALDH⁺，CD133⁺，CD123⁺，CD34⁺CD38^-，CD44⁺CD24^-，CD46^hiCD324⁺CD66c^-，CD133⁺CD34⁺CD10^-CD19^-，CD138^-CD34^-CD19⁺，CD133⁺RC2⁺，CD44⁺α₂β₁ ^hiCD133⁺，CD44⁺CD24⁺ESA⁺，CD271⁺，ABCB5⁺And other cancer stem cell surface phenotypes known in the art. See, e.g., Schulenburg et al, 2010, supra, Visvader et al, 2008, PMID: 18784658 and U.S. patent No. 2008/0138313, each of which is incorporated herein by reference in its entirety. One skilled in the art will appreciate that marker phenotypes, such as those listed above, can be used in conjunction with standard flow cytometric analysis and cell sorting techniques to characterize, isolate, purify, or enrich TIC and/or TPC cells or cell populations for further analysis. Of interest with respect to the present invention, CD46, CD324, and optionally CD66c are highly or heterogeneously expressed on the surface of many human colorectal ("CR"), breast ("BR"), non-small cell lung (NSCLC), Small Cell Lung (SCLC), pancreas ("PA"), melanoma ("Mel"), ovarian ("OV"), and head and neck cancer ("HN") tumor cells, whether the tumor sample being analyzed is a primary patient tumor sample or a patient-derived NTX tumor.

A cell with negative expression (i.e. "-") is defined herein as a cell that: expression in the presence of a complete antibody staining mixture labeled for other proteins of interest in other fluorescence emission channels was less than or equal to 95 percent of the expression observed with isotype control antibody in the fluorescence channel. One skilled in the art will appreciate that this procedure for defining negative events is referred to as "fluorescence minus one" or "FMO" staining. Using the FMO staining procedure described above, cells expressing greater than 95 percent of the expression observed with the isotype control antibody are referred to herein as "positive" (i.e., "+"). As defined herein, there are various cell populations that are broadly defined as "positive". First, cells with low expression (i.e., "lo") are generally defined as the following cells: the expression observed for it was greater than 95 percent of the expression determined with isotype control antibody using FMO staining and within one standard deviation of 95 percent of the expression observed with isotype control antibody using the FMO staining procedure described above. Cells with "high" expression (i.e., "hi") can be defined as the following: the expression observed for it was more than 95 percent of the expression determined with isotype control antibody using FMO staining and more than one standard deviation above 95 percent of the expression observed with isotype control antibody using the FMO staining procedure described above. In other embodiments, it may be preferable to use 99 percent as the point of determination between negative and positive FMO staining and in particularly preferred embodiments, the percentage may be greater than 99%.

Using techniques such as those described above, colorectal tumor antigens are rapidly identified and ranked based on expression intensity and heterogeneity among several NTX tumors from colorectal cancer patients, and candidate TPC antigens are further evaluated by comparing the tumors to normal adjacent tissues and then selecting based at least in part on the up-or down-regulation of specific antigens in malignant cells. Furthermore, the systematic analysis of various cell surface markers for their ability to increase the capacity to transplant fully heterogeneous tumors (i.e., tumorigenic capacity) into mice and the subsequent combination of these markers significantly improves the resolution of the method and improves the ability to tailor Fluorescence Activated Cell Sorting (FACS) techniques to identify and characterize distinct, highly enriched tumor cell subsets that contain exclusively all tumorigenic capacity (i.e., tumor initiating cells) after transplantation. To reiterate, the terms Tumor Initiating Cell (TIC) or Tumorigenic (TG) cell encompass tumor perpetuating cells (TPC; i.e. cancer stem cells) and highly proliferative tumor progenitor cells (TProg), which together typically constitute a distinct subset (i.e. 0.1-25%) of a large tumor or mass; the characteristics of which are defined above. Most tumor cells characterized in this manner lack this tumor-forming ability and can therefore be characterized as non-tumorigenic (NTG). Surprisingly, it was observed that most of the differential markers identified using proprietary phonoprint arrays do not show the ability to enrich the tumor initiating cell population in colorectal tumors (when standard FACS protocols are used), while differential marker combinations can be used to identify two subpopulations of tumor initiating cells: TPC and TProg. One skilled in the art will recognize that the decisive difference between TPC and TProg (although both are tumor-initiating in primary transplants) is the ability of TPC to permanently provoke tumor growth after a series of transplants at low cell numbers. Furthermore, while others have defined cell surface markers or enzymatic activities that can similarly be used to enrich for tumorigenic cells (Dylla et al 2008, supra), it was not known prior to the present inventors' findings that the markers/proteins used in combination to enrich for TPC and TProg were associated with cells containing such activities in any tissue or tumor. As shown below, the specific tumor cell subpopulations isolated using the cell surface marker combinations described above were then analyzed using whole transcriptome next generation sequencing to identify and characterize the differentially expressed genes.

Example 2

Isolation and analysis of RNA samples from enriched tumor initiating cell populations

Several established colorectal NTX cell lines (SCRX-CR4, CR11, CR33, PA3, PA 6) generated and passaged as described in example 1&PA14) was used to induce tumors in immunocompromised mice. For mice bearing SCRX-CR4, PA3 or PA6 tumors, once the mean tumor load reached-300 mm³Mice were randomized and treated twice weekly with 15mg/kg irinotecan, 25mg/kg gemcitabine or vehicle control (PBS) for at least twenty days prior to euthanasia. Tumors arising from all six NTX lines (including those from mice undergoing chemotherapy) were removed and TPC, TProg and NTG cells were isolated from freshly resected colorectal NTX tumors and similarly TG and NTG cells were isolated from pancreatic NTX tumors, typically using the techniques shown in example 1. More specifically, by FACS sortingThe cell population was isolated, immediately pelleted and lysed in Qiagen RLTplus RNA lysis buffer (Qiagen, Inc.). The lysate was then stored at-80 ℃ until use. After thawing, total RNA was extracted using Qiagen RNeasy isolation kit (Qiagen, Inc.) according to the supplier's instructions and quantified again using the supplier's protocol and recommended equipment settings on nanodrop (thermo scientific) and Bioanalyzer 2100(Agilent Technologies). The total RNA preparations produced were suitable for genetic sequencing and analysis.

Total RNA samples obtained from each cell population isolated from vehicle or chemotherapeutic treated mice as described above were prepared for whole transcriptome sequencing using an applied biosystems SOLiD 3.0 (sequencing by oligonucleotide ligation/detection) next generation sequencing platform (life technologies) starting from 5ng total RNA/sample. The data generated by the SOLiD platform mapped to 34,609 genes from the human genome and was able to detect ephrin-a ligands (including EFNA4) in most samples and provided a verifiable measure of ENFA levels.

Generally, the SOLiD3 next generation sequencing platform enables parallel sequencing of clonally amplified RNA/DNA fragments attached to beads. Sequencing by ligation with dye-labeled oligonucleotides was then used to generate 50 base reads for each fragment present in the sample, for a total of greater than 5 million reads, which produced a much more accurate representation of the expression of mRNA transcript levels of proteins in the genome. The SOLiD3 platform is capable of capturing not only expression, but also SNPs, known and unknown alternative splicing events, and potential new exon discovery based on read coverage alone (reads uniquely localized to genomic locations). Thus, the use of this next generation platform allows for the determination of differences in expression at the transcript level as well as differences or preferences for particular splice variants in those expressed mRNA transcripts. Furthermore, analysis with the SOLiD3 platform using a modified whole transcriptome protocol from Applied Biosystems required only about 5ng of starting material to be pre-amplified. This is significant because total RNA is then extracted from sorted cell populations where TPC cell subsets are, for example, much smaller in number than NTG or large tumors, and thus produce very small amounts of usable starting material.

Duplicate runs of sequencing data from the SOLiD3 platform were normalized, transformed, and fold ratios calculated as standard industry practice. As seen in fig. 2, EFNA1, EFNA3 and EFNA4 levels and Eph receptors EPHA1, EPHA2 and EPHA10 levels from tumors were measured. Analysis of the data showed that EFNA4 was up-regulated at the transcript level by 1.9-3 fold in SCRx-CR4NTX tumor TPC and 1.2-1.4 fold in TPC relative to TProg population, relative to NTG population, whether the cells were obtained from vehicle treated (figure 2A) or from mice treated with 15mg/kg irinotecan (figure 2B). It will also be appreciated that EFNA1 also rose in TPC, albeit to a lesser extent than EFNA4, relative to TProg and NTG cells respectively. Furthermore, when additional colorectal (SCRx-CR11& CR33) and pancreatic (SCRx-PA3, PA6& PA14) tumor samples were analyzed by SOLiD3 whole transcriptome sequencing, expression of EFNA4 gene was similarly elevated in the TPC (relative to TProg and NTG cells) (fig. 3A) in the colorectal and in the TIC (or TG) cell subsets from pancreatic tumors (fig. 3B), as determined using the unique set of cell surface markers found as shown above (TPC and TProg cell subsets, which constitute the as yet undefined TIC population in pancreatic tumors).

Expression of EPHA2 receptor (to which both EFNA4 and EFNA1 ligands interact) was also observed, which inversely reflects expression of EFNA4 and EFNA 1in the progression of differentiation from TPC to NTG cells. This opposite expression pattern of EFNA1/EFNA4 ligand and EPHA2 receptor suggests that cross-talk (crossbar) between these ligand/receptor pairs may play a role in cell fate determination during colorectal cancer stem cell differentiation, and that neutralization of these responses may negatively impact tumor growth. Specifically, by blocking the interaction of EphA2 with EFNA1 and/or EFNA4 using neutralizing antibodies against the latter ephrin-a ligand pair, TPC can be sensitized to chemotherapeutic agents, or can be forced to differentiate, for example. Furthermore, by targeting TPC using EFNA1 and/or EFNA4 internalizing antibodies, TPC can be killed directly by naked modulators or by using toxins or antibody drug conjugates.

The observations detailed above show that EFNA1 and/or EFNA4 expression is generally elevated in the TPC population and suggest that these membrane-tethered ligands may play an important role in tumorigenesis and tumor maintenance and therefore constitute excellent targets for new therapeutic approaches.

Example 3

Real-time PCR analysis of ephrin-A ligands in enriched tumor initiating cell populations

To verify the differential ephrin-a ligand expression of the TPC population relative to TProg and NTG cells in colorectal cancer, and TG relative to NTG cells in pancreatic cancer, as observed by whole transcriptome sequencing, use was made ofQuantitative real-time PCR was used to measure gene expression levels in each cell population isolated from various NTX lines as shown above. It will be appreciated that such real-time PCR analysis allows for more direct and rapid measurement of gene expression levels of discrete targets using primer and probe sets specific to a particular gene of interest. Carried out on an Applied Biosystems 7900HT apparatus (Life Technologies)Real-time quantitative PCR, which was used to measure EFNA4 gene expression in multiple patient-derived NTX line cell populations and corresponding controls. In addition, the analysis was performed as indicated in the instructions provided with the TaqMan system and using the commercially available EFNA4 primer/probe set (Life Technologies).

As seen in figure 4, quantitative real-time PCR queries for gene expression in NTG, TProg and TPC populations isolated from 3 distinct colorectal NTX tumor lines (SCRx-CR4, CR5& CR14) showed that EFNA4 gene expression was increased by more than 1.4-fold in the TIC subpopulations (TPC and/or TProg) relative to NTG cells. EFNA4 was also elevated approximately 1.8-fold in the TIC population of mice undergoing irinotecan treatment and in the TG cell population of pancreatic tumors (e.g., SCRx-PA 3). Elevated EFNA4 expression (compared to NTG cell controls) was observed in NTX TIC cell preparations from NTX tumors derived from colorectal and pancreatic patients using a more widely accepted real-time quantitative PCR method, confirming the more sensitive SOLiD3 whole transcriptome sequencing data of the previous examples and supporting the observed association of EFNA4 with cells on which tumorigenesis, resistance to treatment, and recurrence are based.

