MOLECULES

FIELD OF THE INVENTION

The present invention is directed to a pharmaceutical composition comprising a binding molecule having binding affinity to one or more ErbB ligands and at least one therapeutic agent. The present invention further provides uses of the binding molecule in combination with therapy for inhibiting cell proliferation, increasing sensitivity to therapy, attenuating or preventing resistance to therapy and treating diseases associated with the one or more ErbB ligands. The present invention also provides uses of the binding molecules of the invention for preventing a disease associated with ErbB ligands, including preventing recurrence of said disease.

BACKGROUND OF THE INVENTION The ErbB family of receptors and cognate growth factors, all sharing an epidermal growth factor (EGF) module, play important roles in embryonic development and in tissue remodeling throughout adulthood. The family includes four receptors: ErbB-1 (EGFR), which binds EGF, transforming growth factor a (TGFa), heparin-binding EGF like growth factor (HB-EGF), amphiregulin (AR), betacellulin (BTC), epiregulin (EPR) and epigen, ErbB-2 (also called HER2), which has no known ligand, and two neuregulin (NRG) receptors, ErbB-3 and ErbB-4.

Several clinical studies indicate that overexpression of one or more EGF-like ligands correlates with decreased patient survival. For example, in colorectal tumors enhanced expression of TGFa is associated with over 50-fold increased risk of developing liver metastases, and TGFa levels in liver metastases associate with poor patient outcome. Furthermore, increased expression of TGFa in head and neck tumors correlates with decreased patient survival (Grandis et al, J Cell Biochem., 69:55-62, 1998). In bladder cancer, the elevated expression of a number of ligands is linked to decreased patient survival (Thogersen et al, Cancer Res., 61 : 6227-33, 2001). Moreover, overexpression of neuregulins (NRGs) in mammary tissue, in vivo, accelerates adenocarcinoma development (Krane and Leder, Oncogene, 12: 1781-1788, 1996) and metastatic spread of breast cancer cells (Atlas et al, Mol Cancer Res., 1 : 165-175, 2003). In addition, ErbB receptors and their ligands are also involved in resistance to endocrine and cytotoxic therapy, as well as to radiotherapy.

Therapeutics that interfere with ligand binding to ErbB family are known in the art. These include monoclonal antibodies directed at ErbB-1 or at ErbB-2/HER2 (e.g. cetuximab and trastuzumab, respectively) or at ErbB-3 and small-molecule tyrosine kinase inhibitors (TKIs; e.g., lapatinib, gefitinib, AG1478 and erlotinib).

A binding molecule having binding affinity for an ErbB ligand is disclosed in WO 2006/096663.

A bivalent binding molecule, also termed 'double trap', having binding affinity for two ErbB ligands at separate binding sites is disclosed in WO 2007/092932, by one of the inventors of the present invention.

A bivalent molecule comprises portions of a first and a second binding sites of ErbB ligands is disclosed in WO 2011/0171159. According to this publication, the bivalent molecule consists of LI domain of a first ErbB receptor, L2 domain of a second ErbB receptor and a modified CI domain. The modified CI domain contains only parts (modules) of the CI domain of each ErbB receptor, rather than the complete native CI domain. According to the disclosure of WO 2011/0171159 the resulting bivalent molecule binds only a single ErbB ligand (HRGlp).

The inventor of the present invention also disclosed cancer immunotherapy comprising combinations of chemotherapy with one or more antibodies against EGF-receptor ligands (Lindzen et al, PNAS, 107(28): 12559-12563, 2010).

There remains an unmet need for improved therapeutic platforms for inhibiting tumor growth and metastasis. SUMMARY OF THE INVENTION

The present invention is directed to a pharmaceutical composition comprising a binding molecule having binding affinity to an ErbB ligand or to a plurality of ErbB ligands and at least one therapeutic agent and uses thereof for treating a disease associated with the ErbB ligands. These uses include inhibiting cell proliferation, increasing sensitivity to therapy, and attenuating or preventing resistance to therapy. In addition, the present invention provides methods for preventing a disease associated with ErbB ligands, such as a malignant disease, using the binding molecules of the invention in combination with one or more therapeutic agents.

The present invention is based in part on the unexpected discovery that combining a bivalent binding molecule (also termed hereinafter "TRAP-Fc" or "TRAP-His"), having a binding affinity to two different ErbB ligands, with anti cancer therapy, results with a synergistic therapeutic effect. Specifically, as exemplified hereinbelow, combining TRAP-Fc with various anti cancer therapeutic agents prolonged the survival of mice having pancreatic cancer in a synergistic manner, compared to the effect obtained by each of TRAP-Fc or the therapeutic agent alone. Moreover, the combined treatment of the invention overcomes major drawbacks associated with anti cancer therapy, such as, weight loss and resistance to the therapeutic agent(s). Advantageously, the therapeutic effect of the bivalent binding molecule is not accompanied by weight loss. Moreover, the combined therapy of the invention prevents or attenuates resistance to the anti cancer therapeutic agent and increases sensitivity thereto. Furthermore, in the attempts to decrease the size of the bivalent molecule, it was surprisingly found that the molecule has to contain at least the LI, CI and L2 subdomains and part of the C2 domain of each ErbB receptor, in order to have an effective affinity and therefore a therapeutic effect.