Example 4

Expression of ephrin-a ligand in unfractionated colorectal tumor samples

In view of the fact that increased expression of the ephrin-a ligand gene was found in a TPC population from the same tumor as compared to TProg and NTG cells from colorectal tumors, experiments were performed to determine if increased ephrin-a ligand (i.e., EFNA4) expression could also be detected in unfractionated colorectal tumor samples relative to Normal Adjacent Tissues (NAT). Similarly, measurements were also made to determine how ephrin-a ligand expression in tumors compared to levels in normal tissue samples. A custom tumor scan qpcr (origene technologies) 384-well array containing 110 colorectal patient tumor samples, normal adjacent tissue, and 48 normal tissues was designed and fabricated using techniques known in the art. TaqMan real-time quantitative PCR was performed in the wells of the custom plates using the procedure detailed in example 3 and the same EFNA4 specific primer/probe set.

Fig. 5A and 5B graphically show the results of the expression data normalized to the mean expression in normal colon and rectal tissue. More specifically, fig. 5A summarizes data generated using 168 tissue samples obtained from 110 colorectal cancer patients, 35 of which were Normal (NL) adjacent tissue from colorectal cancer patients, and 48 normal tissues from other locations (other NL). In the graph, data from each tissue sample/patient is represented by dots, with the geometric mean of each population being plotted on the X-axis represented by a line. Similarly, fig. 5B contains data from 24 matched colorectal patient samples obtained from tumors (T) or normal adjacent tissues (N) at various stages of the disease (I-IV). Here, the plotted data is presented on a sample-by-sample basis with a link between the corresponding tumor from a single patient and normal adjacent tissue. Expression of EFNA4 was significantly higher in most matched tumors relative to normal adjacent tissues, with differential expression reaching statistical significance (n.gtoreq.4, P.ltoreq.0.047) in stages 2, 3 and 4. Figures 5A and 5B both show that the expression level of EFNA4 gene was elevated in most colorectal tumors and in matched tumor samples (relative to normal adjacent tissue) during all four phases presented. Furthermore, mean EFNA4 gene expression in any stage of colorectal cancer appeared to be at least equal to (if not greater than) the highest level of EFNA4 gene expression in any normal tissue queried in these experiments (fig. 5A). These results show that EFNA4 expression is increased in colorectal cancer, when coupled with the above observed expression of EFNA4 being highest in colorectal TPC and pancreatic TIC, suggesting that therapeutic targeting of tumor-initiating cells expressing EFNA4 may provide great therapeutic benefit to cancer patients.

Example 5

Differential expression of ephrin-a ligands in exemplary tumor samples

To further assess the gene expression of ephrin-A ligand in tumor samples from other colorectal cancer patients and tumor samples from patients diagnosed with one of 17 other different solid tumor types, tissue Scan was used^TMqPCR (origin technologies)384 well arrays (custom fabricated as described in example 4) were performedTaqman qRT-PCR. The results of the measurements are presented in fig. 6 and show that gene expression of EFNA4 is significantly elevated or inhibited in many tumor samples.

In this regard, FIGS. 6A and 6B show the relative and absolute gene expression levels of human EFNA4 in a whole tumor sample (gray dot) or matched normal adjacent tissue (NAT; white dot) from a patient with one of eighteen different solid tumor types, respectively. In fig. 6A, the data were normalized to the mean gene expression in NAT for each tumor type analyzed. In fig. 6B, the absolute expression of EFNA4 was evaluated in each tissue/tumor, with the data plotted as the number of cycles (Ct) required to achieve exponential amplification by quantitative real-time PCR. The unamplified samples were assigned a Ct value of 45, which represents the last amplification cycle in the protocol. Each point represents a single tissue sample, with the average represented as a black line.

Using the customized array, it was observed that most patients diagnosed with colorectal cancer and most patients diagnosed with endometrial, esophageal, liver, lung, prostate, bladder and uterine cancers had significantly more EFNA4 gene expression in their tumors (relative to NAT), indicating that EFNA4 may play a role in tumorigenesis and/or tumor progression in these tumors. In contrast, expression of EFNA4 appears to be significantly inhibited in tumors from patients with adrenal and pancreatic cancer. It is also clear from these studies that EFNA4 gene expression is generally low to moderate in most NAT samples; among them, the highest expression was observed in adrenal gland, breast, cervix and ovary. Again, these data indicate that differential EFNA4 expression (high or low) is indicative and potentially crucial with respect to tumorigenesis or perpetuation in patients presenting with selected hyperproliferative disorders.

EFNA4 expression was also assessed and quantified in relation to normal tissue expression as discussed above using proprietary non-traditional xenografts (NTX). Quantitative real-time PCR was performed on commercial normal tissue RNA samples (breast, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, skeletal muscle, small intestine) and NTX tumors from breast cancer (BR), colorectal Cancer (CR), kidney cancer (KDY), liver cancer (LIV), Melanoma (MEL), non-small cell lung cancer (NSCLC), ovarian cancer (OV), pancreatic cancer (PA) and Small Cell Lung Cancer (SCLC). The results (shown in fig. 6C) indicate elevated expression of EFNA4 in breast, colon, and liver NTX lines relative to expression in normal tissue. In contrast, figure 6D recorded the expression of related family member EFNA 1in many of the same normal and NTX lines and showed little differential expression between normal and tumor tissues. Despite this expression profile, EFNA modulators of the invention that react with EFNA1 (including those that react with other EFNAs) can be effectively used to eliminate tumorigenic cells, as shown in the examples that follow.

In any case, western blot analysis was performed in order to confirm that the elevated mRNA expression detected by quantitative real-time PCR also translated to elevated protein levels of EFNA 4. Cell lysates of NTX and cell lines (293 na4 overexpressing cells) were generated using a total protein extraction kit (Bio ChainInstitute # K3011010) following the protocol provided to match commercially available normal tissue lysates (Novus Biologicals). Protein concentrations of the lysates were determined using a BCA protein assay (Pierce/Thermo Fisher # 23225). Equal amounts of cell lysate were run on NuPAGE Novex 4-12% Bis-Tris gels (Life technologies) under reducing conditions in MES buffer. EFNA4 protein expression was detected using a commercially available antibody against human EFNA4A (R & D Systems-AF 369). In the top panel of fig. 6E, 293 cells engineered to overexpress EFNA4 showed high expression compared to naive 293 cells. Furthermore, in the top column, several breast, colon and non-small cell lung cancers NTX showed relatively high expression of EFNA 4. Under similar conditions, the western blot in the bottom column of fig. 6E shows that normal tissues express low or undetectable levels of EFNA4 when compared to high EFNA4 expression in NTX cell line CR 11. anti-GAPDH control antibody was used to show equal cell lysate loading in both columns.

Example 6

Generation of anti-EFNA antibodies using EFNA immunogens

According to the teachings herein, EFNA modulators in the form of murine antibodies were generated by seeding whole cell BALB/c 3T3 cells overexpressing EFNA4 or a cytoplasmic preparation (ECD-extracellular domain) prepared as shown herein with hEFNA4-ECD-Fc, hEFNA4-ECD-His, hEFNA 1-ECD-His. Immunogens were prepared using commercially available starting materials (e.g., recombinant human ephrin-A4 Fc chimera, CF R & D system #369-EA-200) and/or techniques well known to those skilled in the art.

More specifically, murine antibodies were generated by immunizing 9 female mice (Balb/c, three each of CD-1, FVB) with various preparations of EFNA4 or EFNA1 antigens. The immunogen comprises Fc construct or His-tagged human EFNA4 or EFNA1 from 10⁷Membrane fraction extracted from 293 cells overexpressing EFNA4 or whole 3T3 cells overexpressing human EFNA4 on the surface. For all injections, mice were immunized by the footpad route. Using 10. mu.g of EFNA4 or EFNA1 immunogen or 1X10⁶Cells or with equal volumes of TITERMAX^TMOr aluminum-adjuvanted cell equivalents. Following immunization, mice were euthanized, and draining lymph nodes (popliteal and inguinal, if enlarged) were dissected and used as a source of antibody-producing cells. Lymphocytes are released by mechanical disruption of the lymph nodes using a tissue grinder.

One of two fusion schemes is used. In the first, electrofusion was performed with a Genetronic device, followed by plating and screening of polyclonal hybridomas, followed by subcloning to generate monoclonal hybridomas. In the second, electrofusion is performed with a BTX device, followed by culture of hybridoma libraries in large and single cell deposits of hybridomas, followed by screening of clones.

Genetronic device fusion protocol: fusion was performed by mixing single cell suspensions of B cells with non-secreting P3x63Ag8.653 myeloma cells purchased from (ATCC CRL-1580; Kearney et al, J.Immunol.123:1548-1550(1979)) in a ratio of 1: 1. The cell mixture was gently pelleted by centrifugation at 800 g. After complete removal of the supernatant, the cells were treated with 2-4mL of pronase protease solution for no more than 2 minutes. Electrofusion was performed using fusion generator model ECM2001(Genetronic, Inc.).

2X10 on flat-bottomed microtiter plates⁴Cells were plated and then incubated in selective HAT medium (Sigma, CRL P-7185) for two weeks. Individual wells were then screened by ELISA and FACS for anti-human EFNA4 monoclonal IgG antibody.

ELISA microtiter plates were coated with purified recombinant EFNA4His fusion protein (100 ng/well in carbonate buffer) from transfected 293 cells. The plates were incubated overnight at 4 ℃ and then blocked with 200. mu.l/well of 3% BSA in PBS/Tween (0.05%). Supernatants from hybridoma plates were added to each well and incubated at ambient temperature for 1-2 hours. The plates were washed with PBS/Tween and then incubated with goat anti-mouse IgG (Fc fragment specifically conjugated with horseradish peroxidase (HRP)) (Jackson ImmunoResearch) for one hour at room temperature. After washing, the plates were developed with TMB substrate (Thermo Scientific 34028) and analyzed with a spectrophotometer at OD 450.

EFNA4 secreting hybridomas from positive wells were rescreened and subcloned by limiting dilution or single cell FACS sorting.

Subcloning was performed on selected antigen positive wells using limiting dilution plating. The plates were visually observed for the presence of single colony growth and then screened for supernatants from single colony wells by antigen-specific ELISA as described above and FACS demonstration as described below. The resulting clonal populations were expanded and cryopreserved in freezing medium (90% FBS, 10% DMSO) and stored in liquid nitrogen. This fusion from mice immunized with EFNA4 produced 159 murine monoclonal antibodies reactive to EFNA4 using the ELISA protocol described above.

BTX device fusion protocol: single cell suspensions of B cells were fused by electrofusion to nonsecretory p3x63ag8.653 myeloma cells at a 1:1 ratio. Electrofusion was performed using the Hybrimune System model 47-0300(BTXHarvard Apparatus). The fused cells were resuspended in hybridoma selection medium (DMEM (Cellgro cat #15-017-CM) supplemented with azaserine (Sigma # A9666) containing 15% fetal clone I serum (Hyclone), 10% BM conditioned (Roche Applied Sciences), 1mM sodium pyruvate, 4mM L-glutamine, 100IU penicillin-streptomycin, 50. mu.M 2-mercaptoethanol, and 100. mu.M hypoxanthine) and plated in four T225 shake flasks at 90ml selection medium/shake flask. The shake flask was then placed in a 5% CO₂And 95% air in a humidified 37 ℃ incubator for 6-7 days.

At 6-7 days of growth, the library was plated in 48 Falcon 96-well U-plates at 1 cell/well using an Aria I cell sorter. Briefly, media containing 15% fetal clone I serum (Hyclone), 10% BM-conditioned (Roche Applied sciences), 1mM sodium pyruvate, 4mM L-glutamine, 100IU penicillin-streptomycin, 50. mu.M 2-mercaptoethanol, and 100. mu.M hypoxanthine were plated at 200. mu.l/well in 48 Falcon 96-well U-plates. Surviving hybridomas were plated and cultured at 1 cell/well for 10-11 days using an Aria I cell sorter, and supernatants were assayed for antibody reactivity to EFNA4 or EFNA1 by FACS or ELISA.

Growth positive hybridoma wells secreting mouse immunoglobulin were screened for specificity for murine EFNA4 using an ELISA assay similar to that described above. Briefly, 96-well plates (VWR, 610744) were coated overnight at 4 ℃ with 1. mu.g/mL of murine EFNA4-His in sodium carbonate buffer. The plates were washed and blocked with 2% FCS-PBS for 1 hour at 37 deg.C and used immediately or stored at 4 deg.C. Undiluted hybridoma supernatants were incubated on the plates for 1 hour at RT. The plates were washed and probed with HRP-labeled goat anti-mouse IgG (diluted 1:10,000 in 1% BSA-PBS) for one hour at RT. The plates were then incubated with substrate solution as described above and read at OD 450.

Growth positive hybridoma wells secreting mouse immunoglobulin were also screened for specificity for human EFNA1 using FACS assays as described below. Briefly, 1 × 10 was washed with 25-100 μ l hybridoma supernatant⁵Jurkat cells expressing human EFNA 1in well were incubated for 30 min. Cells were washed twice with PBS/2% FCS and incubated with 50. mu.l/sample of DyeLight 649 labeled goat-anti-mouse IgG (Fc fragment specific secondary antibody diluted 1:200 in PBS/2% FCS). After 15 min incubation, cells were washed twice with PBS/2% FCS and resuspended in DAPI-containing PBS/2% FCS and analyzed by FACS Canto II (BD Biosciences) under standard conditions using HTS-ligation. The resulting EFNA 1-specific clonal hybridomas were expanded and cryopreserved in CS-10 cryoculture medium (Biolife Solutions) and stored in liquid nitrogen. This fusion from mice immunized with EFNA1 produced 1 hybridoma reactive with EFNA4 as determined using FACS analysis. In addition, FACS analysis demonstrated that antibodies purified from most or all of these hybridomas bind to EFNA4 or EFNA 1in a concentration-dependent manner.

Example 7

Sequencing and humanization of ephrin-a ligand modulators

7(a) sequencing:

based on the foregoing, a number of exemplary discriminatory monoclonal antibodies were selected that bind immobilized human EFNA4 or EFNA1 with significantly high affinity. As shown in tabular form in fig. 7A, sequence analysis of DNA encoding mabs from example 6 confirmed that many had unique VDJ rearrangements and displayed novel complementarity determining regions. It should be noted that the complementarity determining regions shown in FIG. 7A (SEQ ID NOS: 8-59 and 70-95) were derived from VBASE2 analysis.

For initial sequencing, TRIZOL reagent was purchased from invitrogen (life technologies). One-step RT PCR kit and QIAquick PCR purification kit were purchased from Qiagen, Inc, wherein the rnase inhibitor was from Promega. Custom oligonucleotides were purchased from Integrated DNA Technologies.

Hybridoma cells were lysed in TRIZOL reagent for RNA preparation. Will 10⁴mu.L to 10⁵The cells were resuspended in 1ml TRIZOL. The tube was shaken vigorously after addition of 200. mu.l of chloroform. The samples were centrifuged for 10 minutes at 4 ℃. The aqueous phase was transferred to a fresh microcentrifuge tube and an equal volume of isopropanol was added. The tube was shaken vigorously and allowed to incubate for 10 minutes at room temperature. The samples were then centrifuged for 10 minutes at 4 ℃. The pellet was washed once with 1ml 70% ethanol and dried briefly at room temperature. The RNA pellet was resuspended in 40. mu.L of DEPC-treated water. The quality of the RNA preparation was determined by fractionating 3. mu.L in a 1% agarose gel. The RNA was stored in a freezer at-80 ℃ until use.

Variable DNA sequences of hybridomas amplified with a consensus primer pair specific for murine immunoglobulin heavy and kappa light chains were obtained using a mixture of variable domain primers. A one-step RT-PCR kit was used to amplify VH and VK gene segments from each RNA sample. The Qiagen one-step RT-PCR kit provides a blend of Sensiscript and Omniscript reverse transcriptases, HotStarTaq DNA polymerase, Qiagen one-step RT-PCR buffer, dNTP mixtures and Q solution (a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates).

A reaction mixture was prepared comprising 3. mu.L of RNA, 0.5 of 100. mu.M heavy or kappa light chain primers, 5. mu.L of 5 XTR-PCR buffer, 1. mu.L of dNTPs, 1. mu.L of an enzyme cocktail (containing reverse transcriptase and DNA polymerase) and 0.4. mu.L of the carbonuclease inhibitor RNase (1 unit). The reaction mixture contains all reagents required for reverse transcription and PCR. The thermal cycling procedure was: the RT step was performed at 50 ℃ for 30 minutes and at 95 ℃ for 15 minutes, followed by 30 cycles of: at 95 ℃ for 30 seconds, at 48 ℃ for 30 seconds and at 72 ℃ for 1.0 minute. Then finally incubation at 72 ℃ for 10 min.

To prepare PCR products for direct DNA sequencing, the manufacturer's protocol was followedBy QIAquick^TMThey were purified by PCR purification kit. DNA was eluted from the spin column using 50. mu.L of sterile water and then sequenced directly from both strands. The PCR fragment was sequenced directly and the DNA sequence was analyzed using VBASE2(Retter et al, Nucleic Acid Res.33; 671-674, 2005).

As briefly described above, the genetic arrangement and derived CDRs (from VBASE2 analysis) of several exemplary anti-hENFA 4/hENFA 1 antibodies are shown in tabular form in FIG. 7A (SEQ ID NOS: 8-59 and 70-95). In addition, the nucleic acid and amino acid sequences of these same exemplary antibody heavy and light chain variable regions are shown in FIGS. 7B-7N (SEQ ID NOS: 96-147).

7(b) humanization:

four of the murine antibodies from example 6 were humanized using Complementarity Determining Region (CDR) grafting. Human frameworks for heavy and light chains were selected based on sequence and structural similarity with respect to functional human germline genes. In this regard, structural similarity was assessed by comparing mouse conventional CDR structures with human candidates having the same conventional structure (derived from VBASE2 analysis).

More specifically, murine antibodies SC4.5, SC4.15, SC4.22 and SC4.47 were humanized using computer-assisted CDR grafting methods (Abysis database, UCL Business Plc.) and standard molecular engineering techniques to provide hSC4.5, hSC4.15, hSC4.22 and hSC4.47 modulators (note: the addition of a subsequent number (i.e., SC4.47.3) after the clone or antibody name refers to a particular subclone and is not critical for the purposes of this disclosure unless the context indicates otherwise or requires otherwise). The human framework regions of the variable regions were selected based on their highest sequence homology to the mouse framework sequence and its regular structure. For analytical purposes, the assignment of amino acids to each CDR domain is according to Kabat et al. Several humanized antibody variants were made to produce optimal humanized antibodies that generally retain antigen binding Complementarity Determining Regions (CDRs) from a mouse hybridoma bound to human framework regions. Humanized SC4.15, SC4.22 and sc4.471mabs bound EFNA4 antigen with similar affinity as their murine counterparts, while hsc1.5 bound with slightly lower affinity as measured using the Biacore system.

Molecular engineering procedures were performed using art-recognized techniques. For this purpose, according to the manufacturer's protocol (Plus RNA purification system, Life Technologies) extracted total mRNA from the hybridomas. A primer mixture comprising thirty-two mouse-specific 5 'leader primers (designed to target the entire mouse repertoire) was used in combination with the 3' mouse C γ 1 primer to amplify and sequence the variable region of the antibody heavy chain. Similarly, thirty-two 5' Vk leader primer mixtures (designed to amplify each of the Vk mouse family) in combination with a single reverse primer specific for the mouse kappa constant region were used to amplify and sequence the kappa light chains. Amplification of V from 100ng Total RNA Using reverse transcriptase polymerase chain reaction (RT-PCR)_HAnd V_LA transcript.

A total of eight RT-PCR reactions were performed for each hybridoma: four for the V κ light chain and four for the V γ heavy chain (γ 1). Amplification was performed using the QIAGEN one-step RT-PCR kit (QIAGEN, Inc.). The extracted PCR products were directly sequenced using specific V region primers. Nucleotide sequences were analyzed using IMGT to identify germline V, D and J gene members with the highest sequence homology. Using V-BASE2(Retter et al, supra) and by mixing V_HAnd V_LThe genes were aligned to the mouse germline database and the derived sequences were compared to the known germline DNA sequences of the Ig V and J regions.

From the nucleotide sequence information, data were obtained for the V, D and J gene segments of the heavy and light chains of SC4.5, SC4.15, SC4.22 and SC4.47. Based on the sequence data, Ig V specific for antibodies were designed_HAnd V_KThe new primer set for the leader sequence of the strand is used to clone the recombinant monoclonal antibody. Subsequently, the V- (D) -J sequence was aligned with the mouse Ig germline sequence. The heavy chain genes of SC4.5 were identified as IGHV2-6(V) and JH 3. Analysis of the short CDR3 of the heavy chain of the E5 monoclonal antibody did not identify a specific mouse D gene. The heavy chain gene of SC4.15 was identified as IGHV5-6(V), DSP2.9(D) and JH 3. The heavy chain gene of SC4.22 was identified as VHJ558(V), and the D segment was identified as dfl16.1e and JH4 (J). The heavy chain genes of SC4.47 were identified as IGHV1-26(V), P1inv (D) and JH2 (J). All four light chains are of class K. The light chain genes were identified as IGKV6-15, JK2 (for sc4.5mab), IGKV6-b and JK5 (for sc4.15mab), IGKV1-110 and JK1 germline sequences (for sc4.22mab) and IGKV21-7, JK1 germline sequences (for SC4.47 kappa light chain). These results are summarized in table 1 below.

TABLE 2

Cloning

Mouse isotype

VH

DH

JH

VL

JL

SC4.5

IgG1/K

IGHV2-6

Is free of

JH3

IGKV6-15

JK2

SC4.15

IgG1/K

IGHV5-6

DSP2.9

JH3

IGKV6-b

JK5

SC4.22

IgG2b/K

VHJ558

DFL16.1e

JH4

IGKV1-110

JK1

SC4.47

IgG1/K

IGHV1-26

P1inv

JH2

IGKV21-7

JK1

The heavy and light chain sequences obtained from all four clones were aligned with functional human variable region sequences and evaluated for homology and general structure. The results of the heavy and light chain analyses are shown in tables 3 and 4 below, respectively.

TABLE 3

TABLE 4

Cloning	Human VK	Human JK	Homology to human germline sequence%	Homology to mouse sequence%
					SC4.5	L1	JK2	86	79
SC4.15	A27	JK4	89	76
					SC4.22	A18b	JK1	89	91
SC4.47	L6	JK4	87	84

Since the germline selection and CDR grafting processes appear to provide antibodies that generally retain their binding characteristics, it is apparent that little murine residues need to be inserted in most constructs. However, in hsc4.15, heavy chain residue 68 was back mutated from thr (t) to lys (k) to improve antibody characteristics.

The amino acid sequences (and related nucleic acid sequences) of the humanized heavy chain variable regions and the humanized kappa light chains of all four antibodies are shown in FIGS. 7O-7R (SEQ ID NO: 148-.

More specifically, the nucleic acid sequences and corresponding amino acid sequences of the humanized SC4.5 heavy chain (SEQ ID NOS: 148 and 149) and the humanized light chain (SEQ ID NOS: 150 and 151) are shown in FIG. 7O. Similarly, the nucleic acid sequences and corresponding amino acid sequences of the humanized SC4.15 heavy chain (SEQ ID NOS: 152 and 153) and the humanized light chain (SEQ ID NOS: 154 and 155) are shown in FIG. 7P. Another embodiment of the invention is illustrated in FIG. 7Q, where the nucleic acid sequences and corresponding amino acid sequences of the humanized SC4.22 heavy chain (SEQ ID NOS: 156 and 157) and the humanized light chain (SEQ ID NOS: 158 and 159) are shown. In another embodiment, FIG. 7R shows the nucleic acid sequences and corresponding amino acid sequences of the humanized SC4.47 heavy chain (SEQ ID NOS: 160 and 161) and humanized light chain (SEQ ID NOS: 162 and 163). As shown in the examples below, each of the aforementioned humanized antibodies functions as an effective EFNA modulator according to the teachings herein.