According to a first aspect, the present invention provides a pharmaceutical composition comprising a binding molecule comprising a first and a second binding sites on a single amino acid chain, and at least one therapeutic agent, wherein each binding site comprises subdomains LI, CI, L2 subdomain C2 or a portion thereof, of an ErbB receptor.

According to another embodiment, the pharmaceutical composition further comprises a therapeutically acceptable carrier. According to yet another embodiment, the pharmaceutical composition is for inhibiting cell proliferation. According to yet another embodiment, the pharmaceutical composition is for treating a disease. According to yet another embodiment, the disease is selected from the group consisting of: atherosclrosis, cancer, a malignant disease, psoriasis, a skin disorder, a coronary disease, a disease associated with ErbB ligands and a neurodegenerative disease. Each possibility represents a separate embodiment of the invention. According to yet another embodiment, the malignant disease is cancer. According to yet another embodiment, the malignant disease is selected from the group consisting of: pancreatic cancer, bladder cancer, breast cancer, prostate cancer, lung cancer, colorectal cancer, head and neck cancer, ovarian cancer, and cervical cancer. Each possibility represents a separate embodiment of the invention.

According to yet another embodiment, the pharmaceutical composition is for augmenting sensitivity to the at least one therapeutic agent, attenuating resistance to the at least one therapeutic agent, for preventing resistance to the at least one therapeutic agent, or for preventing recurrence of said disease. Each possibility represents a separate embodiment of the invention.

According to yet another embodiment, the pharmaceutical composition is for augmenting sensitivity to the at least one therapeutic agent.

According to yet another embodiment, the pharmaceutical composition is for attenuating resistance to the at least one therapeutic agent.

According to yet another embodiment, the pharmaceutical composition is for preventing resistance to the at least one therapeutic agent.

According to another aspect, the present invention provides a method for treating a disease in a subject in need thereof comprising administering to said subject a binding molecule having binding affinity for a first and a second ErbB ligand at separate binding sites on a single amino acid chain, in combination with therapy directed to inhibition of cell proliferation, wherein each binding site comprises subdomains LI, CI, L2 and subdomain C2 or a portion thereof, of an ErbB receptor.

According to some embodiments, treating the disease comprises inhibiting cell proliferation, augmenting the sensitivity of said subject to said therapy, attenuating resistance of said subject to said therapy, and preventing resistance of said subject to said therapy. Each possibility represents a separate embodiment of the invention.

According to one embodiment, the binding molecule and said therapy are administered concomitantly. According to another embodiment, the binding molecule is administered prior to applying said therapy. According to yet another embodiment, the binding molecule is administered after administration of said therapy. According to yet another embodiment, said therapy is selected from the group consisting of: at least one therapeutic agent, radiation therapy, organ transplantation, and surgery. According to yet another embodiment, said therapy comprises administering to said subject the at least one therapeutic agent selected from the group consisting of: chemotherapeutic agents, including, but not limited to, alkylating agents, antimetabolites, anthracyclines, plant alkaloids and topoisomerase inhibitors, targeted therapy, tyrosine kinase inhibitors and antitumor agents. Each possibility represents a separate embodiment of the invention.

According to further embodiments, the at least one therapeutic agent is selected from the group consisting of: gemcitabine, erlotinib, imatinib mesylate (Gleevec® or Glivec®), gefitinib, lapatinib, CI-1033, AG-1478, cetuximab, trastuzumab (Herceptin®/ Anti-ErbB-2), anti-ErbB3 monoclonal antibody, bevacizumab, docetaxel and panitumumab. Each possibility represents a separate embodiment of the invention.

According to further embodiments, the at least one therapeutic agent is gemcitabine. According to other embodiments, the at least one therapeutic agent is gefitinib. According to yet other embodiments, the at least one therapeutic agent is cetuximab.

According to yet another aspect, the present invention provides a kit for treating a disease associated with an ErbB ligand in a subject in need thereof, comprising a pharmaceutical composition comprising a binding molecule having binding affinity for at least one ErbB ligand; and at least one therapeutic agent.

According to one embodiment the kit comprises (a) a first container comprising the pharmaceutical composition; and (b) a second container comprising the at least one therapeutic agent.

According to another embodiment, the kit further comprises instructions for using said kit.