In any event, the disclosed modulators are expressed and isolated using techniques recognized in the art. For this purpose, synthetic humanized variable DNA fragments (Integrated DNA Technologies) of both heavy chains were cloned into the human IgG1 expression vector. The variable light chain fragment was cloned into a human C-kappa expression vector. The antibody was expressed by co-transfection of the heavy and light chains into CHO cells.

More specifically, to generate antibodies, PCR products of murine and humanized variable genes were performedDirectly cloned into a human immunoglobulin expression vector. All primers used in Ig gene specific PCR included restriction sites (ageni and XhoI for IgH, XmaI and DraIII for IgK, this allows direct cloning into expression vectors containing human IgG1 and IGK constant regions, respectively briefly, PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Inc.), followed by digestion with AgeI and XhoI (IgH), XmaI and DraIII (IgK), respectively, the digested PCR products were purified, ligation was then performed with 200U T4-DNA ligase (New England Biolabs), 7.5. mu.L of digested and purified gene-specific PCR product and 25ng of linearized vector DNA in a total volume of 10. mu.L, competent E.coli DH10B bacteria (Life Technologies) were transformed with 3. mu.L of the ligation product by heat shock at 42 ℃ and plated on ampicillin plates (100. mu.g/mL)._HThe AgeI-EcoRI fragment of the region was inserted into the same site of the pEE6.4HuIgG1 expression vector and the synthetic XmaI-DraIII V_KThe insert was cloned into the XmaI-DraIII site of the corresponding pEE12.4Hu-Kappa expression vector.

Humanized antibody producing cells were generated by transfecting HEK293 cells with the appropriate plasmid using 293 fectin. In this regard, plasmid DNA was purified using QIAprep spin columns (Qiagen). Embryonic Kidney (HEK)293T (ATCC No CRL-11268) cells were cultured on 150mm plates (Falcon, Becton Dickinson) in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated FCS, 100. mu.g/mL streptomycin, 100U/mL penicillin G (both from Life Technologies) under standard conditions.

For transient transfection, cells were grown to 80% confluence. Equal amounts of IgH and corresponding IgL chain vector DNA (12.5. mu.g of each vector DNA) were added to 1.5mL of Opti-MEM mixed with 50. mu.L of HEK293 transfection reagent in 1.5mL of Opti-MEM. The mixture was incubated at room temperature for 30min and evenly distributed into the plates. Supernatants were harvested 3 days post-transfection, replaced with 20mL fresh DMEM supplemented with 10% FBS, and harvested again 6 days post-transfection. Cell debris in the culture supernatant was removed by centrifugation at 800 Xg for 10min and stored at 4 ℃. Recombinant chimeric and humanized antibodies were purified using protein G beads (GEHealthcare).

Example 8

Characterization of the EFNA Modulator

8(a) general regulator characteristics

Various methods were used to analyze the binding characteristics of selected ephrin-a 4 modulators produced as described above. In particular, byMany EFNA4 antibodies were characterized for affinity, kinetics, binning, and cross-reactivity with cynomolgus and mouse homologues (generated internally). Western reactivity was also measured and epitopes were determined for both antibodies (SC4.22 and SC4.91) bound under reducing conditions. In addition, the antibodies were tested for their ability to neutralize (i.e., block receptor ligand interactions), internalize, and the relative EC to kill them was determined by in vitro cytotoxicity using the procedures shown in these examples (see, e.g., examples 12 and 16)₅₀Benchmarking is performed. The results of this characterization are shown in tabular form in fig. 8A.

With respect to the data, affinity was measured in three ways to ensure accuracy. First, binding signals were measured in ELISA for a fixed amount of antibody (probed against serial dilutions of antigen) to determine relative modulator activity (data shown for cyno binding only). Second, the affinity and kinetic constant k of the selected modulator was then measured on a ForteBIO RED (ForteBIO, Inc.) using a standard antigen concentration series, using biolayer interferometry analysis_onAnd k_off. Finally, the affinity of the selected regulons was measured by surface plasmon resonance (Biacore System, GE Healthcare). K for antibody binding to antigen was determined based on a standard antigen concentration series and using a 1:1Langmuir binding model_dAnd kinetic constant k_onAnd k_off. In general, the selected adjustmentThe molecules show relatively high affinity in the nanomolar range.

As for antibody binning, ForteBIO was used to identify antibodies that bound the same or different bins (bins) according to the manufacturer's instructions. Briefly, antibody (Ab1) was captured onto an anti-mouse capture chip, which was then blocked with a high concentration of non-bound antibody and a baseline was collected. The monomer, recombinant ephrin-a 4-His was then captured by specific antibody (Ab1) and the head was immersed in wells containing the same antibody (Ab1) as a control or in wells containing different antibody antibodies (Ab 2). If additional binding was observed with the new antibody, Ab1 and Ab2 were determined to be in different bins. If no other binding occurred, similar to control Ab1, Ab2 was determined to be in the same bin. This process can be extended to screen large libraries of unique antibodies in 96-well plates using full rows of antibodies representing unique bins. This experiment shows that the screened antibodies bind to at least three different epitopes on the box or EFNA4 protein.

To determine whether the epitope recognized by the ephrin-a 4 regulon comprises consecutive amino acids or is formed by non-consecutive amino acids juxtaposed by the secondary structure of the antigen, western blot analysis was performed under reducing and non-reducing conditions. More specifically, ephrin-a 4 antigen in both states is exposed to the selected modulator using standard electrophoretic techniques well known in the art. As shown in fig. 8A, most ephrin-a 4 modulators react substantially with antigen only when the disulfide bond is intact (NR), while both modulators react with non-reduced and reduced antigen (NR/R). For these antibodies, pepspot (jpt) membranes were used to determine the restriction of recognition of the antibody by the peptide. SC4.22 and SC4.91 were found to recognize sequences QRFTPFSLGFE (SEQ ID NO: 164) and RLLRGDAVVE (SEQ ID NO: 165), respectively. Retesting the ability of these peptides to bind the peptide of interest by ELISA confirms that the antibodies are indeed specific for these epitopes.

Finally, cross-reactivity with respect to cynomolgus ephrin-a 4 homologues was evaluated in ForteBIO using a concentration series of recombinantly expressed monomeric ephrin-a 4 antigen. As shown in fig. 8A, the selected modulators react with the homologs. In particular, SC4.5, SC4.15, SC4.91 and SC4.105 cross-reacted with mouse ephrin-a 4, while all antibodies cross-reacted with highly similar cynomolgus ephrin-a 4. ND in the table indicates that the data is not determined.

8(b) humanization Modulator characteristics

The humanized constructs hsc4.15, hsc4.22 and hsc4.47 were analyzed to determine their binding characteristics using the techniques shown above in this example. In addition, the humanized antibody binding was directly compared to the parent murine antibody for both antibodies to identify any subtle changes in rate constants brought about by the humanization process.

More specifically, the affinity of murine SC4.47 was determined by Biacor using Surface Plasmon Resonance (SPR) to provide the results shown in fig. 8B. Based on concentration series 25, 12.5 and 6.25nM (resulting in the top to bottom curves in FIGS. 8B and 8C) and using the 1:1Langmuir binding model, K for antibody binding to antigen was estimated_dIt was 1.1 nM. Similar experiments with the humanized constructs then showed equivalent results (fig. 8C), indicating that the humanization process did not adversely affect the affinity. In this regard, measurements indicate that the humanized construct has a K_dIs composed of<1x10^-10This is essentially the same as the parent murine antibody.

Together with the other techniques shown in this example, these measurements show that all of the humanized ephrin-a 4 effectors from example 7 have the desired qualities. As shown in fig. 8D, SC4.15 strongly cross-reacted with the murine ephrin-a 4 homolog, facilitating toxicology studies. The reactivity of all antibodies to cynomolgus antigens by ELISA was indistinguishable from human EFNA and thus expected to be very similar.

Example 9

Epigrin-A ligand modulators display cell surface binding

Supernatants from hybridomas producing antibodies to hEFNA4-Fc as shown above were screened for cell surface binding as measured in a flow cytometry assay. To demonstrate the binding properties of the antibody, two cell lines, JurkatE6 cells and Z138 cells (each of which is known to express high levels of surface ephrin-a 4) were used. More specifically, six million Jurkat E6 cells (stained with the cell labeling dye CFSE (for ease of identification)) and four million unlabeled Z138 cells (incubated with 20 μ g/mL Fc blocking reagent (Trueblock, Biolegend, Inc.) were mixed to a final concentration of 1 million cells/mL. 50 μ L of this cell mixture was added to 50 μ L of antibody-containing supernatant in each well and incubated at 4 ℃ for 60 minutes. The cells were washed once with PBS (wash buffer) containing 2% FBS, 2mM EDTA and 0.05% sodium azide, and then with F (ab) of Fc region-specific goat-anti-mouse IgG polyclonal antibody conjugated with DyLight649 in the dark at 4 ℃₂Fragments (Jackson Immuno Research) were stained for 60 min. Cells were washed twice with wash buffer and counterstained with 2. mu.g/ml DAPI. Negative control samples were mouse IgG1 isotype antibody (10 μ g/ml, Biolegend, Inc.) and supernatant from a hybridoma (H13.2) known not to secrete mouse IgG. A positive control sample was prepared using 10 μ g/ml purified antibody (sc4.76.2aka E76.2) previously identified as specific for EFNA4 by ELISA (left side of figure 9). Samples were collected on a FACS Canto II (BDBiosciences) using HTS ligation under standard conditions. Eighty-four (84) clones out of one hundred and fourteen (114) were judged to show significant cell surface binding (shown by flow cytometry by staining both cell lines) significantly higher than the negative control samples. In this regard, fig. 9 shows the relative binding capacities of fifty exemplary hybridoma supernatants.

Example 10

Selected EFNA4 modulators mediate ephrin-a 4 ligand binding

Supernatants from hybridomas producing antibodies known to bind to cells expressing ENFA4 (example 9) were tested for their ability to block the binding of soluble hENFA 4-Fc to its receptors (EphAs) on the surface of HEK293Td cells. Initially, as seen in fig. 10A, HEK293Td cells were shown to bind hEFNA4-Fc in a dose-dependent manner when compared to the negative control antibody. To show neutralization of this binding, 60 μ L of anti-EFNA 4 hybridoma supernatant was incubated with 200ng/ml hENFA 4-Fc (diluted in wash buffer) for 2 hours at 4 ℃. The mixture was then added to fifty thousand HEK293Td cells and incubated at 4 ℃ for 1 hour. The cells were washed once in wash buffer and then in the dark at 4 ℃ with F (ab) of a goat-anti-mouse IgG polyclonal antibody specific for the Fc region conjugated with DyLight649₂Fragments (Jackson Immuno Research) were stained for 45 min. The cells were then washed twice with wash buffer and counterstained with 2. mu.g/ml DAPI. Negative control samples were unstained cells, cells stained with supernatant from a non-IgG-producing hybridoma (H13.2) and cells stained with human IgG Fc γ 1 fragment. Positive control samples were cells stained with hEFNA4-Fc in the absence of hybridoma supernatant and with hEFNA4-Fc in the presence of non-IgG-producing hybridoma supernatant (left side of fig. 10B). Samples were measured on a FACS Canto II, as discussed previously. As shown in fig. 10B, sixty-two (62) of the eighty-three (83) clones tested showed some ability to neutralize hEFNA4-Fc binding to its cell surface receptors when measured using flow cytometry.

Example 11

EFNA modulators block cell surface EFNA binding in a concentration-dependent manner

To further measure the ability of the ephrin-a ligand modulator of the present invention to neutralize EFNA activity, anti-EFNA 4 antibodies from selected hybridomas were purified and used as sterile reagents in PBS buffer. At first, build in parallelSeparate human and murine EFNA4-Fc (recombinant murine ephrin-A4 Fc chimera, CF R) were established&D system) to show dose-limited binding of EFNA4-Fc to HEK293Td cells (fig. 11A). Once this control was established, serial dilutions of anti-EFNA 4 antibody obtained from three exemplary hybridomas (i.e., SC4.15.3, SC4.47.3, and SC4.76.2) were incubated with limited concentrations (0.1. mu.g/ml and 1.0. mu.g/ml) of hENFA 4-Fc and mEFNA4-Fc in wash buffer, respectively, for 1hr at 4 ℃. The resulting reagent mixture was then transferred to fifty thousand HEK293Td cells and incubated at 4 ℃ for 1 hour. The cells were washed once in wash buffer and then washed with F (ab) of a goat-anti-mouse IgG polyclonal antibody specific for the Fc region conjugated with DyLight649 in the dark at 4 ℃₂Fragments (Jackson Immuno Research) were stained for 45 min. Cells were washed twice with wash buffer and counterstained with 2. mu.g/ml DAPI. Negative control samples were unstained cells and cells stained with human IgG Fc γ 1 fragment. Samples were collected on a FACS Canto II as described previously. Figure 11B shows the activity of mAb SC4.15.3, which partially inhibited the ability of human and mouse EFNA4-Fc to bind cells at relatively high concentrations. Figure 11C illustrates the activity of mAb SC4.47.3, which almost completely blocks the ability of hEFNA4-Fc to bind cells, but does not block the ability of menfa 4-Fc. Similarly, fig. 11D shows the following capabilities of ephrin-a ligand modulator mAb SC4.76.2: it significantly inhibited the ability of hEFNA4-Fc to bind cells but did not significantly affect the ability of mfna 4-Fc to bind cells. These results strongly suggest the ability of selected modulators of the invention to inhibit binding of ephrin-a ligands to cell surface receptors and thus inhibit any activity associated with tumorigenesis.

Example 12

EFNA modulators block binding of EFNA to EphA receptor in a concentration-dependent manner

As discussed above, EphA2 is a known binding partner for EFNA 4. To exploit this known relationship, the extracellular domain of EphA2 was fused to the Fc portion of human IgG using standard techniques, transiently expressed in HEK293Td cells and purified from the supernatant of the culture using protein a affinity chromatography. As seen in figure 12A, EphA2-Fc homodimers bound Jurkat cells (known to express EFNA) in a dose-dependent manner while the Fc portion of human IgG alone did not show any binding. Such binding of EphA2-Fc to Jurkat cells can be inhibited using the ephrin-A modulator of the present invention, particularly by using a monoclonal antibody to ephrin-A4. For this purpose, fifty thousand Jurkat cells/well were incubated with 10. mu.g/ml of the four selected anti-ENFA 4 antibodies (i.e., SC4.22, SC4.31.3, SC4.47.3 and SC4.73, all prepared as described above) in wash buffer for 1hr at 4 ℃. Mouse IgG and no antibody (data not shown) were used as negative controls. After washing, serial dilutions of EphA2-Fc were added to the cells in wash buffer for 1hr at 4 ℃ to provide the results shown graphically in figure 12B. Review of figure 12B shows that modulators sc4.31.3 and SC4.47.3 significantly inhibited EphA2-Fc binding to EFNA4, while modulators SC4.22 and SC4.73 show relatively less inhibition. To further illustrate the ability of the disclosed modulators to inhibit interaction with a receptor, Jurkat cells were first incubated with serial dilutions of the antibody, followed by incubation with 10 μ g/ml EphA 2-Fc. The cells were then washed twice with wash buffer, counterstained with 2 μ g/ml DAPI, and then analyzed on a FACS Canto II (BD Biosciences) using HTS ligation under standard conditions to provide the data shown in fig. 12C. Like fig. 12B, fig. 12C shows that modulator mAb SC4.47.3 is a relatively potent inhibitor and effectively blocks binding of EphA2-Fc to EFNA4 expressed on Jurkat cells. By comparison, the other modulators showed somewhat less activity, with sc4.31.3 providing a moderate amount of inhibition at higher concentrations.

To extend these findings, the interaction between other EFNA4 modulators and EphA receptors was explored. Experiments were performed similarly to the above except HEK293T cells overexpressing EFNA4 (referred to as HEK293t. hefna4 cells) were transduced by retroviruses (fig. 12D) or HEK293T cells overexpressing EFNA1 were transduced by retroviruses (fig. 12E) were used. In addition, the assay was performed at a single EphAx-Fc concentration (10. mu.g/ml). The data show that SC4.2, SC4.31 and SC4.47 are able to block the binding of all EphA receptor binding partners tested to ephrin-a 4 ligands (i.e., EphA2, EphA3, EphA4, EphA6, EphA7, EphA8 and EphA 10). In addition, it was established that EFNA4 modulator SC9.65, which was generated in the vaccination campaign against EFNA1 (as per example 6), has the ability to interfere with the binding of EphA1, EphA2, EphA4 and EphA7 to ephrin-a 1 ligand. These data, when combined with the results of the other examples herein, indicate that this modulator ability to antagonize the binding of various receptors can be significant in providing the observed therapeutic effects of the present invention.

Example 13

Human ephrin-A regulon cross-reacts with mouse ortholog

In view of the fact that the extracellular domains of human and mouse ephrin-a 4 ligands share 80% sequence identity at the protein level, the disclosed modulators of human EFNA4 were tested to see if they bound to mouse homologues. More specifically, an antibody sandwich ELISA was used to determine the level of cross-reactivity of hEFNA 4-specific monoclonal antibodies with their mouse homologues. High protein binding 96 well assay plates were coated with 0.5 μ g/ml of donkey anti-human IgG polyclonal antibody molecule specific for the Fc portion of the IgG molecule. Protein coating of the plates occurred during incubation at 4 ℃ for 16 hours using 50mM sodium carbonate buffer (ph9.6) in 100 μ L volumes per well. Human and mouse EFNA4 molecules fused to the Fc γ 1 portion of human IgG molecules (EFNA4-Fc) were serially diluted in PBS Buffer (PBSA) containing 2% (w/v) bovine serum albumin. After washing the coated plates in PBS Buffer (PBST) containing 0.05% Tween20, 100 μ L/well of mouse or human EFNA4-Fc diluted in PBSA was added to the wells for 3 hours at ambient temperature. The plates were then washed again with PBST and 100 μ L/well of PBSA containing 10% spent hybridoma supernatant or 1 μ g/ml purified monoclonal antibody (as a positive control) was added to the plates for 1 hour at ambient temperature. After washing the plates with PBST, 100 μ L/well of PBSA (containing a 1:5000 dilution of goat anti-mouse IgG polyclonal antibody specific for the Fc portion of mouse IgG and conjugated with horseradish peroxidase (Jackson ImmunoResearch)) was added to the plates for 30 minutes at ambient temperature. After the plates were thoroughly washed with PBST, 100. mu.L/well of TMB substrate (Thermo Fisher) was added to the wells for 15 minutes. The enzymatic reaction was stopped by adding 100. mu.L/well of 2M sulfuric acid. The absorbance of this colorimetric assay was measured at 450nm using a Victor plate reader (Perkin Elmer). Using two replicate assays, data are presented as mean absorbance readings plus standard deviation. Figure 13A shows an exemplary monoclonal antibody sc4.31.3, which recognizes hEFNA4 but not mufna 4. In contrast, fig. 13B shows binding of the exemplary monoclonal antibody sc4.91.4, which recognizes human and mouse EFNA 4.

To confirm these results, an assay was performed using the humanized ephrin-a 4 modulator hsc 4.15. More specifically, titrated amounts of human and mouse ephrin-A4-His were coated in PBS on high protein-binding 96-well plates for 16 hours at 4 ℃. After blocking the plates in PBSA at ambient temperature for 2hr, 0.5. mu.g/ml of hSC4.15 modulator was added to PBSA for 2 hours. The ELISA was developed using donkey anti-human IgG polyclonal antibody conjugated to horseradish peroxidase (Jackson Immuno Research) as described above. Figure 13C shows that the hsc4.15 modulator recognizes human and mouse ephrin-a 4 ligands equally well, indicating that the disclosed humanized modulators are fully compatible with the teachings herein.

Example 14

Expression of ephrin-A ligands in exemplary tumor samples, tumor cell subsets and hematopoietic cells

After recording elevated gene expression levels and generation of antibodies against EFNA4 in the previous examples, evidence was sought for the corresponding expression of EFNA4 protein in selected cell populations. In this regard, a reverse phase oncoprotein lysate array (ProteoScan array; OriGene Technologies) is provided comprising 4 dilutions of 432 tissue lysates from 11 tumor types or their respective normal adjacent tissues, together with a control consisting of HEK293 cells (with no or with TP53 overexpression driven by an exogenous promoter). EFNA4 protein expression in the lysates described on this array was examined by western blotting using a mouse monoclonal EFNA4 antibody recognizing EFNA4 protein (e.g., clone ej47.3aka SC4.47.3) of the invention. Colorimetric detection reagents and protocols are provided by the manufacturer of ProteoScan arrays, and spots on the manufactured arrays are converted into digital images using a flatbed scanner (using BZScan2 Java software (INSERM-TAGC)) to quantify spot intensities.

Selected results of such assays are shown in fig. 14, and show that expression of EFNA4 protein is up-regulated in colorectal tumor samples. More specifically, fig. 14A shows that EFNA4 protein expression appears to be significantly elevated in a subset of colorectal tumor samples; particularly in patients with stage IV disease (when compared to normal adjacent tissue or tumor tissue from samples obtained from earlier disease stages). Data was generated as described above and presented as mean pixel intensity/point (point intensity). The horizontal black bars in each sample represent the average of the samples in each respective category.

After confirming that EFNA4 protein is upregulated in certain colorectal whole tumor cell lysates, tests were performed to establish that the same target is expressed on tumor initiating cells. More specifically, to determine whether EFNA4 protein expression could be detected on the cell surface of tumor initiating cells, tumors were dissociated for flow cytometric analysis as described above. After dissociation of the tumor samples (e.g. colorectal cell line CR33, as according to example 2) into single cell suspensions, they were incubated for 24 hours at 37 ℃ to facilitate antigen re-expression (due to the enzymatic sensitivity of the EFNA4 antigen to collagenase/hyaluronidase) and then stained with Phycoerythrin (PE) conjugated monoclonal antibodies capable of recognizing EFNA 4. Then, as in the previous examples, the HTS connection F was used under standard conditionsACS Canto II (BD Biosciences) analyzed the cells. In performing these experiments, it was observed that EFNA4 was present in a subpopulation of TIC cells (as determined by co-staining said cells with antibodies recognizing TIC-definitive cell surface markers; e.g. 46) compared to expression on NTG cells⁺，324⁺，66^-) The expression above is significantly higher. Representative results from experiments using SCRx-CR33 colorectal NTX tumor cells and EFNA4 modulator SC4.47.3 showed that expression of EFNA4 was more than 2-fold higher on TIC than on NTG cells (fig. 14B).

To further confirm that EFNA4 was relatively highly expressed on TIC cells, LU86 and LU64 cells were cultured in vitro for 10 days and expression was measured by flow cytometry using PE-conjugated SC4.47 antibody as shown herein. The resulting colonies were harvested and stained as described above. As shown in fig. 14D, the TIC population of LU86 cells (solid black line) expressed EFNA4 well above the isotype control (shaded gray) and the NTG population from the same tumor line (dashed black line). Furthermore, LU86 cells cultured in vivo can be killed by EFNA4 antibody (as shown in example 16 below). In contrast, LU64 cells were found not to express elevated levels of EFNA4 (fig. 14D) and were subsequently not killed by anti-EFNA antibodies.