According to some embodiments, the pharmaceutical composition comprises a binding molecule having a binding affinity for a first and a second ErbB ligand at separate binding sites on a single amino acid chain. According to another embodiment, the binding molecule comprises the extracellular domains of first and second ErbB receptors on a single amino acid chain, each having a binding affinity for a first and a second ErbB ligand. According to another embodiment, the binding molecule comprises a first and a second binding site, wherein each binding site comprises subdomains LI, CI, L2 and subdomain C2 or a portion thereof, of an ErbB receptor. According to further embodiments, the ErbB receptor is selected from the group consisting of: ErbBl, ErbB3 and ErbB4. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, the ErbB ligand is selected from the group consisting of EGF, TGFa, HB-EGF, betacellulin, amphiregulin, epiregulin, epigen, neuregulin-1, neuregulin-2, neuregulin-3 and neuregulin-4.

According to further embodiments, the binding molecule comprises the extracellular domains of ErbBl and ErbB3 receptors on a single amino acid chain.

According to further embodiments, the binding molecule comprises the extracellular domains of ErbB 1 and ErbB4 receptors on a single amino acid chain. According to further embodiments, the first binding site comprises the extracellular domain of ErbBl or a portion thereof. According to further embodiments, the first binding site comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 18. Each possibility represents a separate embodiment of the invention.

According to further embodiments, the second binding site comprises the extracellular domain of ErbB3, the extracellular domain of ErbB4 or a portion thereof. According to further embodiments, the second binding site comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. Each possibility represents a separate embodiment of the invention.

According to further embodiments, the binding molecule comprises a first binding site comprising a portion of the ErbBl receptor, consisting of SEQ ID NO: l and a second binding site comprising a portion of the ErbB3 receptor, consisting of SEQ ID NO: 2. According to further embodiment, the binding molecule further comprises a six-histidine tag. According to further embodiment, the binding molecule further comprises linker consisting of SEQ ID NO:6.

According to further embodiments, the binding molecule comprises a first binding site comprising a portion of the ErbBl receptor, consisting of SEQ ID NO: l and a second binding site comprising a portion of the ErbB4 receptor, consisting of SEQ ID NO: 3. According to further embodiment, the binding molecule further comprises a six-histidine tag. According to further embodiment, the binding molecule further comprises linker consisting of SEQ ID NO:6.

According to further embodiments, the binding molecule comprises a first binding site comprising a portion of the ErbB4 receptor, consisting of SEQ ID NO:4 and a second binding site comprising a portion of the ErbBl receptor, consisting of SEQ ID NO: 5. According to further embodiment, the binding molecule further comprises a portion of the Fc domain of human immunoglobulin G2. According to yet another embodiment, the binding molecule further comprises a portion of the Fc domain of human immunoglobulin consisting of SEQ ID NO:8. According to further embodiments, the binding molecule further comprises linker consisting of SEQ ID NO:6. According to further embodiment, the binding molecule further comprises a signal peptide comprising the amino acid sequence as set forth in SEQ ID NO:7. According to a further embodiment, the signal peptide is encoded by the nucleotide sequence as set forth in SEQ ID NO: 20.

According to some embodiments, the signal peptide corresponds to any one of the signal peptides set forth in WO 2007/092932.

Other objects, features and advantages of the present invention will become clear from the following description.

BRIEF DESCRIPTION OF THE INVENTION

Figure 1A is a schematic presentation of four TRAP-His proteins.

Figure IB presents immunoblots, with an anti-EGFR antibody, of conditioned medium from cells (upper panel) and cell lysates (lower panel) of HEK-293 cells stably expressing the TRAP-His recombinant proteins.

Figure 1C shows a gel autoradiogram of TRAP-His molecules and IgB-1 (as positive control) incubated for 60 minutes at 4°C with ¹²⁵ I-NRG1- β (upper panel) or ¹²⁵ I-EGF (lower panel), in the presence or absence of the respective unlabelled ligand, and the cross-linking molecule BS ₃ (2 mM). Figure ID exhibits the percentage uptake of ¹²⁵ I- EGF in HeLa cells incubated for 10 min (in triplicates) with ¹²⁵ I-EGF (3 ng/ml) in the presence of TRAP-His molecules or IgB-1 (20 ng/ml).

Figure IE presents immunoblots with an anti-phosphotyrosine antibody of extracts obtained from HeLa cells and T47D cells incubated for 10 min with the ligands (5 ng/ml): EGF, TGF-a, HB-EGF, BTC, EPG or mTGF (HeLa), NRG1, HB-EGF, BTC or mNRG-1 (T47D), in the absence or presence of the IgBl, IgB4 or TRAP-Fc proteins (each at 60 μg/ml).