While it is believed that EFNA4 protein expression has not been evaluated in solid tumor samples prior to the present disclosure, it is reported that the protein is expressed at relatively low levels on B cells and elevated on B cells from Chronic Lymphocytic Leukemia (CLL) patients. To confirm the expression of EFNA4 protein on normal Peripheral Blood Mononuclear Cells (PBMCs), assays were performed as previously described in this example to provide the data shown in figure 14C. Examination of the graph presented in FIG. 14C shows that when determining expression of EFNA4 on PBMCs from normal donors, only CD19⁺B cells were weakly positive confirming reports in the literature on where EFNA4 is expressed.

These data support that the overexpression of EFNA4 observed in the above examples is associated with TIC and/or TPC in colorectal cancer and may be involved in proliferation and/or survival. The data also show that EFNA4 is not expressed on most normal PBMCs, and expression on normal B cells is minimal. In view of the foregoing examples it was shown that a) EFNA4 gene expression correlates with a subpopulation of TPC cells in colorectal cancer and a subpopulation of tumor-initiating cells in pancreatic tumors; b) EFNA4 protein expression was higher on TIC cell subpopulations; c) EFNA4 protein expression was elevated in whole tumor samples from advanced colorectal cancer; and d) general observations that TIC is more frequent in advanced tumors, it appears that EFNA4 is associated with those cells that underlie tumor growth, resistance to treatment, and tumor recurrence, suggesting that EFNA4 may play an important role in supporting TPC and/or TIC in the above-mentioned tumors.

Example 15

Epigrin-A ligand modulator is internalized by K562 cells

In view of the expression profile of ephrin-a ligands established in the previous examples, assays were performed to see if the modulators of the invention were internalized upon binding to cell surface antigens. In this regard, supernatants from the hybridomas producing the anti-EFNA 4-Fc antibodies of the examples were screened for their ability to internalize in K562 cells expressing EFNA4 at low levels on the cell surface. Blocking with human TruStain (BioLegend, Inc.) at a starting concentration of 10 at room temperature⁶K562 cells/ml (single cell suspension) for 10min, then diluted to 5X10⁴Cells/well. Duplicate samples were then stained with a final volume of 50. mu.l of anti-EFNA antibody (containing supernatant) on ice for 30 minutes, followed by FACS staining medium (FSM; 2% fetal bovine serum/Hank's buffered saline/25 mM HEPES [ pH7.4 ]]) The cells are washed to remove unbound antibody. This was followed by a second staining with donkey anti-mouse Alexa647(Life Technologies) for 30 minutes on ice. The samples were washed again to remove unbound antibody and then resuspended in internalization medium (2% fetal bovine serum/Iscove's modified Dulbecco's medium). To allow internalization, samples were incubated at 37 deg.C (or 4 deg.C for controls) in 5% CO₂And incubated for 1 hour. By mixingSamples were transferred to ice and excess ice-cold FSM was added to terminate internalization. To remove any antibody that remains on the cell surface without internalization, low pH phosphate buffered saline (PBS [ pH2.0 ]]) The samples were treated on ice for 10 minutes. After this "acid removal (strip)" procedure, the samples were washed extensively with FSM, resuspended in 150 μ l FSM containing 2 μ g/ml DAPI, and analyzed by flow cytometry (again using FACS Canto II (BDBiosciences), using HTS ligation under standard conditions). Any signal detected above background is due to antibody internalization, a process that protects the fluorescent conjugate from being removed from the cell surface during acid removal. All incubations were performed in FSM unless otherwise stated.

Screening 159 hybridoma supernatant clones containing EFNA4 antibody using the acid removal protocol described above showed that many supernatants showed positive shifts in fluorescence compared to the IgG negative control antibody (data not shown). For example, exemplary SC4.5, SC4.22, and SC4.73 clones showed internalization for: supernatants from these clones were able to internalize and protect Alexa647 secondary antibody from acid removal (fig. 15A). Approximately 15% of supernatants containing EFNA4 antibody induced internalization to a different extent compared to IgG controls, of which the first nineteen (19) showed mean fluorescence intensities (MFI at 37 ℃ vs.4 ℃) in excess of 150 (fig. 15B). This data indicates that a subset of regulators produced by anti-human EFNA4 ECD bind antigen and are capable of internalization when presented on a cell. Such results underscore the potential therapeutic value of ephrin-a ligands as targets (with or without cytotoxic payloads) for modulators of the invention.

The assay was repeated using selected purified EFNA4 modulators (at a concentration of 10 μ g/ml) and HEK293T (fig. 15C) and HEK293t.hefna4 (fig. 15D) cells as target cells. The parent HEK293T expressed low levels of ephrin-a 4 ligand on their cell surface. Following the protocol described above, the data show that all tested ephrin-a 4 modulators are internalized upon binding ephrin-a 4 ligand expressed on the cell surface. The Mean Fluorescence Intensity (MFI) recorded for each sample was compared to standard beads containing eight different known amounts of encapsulated fluorophore (becton dickenson spoherech 8 color rainbow beads) (data not shown). This allowed conversion of MFI values into linear values and calculation of relative receptor numbers per cell.

Example 16

EFNA4 modulators as targeting moieties

Targeting of cytotoxic drugs stably linked to antibodies represents an enhanced (empowered) antibody approach that may have great therapeutic benefit for patients with solid tumors. To determine whether EFNA 4-specific antibodies described above were able to mediate the delivery of cytotoxic agents to hepatocytes, in vitro cell killing assays were performed in which streptavidin conjugated to ribosome-inactivating protein saponins (Advanced Targeting Systems) was bound to biotinylated EFNA4 antibodies, and the ability of these saponin complexes to internalize and kill cells was measured after 72 hours by measuring cell viability.

Specifically, 10 is⁵Z138 cells/well were plated in wells of a 96-well plate. The anti-EFNA 4 antibody described above was purified from the supernatant, biotinylated, and then diluted to 20 μ g/mL. The Z138 cell line (ATCC CRL-3001) was derived from a patient with mantle cell lymphoma and expressed moderate amounts of EFNA 4. Aliquots of each antibody were mixed with streptavidin-zap (advanced Targeting systems)1:1, vortexed for 5 seconds, and then incubated at room temperature for 1 hour. Two additional serial 10-fold dilutions of the antibody-saponin complex were then prepared and 50 μ L of each mixture was added to wells containing Z138 cells, respectively. Then at 37 deg.C/5% CO₂The cell/antibody-saponin mixture was incubated for 24 hours. After the incubation, cells were spun down in round-bottomed 96-well plates, the supernatant was removed, and 100 μ Ι _ of fresh medium was added to each well. The cells were then incubated for a further 72 hours and then counted using CellTiter-Glo (Promega Corp.) according to the manufacturer's protocolThe number of cells that survived.

Using this protocol, several antibodies capable of internalization as described in the previous examples were also capable of mediating cell killing in vitro (data not shown), whereas biotinylated isotype control antibodies were not capable of killing cells. That is, several of these internalization modulators are capable of mediating saponin toxin internalization leading to cell death. This cell killing ability for the exemplary internalization modulator SC4.5.3 is demonstrated in fig. 16A, where the downward slope of the curve represents cell death in a concentration-dependent manner (compared to control). These data clearly demonstrate the effectiveness of the disclosed modulators as vectors for the selective internalization of cytotoxic payloads in tumorigenic cells expressing ephrin-a ligands.

To corroborate these results with each other and to determine whether the EFNA4 effector was able to mediate toxin internalization and cell killing of primary human tumor cells, mouse lineage-depleted NTX cells (i.e., human tumor cells propagated as low passage xenografts in immunocompromised mice) were plated and subsequently exposed to anti-EFNA 4 antibodies and Fab-ZAPs.

Specifically, NTX tumors representing lung and skin tumor samples were dissociated into single cell suspensions and plated on BD Primaria, as known in the art^TMOn plates (BD Biosciences) in serum-free medium supplemented with growth factors. At 37 deg.C/5% CO₂/5％O₂After 3-5 days of incubation, cells were contacted with either control (IgG1 or IgG2b) or murine EFNA4 modulator (1nM of SC4.5, SC4.22, SC4.47 or SC4.91) and Fab-ZAP (40 nM). After 5-7 days, modulator-mediated saponin cytotoxicity was assessed by quantifying the number of cells remaining using CellTiter Glo. As seen in fig. 16B, exposure to EFNA4 antibody caused a reduced number of LU86 cells, while IgG2B and IgG1 isotype control antibodies did not affect the number of viable cells after treatment. In fig. 16C, exposure to SC4.5, SC4.47, SC4.91 antibody produced a reduced number of SK19 cells, whereas isotype control and SC4.22 were ineffective. This data shows not only the exemplary antibodies described hereinThe above data also show that various anti-EFNA 4 antibodies are capable of mediating killing of various NTX tumor cells, specific for EFNA4, capable of binding to EFNA4 antigen on the cell surface and promoting the delivery of cytotoxic payloads that produce cell death.

In a variant of the aforementioned killing assay, delivery of cytotoxic payloads by EFNA modulators is shown for other antibodies and in other cells. The day before antibody and toxin addition, 2000 cells/well of the following cell types were plated in their respective media in 96-well tissue culture plates: HEK293T cells (fig. 16C), HEK293t. Purified ("naked") mouse monoclonal antibodies at various concentrations and a fixed concentration of 10nM anti-mouse IgG Fab fragment covalently attached to saponin (Advanced Targeting Systems, # IT-48) were added to the cultures for 72 hr. The number of surviving cells was counted as described above. The raw luminosity of the cultures containing cells with the saponin Fab fragments was set to 100% reference value and all other counts were calculated accordingly (called "normalized RLU").

Using this assay, we were able to show that all tested EFNA antibodies (but not isotype control antibodies) were able to kill target cells. This assay also showed that internalization occurred solely due to binding of the EFNA4 antibody to the cell surface without additional cross-linking. Finally, the data show that only cells expressing a sufficient number of EFNAs on their surface are killed by EFNA modulators. Parental HEK293T cells expressed a low number of EFNAs on their cell surface, while HEK293t. hefna4 cells strongly expressed this ligand (see fig. 15C and 15D from previous examples). Table 5 below lists the half-maximal effective concentrations (commonly referred to as "EC 50") for all antibody/target cell combinations tested. In addition to the aforementioned cell line PC3 cell (ATCC CRL-1435), a cell line derived from human adenocarcinoma was used as a target cell.

TABLE 5

EFNA modulators deliver cytotoxic payloads

EC50(pM)	HEK293T	HEK293T.hEFNA4	PC3	Z138
					Isoforms	No damage	No damage	No damage	No damage
SC4.2.1	No damage	10.1	N.T.	N.T.
					SC4.5.1	No damage	15.0	N.T.	4.6
hSC4.15	N.T.	13.7	5.4	N.T
					SC4.22.1	No damage	28.6	5.4	18.7
SC4.31.3	No damage	14.2	N.T.	33.8
					SC4.47.3	201	23.2	2.5	9.6
SC4.91.4	No damage	7.8	N.T.	15.8
					SC4.105.4	No damage	17.3	N.T.	65.2
SC9.65	No damage	28.9	N.T.	N.T.

(n.t. ═ untested)

In another variant of the in vitro killing assay, humanized EFNA modulators were tested for their ability to internalize and deliver cytotoxic payloads. The assay was performed as described above, except that only 500 cells/well were plated and an anti-human IgG Fab fragment covalently linked to saponin (Advanced Targeting Systems, # IT-51) was added to the culture. Figure 16E shows that the humanized (Hz in figure 16E) EFNA modulator described in example 7 is capable of binding ephrin-a 4 ligand expressed on the surface of target cells and inducing EFNA internalization with the bound antibody and cytotoxic payload.

In another variant of the in vitro killing assay, the humanized EFNA modulator hsc4.15 (see fig. 13C) that showed equally good binding to mouse and human EFNA was tested for its ability to internalize and deliver cytotoxic payloads to HEK293T cells overexpressing human or mouse EFNA. To ensure direct comparability, lentiviral transduced cells were stained with hsc4.15 and sorted by FACS for moderate expression of human or mouse ephrin-a 4 (data not shown). The killing assay was performed as described above. Figure 16F shows that the humanized SC4.15 modulator kills cells expressing mouse or human EFNA equally well.

Example 17

Detection of secreted ephrin-a ligands by EFNA modulators

As discussed in more detail above, EFNA4 may exist as a GPI-linked molecule that binds to cell membranes or as a truncated ligand or isoform that is secreted. Detection of these secreted compounds in biological materials (e.g. body fluids or cell culture media) can be used for diagnostic purposes or as an aid in the management of patients (use as a biomarker). For example, it was shown that secreted EFNA4 can be found at elevated concentrations in B-cell chronic lymphocytic Leukemia (B-CLL) patients (Alonso-C LM et al, 2009, Leukemia Research 33: 395-406). To demonstrate this preferred aspect of the invention, the disclosed modulators are used to identify non-coincident epitopes of purified EFNA4 and to generally detect and quantify secreted EFNA ligands in selected tumorigenic samples. With respect to this latter feature of the invention, EFNA modulators were used to detect and quantify secreted ephrin-a ligands in human serum (data not shown) and human plasma obtained from B-CLL patients, as well as serum from mice bearing human tumor xenografts (e.g., as described in example 1 above). In each case, the modulator is effective to measure ligand levels, as described below.

To detect soluble human EFNA4, antibody SC4.91 was adsorbed to high-protein binding microtiter plates (Greiner bioonemicroclone plates) at 5 μ g/ml during incubation overnight at 4 ℃ in 50mM sodium carbonate buffer (ph 9.6). After washing the plates in Phosphate Buffered Saline (PBS) (PBST) containing 0.05% (v/v) Tween20, the plates were blocked in PBS (PBSA) containing 2% (w/v) bovine serum albumin for 2 hours at ambient temperature. Purified ephrin-a 4-His (transiently expressed in CHO-S cells and purified using nickel NTA resin and gel filtration sequentially) was serially diluted in PBSA and added to the plate for 2 hours. After washing with PBST, biotinylated antibody SC4.47 was added to the plate at 1. mu.g/ml (in PBSA) for 1 hour. The plates were then washed with PBST, after which streptavidin-horseradish peroxidase conjugate (e.g., Jackson Immuno Research) was added to the PBSA at a dilution of 1:5000 for 30 minutes. The treated plates were then washed again in PBST and TMB substrate solution (e.g., Thermo Fisher) was added for 30 minutes. The color reaction was stopped by adding an equal volume of 2M sulfuric acid, after which the plates were read in a standard plate reader with absorbance reading at 450 nm. The results of the experiment are shown in FIGS. 17A-C.

Using the techniques described above, the concentration of soluble h ephrin-a 4-His was plotted against absorbance values to provide the curve shown in figure 17A. More specifically, the original curve shows the results of absorbance measurements at concentrations of soluble EFNA4 between 0 and 40pg/ml, whereas the insert shows the same curve at concentrations between 0 and 1000 pg/ml. Those skilled in the art will appreciate that the standard curve shown in fig. 17A can be used to provide an extremely sensitive assay for measuring the concentration of EFNA4 in a biological sample.

The concentration of ephrin-a 4 in unknown samples was calculated using the aforementioned measurements and using non-linear regression (Prism 5, Graphpad software). In this regard, plasma samples from four healthy adults, four patients diagnosed with B-cell chronic lymphocytic leukemia (B-CLL), and four patients diagnosed with Multiple Myeloma (MM) were analyzed for their secreted ephrin-a 4 concentrations. The data obtained showed that the hEFNA4 analyte was significantly higher in CLL patients compared to healthy adults or other selected B cell derived tumors (fig. 17B). Furthermore, as previously indicated and shown in fig. 17C, secreted hENFA4 was also detectable in mice bearing human colorectal cancer xenografts. In particular, each point in fig. 17C represents the level of secreted hEFNA4 in sera obtained from different mice. In contrast, serum levels of secreted hEFNA4 were essentially undetectable in non-xenografted mice (data not shown). Even more surprisingly, when tumor volume was plotted against the concentration of hEFNA4 in serum samples, a significant correlation was observed, suggesting that secreted analytes may be particularly useful for monitoring tumor growth of certain human solid tumors in vivo. More generally, these results strongly indicate the applicability of the invention in therapeutic and diagnostic settings.

Using the above method, plasma samples from 23 normal human donors obtained from a blood bank were used to determine the concentration range of this analyte in healthy adults. As shown in FIG. 17D, an average concentration of 332pg/ml EFNA4 (standard deviation of 6.2 pg/ml) was found. This means that EFNA is secreted or disseminated in very low and tightly regulated concentrations and makes EFNA an ideal biomarker or diagnostic marker for monitoring disease progression or diagnosing EFNA-associated disorders.

To further explore this possibility, commercially obtained serum samples from 17 patients with colorectal cancer and 10 samples from patients with non-small cell lung cancer were compared to 12 samples from healthy adults and tested for EFNA4 concentration using the method described above. As shown in fig. 17E, patients with both colorectal and non-small cell lung cancers had significantly elevated levels of circulating EFNA4 in their blood. Using the unpaired t-test, the comparison between healthy adults and colorectal cancer patients reached a p-value of 0.0002, whereas the comparison between healthy adults and non-small cell lung cancer patients reached a p-value of 0.01. These data indicate that secreted or disseminated EFNA4 is elevated in patients with solid tumors and indicate value in analytical testing or clinical diagnosis using the disclosed modulators.

Example 18

EFNA4 modulators are capable of targeting cells expressing related EFNA ligands

The ligand specificity of EFNA4 modulators was tested against related EFNA ligands to assess the degree of cross-reactivity. As an example, sc4.2.1 and SC9.65 were tested in an in vitro killing assay using HEK293T cells overexpressing EFNA4 (fig. 17A), EFNA3 (fig. 17B) and EFNA1 (fig. 17C). It should be noted that the modulator SC9.65 was generated by immunizing mice with EFNA1 immunogen (as per example 6). The killing assay was performed as described in example 16. Fig. 17 shows that in addition to cells expressing EFNA4, sc4.2.1 was able to kill cells expressing EFNA3, and SC9.65 was able to kill cells expressing EFNA1 and EFNA 4. These data indicate that selected modulators generated against a particular EFNA family member are able to bind to other family members sufficiently well to bind to, induce internalization, and deliver cytotoxic payloads to ligand-expressing cells. This finding was somewhat unexpected in view of the low degree of homology between EFNA family members (about 34-45% amino acid sequence identity between human EFNA1, 2, 3 and 4), and it exemplifies (as described herein) that pan-EFNA modulators can be generated for diagnostic or therapeutic purposes.

Example 19

EFNA ligands selectively interact with a variety of EphA receptors

As discussed above, ephrin-a ligands are known to bind numerous EphA receptors. To explore which EphA receptors have the potential to interact with EFNA4, a flow cytometric binding assay similar to that described in example 9 was developed. More specifically, soluble EphA receptors expressed as human IgG1Fc fusion constructs were added to fifty thousand HEK293T cells/well (fig. 19A) or HEK293T cells overexpressing EFNA4 (fig. 19B) (referred to as HEK293t. hefna4 cells) by 1 hour in staining buffer via retroviral transduction at 4 ℃. After washing, a second anti-human IgG polyclonal antibody conjugated to Dylight649 (Jackson Immuno Research) was added for 1 hour. After two washes, the samples were resuspended in staining buffer containing 2 μ g/ml DAPI and analyzed on a FACS Canto II (BDBiosciences) using HTS-ligation under standard conditions. Figures 19A and 19B show that EphA2, EphA3, EphA4, EphA6, EphA7, and EphA10, but not EphA1, bind ephrin-a 4 ligands. This again points out the advantages and potential for multifaceted action inherent in the modulators of the invention.

Example 20

EFNA4 binds to EphB2 but not EphB3 and EphB4 receptors

Extending the findings shown in example 20, the ability of ephrin-a 4 ligand to bind EphB receptor was explored. EFNA4 was initially identified as a CSC binding target as shown in examples 2-4 above. In the tissue hierarchy of normal mouse colonic crypts, EphB2 and EphB3 receptors are highly expressed by cells at the base of the colonic crypt but not by cells at the top of the crypt, indicating that EphB expression and forward or reverse signaling through EphB receptors are important in tissue architecture and single cell fate determination (Battle et al; 2002PMID: 12408869). Recently, EphB2 expression by colorectal cancer cells has been linked to tumor initiating and long-term proliferative capacity, indicating that EphB2 can serve as a phenotypic marker of colon cancer stem cells (Merlos-Suarez et al, 2011 PMID: 21419747). Thus, the ability of ephrin-a 4 ligand to bind to any differentially expressed EphB receptors may be of biological importance for colorectal cancer stem cells.

Soluble EphB receptors expressed as human IgG1Fc fusion constructs were added to either pentathousand HEK293T cells/well (fig. 20A) or HEK293t. hefna4 cells (fig. 20B) along with EphA1-Fc (which does not bind EFNA4) and EphA2-Fc (which does not strongly bind EFANA4 ligand) by 1 hour at 4 ℃ in staining buffer using art-recognized techniques. After washing, a second anti-human IgG polyclonal antibody conjugated to Dylight649 (Jackson ImmunoResearch) was added for 1 hour. After two washes, the samples were resuspended in staining buffer containing 2 μ g/ml DAPI and analyzed on a FACS Canto II (BD Biosciences) using HTS-ligation under standard conditions. Figures 20A and 20B show that EphB2, but not EphB3 and EphB4, bind EFNA4 ligands, again underscoring the potential diversity of therapeutic pathways that can be beneficially affected by the disclosed modulators.

It will also be appreciated by those skilled in the art that the invention can be embodied in other specific forms without departing from the spirit or central characteristics thereof. Whereas the foregoing description of the invention discloses only exemplary embodiments thereof, it will be understood that other variations are contemplated as being within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments that have been described in detail herein. Rather, reference should be made to the appended claims as an indication of the scope and content of the present invention.

Some embodiments of the invention are as follows:

1. an isolated EFNA modulator.

2. The isolated EFNA modulator of embodiment 1 wherein the EFNA modulator comprises an EFNA antagonist.

3. The isolated EFNA modulator of embodiment 1, wherein the EFNA modulator comprises an antibody or immunoreactive fragment thereof.

4. The isolated EFNA modulator of embodiment 3 wherein the antibody or immunoreactive fragment thereof comprises a monoclonal antibody.

5. The isolated EFNA modulator of embodiment 4 wherein the monoclonal antibody is selected from the group consisting of: chimeric antibodies, CDR-grafted antibodies, humanized antibodies and human antibodies.

6. The isolated EFNA modulator of embodiment 4 wherein the monoclonal antibody comprises a neutralizing antibody.

7. The isolated EFNA modulator of embodiment 4 wherein the monoclonal antibody comprises an internalizing antibody.

8. The isolated EFNA modulator of embodiment 4 wherein the monoclonal antibody comprises a depleting antibody.

9. The isolated EFNA modulator of embodiment 4 wherein the monoclonal antibody comprises an antibody that binds to EFNA 4.

10. The isolated EFNA modulator of embodiment 9 wherein the monoclonal antibody comprises a light chain variable region having three complementarity determining regions and a heavy chain variable region having three complementarity determining regions, wherein the heavy and light chain complementarity determining regions comprise complementarity determining regions set forth in fig. 7A.

11. The isolated EFNA modulator of embodiment 9 wherein the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in seq id no: SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 163 and wherein said heavy chain variable region comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in seq id no: SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121, SEQ id no: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157 and SEQ ID NO: 161.

12. the isolated EFNA modulator of embodiment 9, 10 or 11 further comprising a cytotoxic agent.

13. A nucleic acid encoding the amino acid heavy chain variable region or the amino acid light chain variable region of embodiment 11.

14. A vector comprising the nucleic acid of embodiment 13.

15. A host cell comprising the vector of embodiment 14.

16. The isolated EFNA modulator of embodiment 1 comprising SEQ ID NO: 2 or a fragment thereof.

17. The isolated EFNA modulator of embodiment 16 wherein the EFNA modulator further comprises at least a portion of an immunoglobulin constant region.

18. The isolated EFNA modulator of embodiment 1, wherein the modulator reduces the frequency of tumor initiating cells upon administration to a subject in need thereof.

19. The isolated EFNA modulator of embodiment 18, wherein the reduction in frequency is determined using flow cytometric analysis of tumor cell surface markers known to be enriched for tumor initiating cells.

20. The isolated EFNA modulator of embodiment 18, wherein the reduction in frequency is determined using immunohistochemical detection of tumor cell surface markers known to enrich for tumor initiating cells.

21. The isolated EFNA modulator of embodiment 18 wherein the tumor initiating cells comprise tumor perpetuating cells.

22. The isolated EFNA modulator of embodiment 1 further comprising a cytotoxic agent.

23. The isolated EFNA modulator of embodiment 1 wherein the EFNA modulator comprises a pan-EFNA modulator.

24. A pharmaceutical composition comprising the isolated EFNA modulator of embodiment 1.

25. An isolated EFNA4 modulator.

26. The isolated EFNA4 modulator of embodiment 25 wherein the EFNA4 modulator comprises a pan-EFNA 4 modulator.

27. A pharmaceutical composition comprising the isolated EFNA4 modulator of embodiment 25.

28. A method of treating an EFNA associated disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of an EFNA modulator.

29. The method of embodiment 28 wherein said EFNA modulator comprises an EFNA antagonist.

30. The method of embodiment 28 wherein said EFNA modulator comprises an antibody or immunoreactive fragment thereof.

31. The method of embodiment 30, wherein said antibody or immunoreactive fragment thereof comprises a monoclonal antibody.

32. The method of embodiment 31, wherein said monoclonal antibody is selected from the group consisting of: chimeric antibodies, CDR-grafted antibodies, humanized antibodies and human antibodies.

33. The method of embodiment 32, wherein said monoclonal antibody comprises a light chain variable region having three complementarity determining regions and a heavy chain variable region having three complementarity determining regions, wherein said heavy and light chain complementarity determining regions comprise complementarity determining regions set forth in fig. 7A.

34. The method of embodiment 31 wherein said monoclonal antibody binds to EFNA 4.

35. The method of embodiment 31, wherein said monoclonal antibody comprises a neutralizing antibody.

36. The method of embodiment 31, wherein said monoclonal antibody comprises an internalizing antibody.

37. The method of embodiment 36, wherein said internalizing antibody comprises a cytotoxic agent.

38. The method of embodiment 28, wherein said EFNA associated disorder comprises a hyperproliferative disorder.

39. The method of embodiment 38, wherein said hyperproliferative disorder comprises a neoplastic disorder.

40. The method of embodiment 39, wherein said neoplastic disorder comprises a solid tumor.

41. The method of embodiment 40, wherein the neoplastic disorder comprises an adrenal gland cancer, a bladder cancer, a cervical cancer, an endometrial cancer, a renal cancer, a liver cancer, a lung cancer, an ovarian cancer, a colorectal cancer, a pancreatic cancer, a prostate cancer or a breast cancer.

42. The method of embodiment 39, wherein said neoplastic disorder comprises a hematological malignancy.

43. The method of embodiment 42, wherein said hematologic malignancy comprises leukemia or lymphoma.

44. The method of embodiment 39, wherein the subject having said neoplastic disorder exhibits a tumor comprising tumor initiating cells.

45. The method of embodiment 44, further comprising the step of reducing the frequency of tumor initiating cells in said subject.

46. The method of embodiment 45, wherein said reduction in frequency is determined using flow cytometric analysis of tumor cell surface markers known to be enriched in tumor initiating cells, or immunohistochemical detection of tumor cell surface markers known to be enriched in tumor initiating cells.

47. The method of embodiment 45, wherein said reduction in frequency is determined using an in vitro or in vivo limiting dilution assay.

48. The method of embodiment 47, wherein said reduction in frequency is determined using an in vivo limiting dilution assay comprising transplantation of live human tumor cells into immunocompromised mice.

49. The method of embodiment 48, wherein said reduction in frequency is determined using an in vivo limiting dilution assay comprising quantifying tumor initiating cell frequency using Poisson distribution statistics.

50. The method of embodiment 47, wherein said reduction in frequency is determined using an in vitro limiting dilution assay comprising limiting dilution deposition of viable human tumor cells into in vitro colony supporting conditions.

51. The method of embodiment 50, wherein said reduction in frequency is determined using an in vitro limiting dilution assay comprising quantifying tumor initiating cell frequency using Poisson distribution statistics.

52. The method of embodiment 28, further comprising the step of administering an anti-cancer agent.

53. The method of embodiment 28 wherein said EFNA modulator comprises SEQ ID NO: 2 or a fragment thereof.

54. The method of embodiment 28, wherein said EFNA modulator comprises a pan-EFNA modulator.

55. A method of reducing tumor initiating cell frequency in a subject in need thereof comprising the step of administering to the subject an EFNA modulator.

56. The method of embodiment 55, wherein said tumor initiating cells comprise tumor perpetuating cells.

57. The method of embodiment 56, wherein said tumor perpetuating cells are CD44⁺Or CD133⁺A cell.

58. The method of embodiment 55, wherein said EFNA modulator comprises an antibody.

59. The method of embodiment 58, wherein said antibody comprises a monoclonal antibody.

60. The method of embodiment 59, wherein said EFNA modulator comprises an anti-EFNA 4 antibody.

61. The method of embodiment 55, wherein said subject has a neoplastic disorder selected from the group consisting of: adrenal gland cancer, bladder cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, prostate cancer and breast cancer.

62. The method of embodiment 55, wherein said subject has a hematological malignancy.

63. The method of embodiment 55, wherein the frequency of tumor initiating cells is reduced by at least 10%.

64. The method of embodiment 55, wherein said reduction in frequency is determined using flow cytometric analysis of tumor cell surface markers known to be enriched in tumor initiating cells, or immunohistochemical detection of tumor cell surface markers known to be enriched in tumor initiating cells.

65. The method of embodiment 55, wherein said reduction in frequency is determined using an in vitro or in vivo limiting dilution assay.

66. A method of treating a subject having a hematological malignancy comprising the step of administering to the subject an EFNA modulator.

67. The method of embodiment 66, wherein said EFNA modulator is an EFNA4 modulator.

68. A method of sensitizing a tumor in a subject for treatment with an anti-cancer agent, the method comprising the step of administering to the subject an EFNA modulator.

69. The method of embodiment 68, wherein said EFNA modulator comprises an antibody.

70. The method of embodiment 68, wherein said tumor is a solid tumor.

71. The method of embodiment 68, wherein said anti-cancer agent comprises a chemotherapeutic agent.

72. The method of embodiment 68, wherein said anti-cancer agent comprises an immunotherapeutic agent.

73. A method of diagnosing a hyperproliferative disorder in a subject in need thereof, comprising the steps of:

a. obtaining a tissue sample from the subject;

b. contacting the tissue sample with at least one EFNA modulator; and

c. detecting or quantifying the EFNA modulator bound to the sample.

74. The method of embodiment 73, wherein said EFNA modulator comprises a monoclonal antibody.

75. The method of embodiment 74, wherein said antibody is operably bound to a reporter.

76. An article of manufacture useful for diagnosing or treating an EFNA associated disorder, comprising a container comprising an EFNA modulator and instructional material for using the EFNA modulator to treat or diagnose an EFNA associated disorder.

77. The article of manufacture of embodiment 76, wherein said EFNA modulator is a monoclonal antibody.

78. The article of embodiment 76, wherein the container comprises a readable plate.

79. A method of treating a subject suffering from a neoplastic disorder, said method comprising the step of administering a therapeutically effective amount of at least one internalizing EFNA modulator.

80. The method of embodiment 79, wherein said EFNA modulator comprises an antibody.

81. The method of embodiment 80, wherein said antibody comprises a monoclonal antibody.

82. The method of embodiment 81, wherein said monoclonal antibody further comprises a cytotoxic agent.

83. The method of embodiment 81, wherein said monoclonal antibody binds to EFNA 4.

84. A method of treating a subject suffering from a neoplastic disorder, said method comprising the step of administering a therapeutically effective amount of at least one neutralizing EFNA modulator.

85. The method of embodiment 84, wherein said EFNA modulator comprises an antibody.

86. The method of embodiment 85, wherein said antibody comprises a monoclonal antibody.

87. The method of embodiment 86 wherein said monoclonal antibody comprises an anti-EFNA 4 antibody.

88. The method of embodiment 87, wherein said EFNA4 antibody comprises a pan-EFNA antibody.

89. The method of embodiment 84, wherein said neoplastic disorder is selected from the group consisting of: adrenal gland cancer, bladder cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, prostate cancer and breast cancer.

90. A method of identifying, isolating, fractionating or enriching a population of tumor initiating cells, said method comprising the step of contacting said tumor initiating cells with an EFNA modulator.

91. The method of embodiment 90, wherein said EFNA modulator comprises an antibody.

92. A composition comprising a humanized antibody variable region substantially similar to a humanized variable region found on an antibody selected from the group consisting of hsc4.5, hsc4.15, hsc4.22 and hsc4.47 and a pharmaceutically acceptable carrier.

93. An anti-EFNA 4 antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 151, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO: 163 and wherein the heavy chain variable region comprises an amino acid sequence having at least 60% identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 149, SEQ ID NO: 153, SEQ ID NO: 157 and SEQ ID NO: 161 having an amino acid sequence of at least 60% identity.

94. A method of inhibiting or preventing metastasis in a subject in need thereof, the method comprising the step of administering a pharmaceutically effective amount of an EFNA modulator.

95. The method of embodiment 94, wherein said subject undergoes a tumor reduction procedure prior to or after administration of said EFNA modulator.

96. The method of embodiment 94, wherein said tumor reduction procedure comprises administering at least one anti-cancer agent.

97. A method of maintenance therapy on a subject in need thereof, the method comprising the step of administering a pharmaceutically effective amount of an EFNA modulator.

98. The method of embodiment 97, wherein said subject is treated for a neoplastic disorder prior to administration of said EFNA modulator.

99. A method of depleting tumor cells in a subject having a hyperproliferative disorder, said method comprising the step of administering an EFNA modulator.

100. The method of embodiment 99, wherein said tumor cells comprise tumor initiating cells.

101. A method of diagnosing, detecting or monitoring an EFNA associated disorder in vivo in a subject in need thereof, said method comprising the step of administering an EFNA modulator.

Claims (10)

1. An isolated EFNA modulator.

2. The isolated EFNA modulator of claim 1 wherein the EFNA modulator comprises an EFNA antagonist.

3. The isolated EFNA modulator of claim 1 wherein the EFNA modulator comprises an antibody or immunoreactive fragment thereof.

4. The isolated EFNA modulator of claim 3 wherein the antibody or immunoreactive fragment thereof comprises a monoclonal antibody.

5. The isolated EFNA modulator of claim 4 wherein said monoclonal antibody is selected from the group consisting of: chimeric antibodies, CDR-grafted antibodies, humanized antibodies and human antibodies.

6. The isolated EFNA modulator of claim 4 wherein said monoclonal antibody comprises a neutralizing antibody.

7. The isolated EFNA modulator of claim 4 wherein said monoclonal antibody comprises an internalizing antibody.

8. The isolated EFNA modulator of claim 4 wherein the monoclonal antibody comprises a depleting antibody.

9. The isolated EFNA modulator of claim 4 wherein said monoclonal antibody comprises an antibody that binds to EFNA 4.

10. The isolated EFNA modulator of claim 9 wherein said monoclonal antibody comprises a light chain variable region having three complementarity determining regions and a heavy chain variable region having three complementarity determining regions, wherein the heavy and light chain complementarity determining regions comprise complementarity determining regions set forth in fig. 7A.