Figure 2A is scheme of the recombinant TRAP-Fc protein, which includes a signal peptide, the three N-terminal extracellular sub-domains of ErbB-4 called LI (domain I), SI (domain II), LII (domain III) and a portion of SII (domain IV), a linker, followed by the corresponding portion of ErbB-1 linked to human immunoglobulin lambda's Fc portion. Residue numbers corresponding to ErbB-4 and ErbB-1 appear in parentheses, other numbers refer to the TRAP's full sequence.

Figure 2B exhibits Coomassie blue staining of an acrylamide gel showing the purified TRAP-Fc protein (2μg) following electrophoresis under non-reducing (NR) or reducing (R) conditions.

Figure 2C presents analyses of ligand binding (using ELISA) to IgB proteins or to TRAP-Fc (each at 8 μg/ml).

Figure 2D exhibits the dissociation constants of TRAP-Fc from TGFa, HB-EGF and NRG 1 ligands measured by surface plasmon resonance.

Figures 2E and 2F present immunoblots (E) and densitometric analyses corresponding thereto (F) of cell extracts from HeLa (upper panel) and T47D cells (lower panel) obtained after incubation (10 min.) with the indicated ligands (5 ng/ml) and increasing concentrations of TRAP-Fc (0, 0.8, 1.6, 3.2, 6, 12, 30, 60 μg/ml). Figure 3A shows cell proliferation (determined in hexaplicates using the MTT assay) of BxPC3 pancreatic tumor cells (2X10 ⁴ ) incubated for 5 days with increasing concentrations of TRAP-Fc.

Figure 3B exhibits cell proliferation (determined in hexaplicates using the MTT assay) of various cancer cells (2X10 ⁴ per well) incubated for 5 days with TRAP-Fc (20 μg/ml) (white bars) or without TRAP-Fc (black bars).

Figure 3C presents cell proliferation (determined in hexaplicates using the MTT assay) of H1437 lung tumor cells (black bars), PC3 prostate tumor cells (white bars) and BxPC3 pancreatic tumor cells (grey bars; 2X10 ⁴ cells per well) incubated with the indicated ligands (5 ng/ml), along with TRAP-Fc (20 μg/ml). Figures 3D and 3E show photographs (D) and no. of colonies vs. treatment (E; mean, SD, and statistically significant difference (p) determined by two-tailed Student t test) of H1437 cells seeded in agarose (top layer: 0.3% agar; bottom layer: 0.6% agar) in 6-well plates (1X10 ⁴ cells per well) and overlaid with medium treated with TRAP-Fc (added to both the soft agar and to the medium at 100 μg/ml). Figure 4 exhibits tumor volume in female nude mice (6 week old) inoculated subcutaneously with the human tumor cells (2X10 ⁶ cells per animal): BxPC3 (upper panel), H1437 (middle panel) and PC3 (lower panel) treated with vehicle (open circle), TRAP-Fc (close circle), or with a combination of anti-TGFa and anti HB-EGF mAbs (open triangles or diamonds). Figure 5A shows cellular proliferation (measured in hexaplicates using the MTT assay) of BxPC3 pancreatic tumor cells (2X10 ⁴ ) following 5 days incubation with TRAP-Fc (30 μg/ml) alone, or in combination with cetuximab (20 μg/ml), trastuzumab (20 μg/ml), panitutumab (20 μg/ml), lapatinib (0.05 nM), erlotinib (0.2 nM), CI-1033 (0.5 nM), AG1478 (0.2 nM), gefitinib (0.004 nM), or gemcitabine (at 0.5 ng/ml). Figure 5B presents Kaplan-Meier analysis of animal survival for 4 groups of female nude mice inoculated subcutaneously with BxPC3 pancreatic cancer cells and treated with TRAP-Fc (small pixels), gemcitabine (full line), a combination of TRAP-Fc with gemcitabine (broken line) or non treated (control; large pixels).

Figure 6A shows phase contrast photomicrographs of RFP (Red Fluorescence Protein) -expressing MDA-MB-231 cells (2,000 cells/well) incubated for 6 days without or with TRAP-Fc (30 μg/ml).

Figure 6B presents representative photographs of the lower part of filters through which MDA-MB-231 cells (1.5xl0 ⁵ ), incubated in Transwell chambers in the absence or presence of TRAP-Fc (30 μg/ml). Figure 6C exhibits snapshots of MCF-IOA mammary cells allowed to migrate for 24 hours after being plated on wound-healing inserts in EGF-deprived medium, treated with TRAP-Fc (100 μg/ml) and twenty- four hours later, after plugs were removed, further treated with TGFa (5 ng/ml).

Figure 7A shows proliferation of MDA-MB-231 cells (2X10 ⁴ ) after 5 days incubation with TRAP-Fc (30 μg/ml). Figure 7B shows immunoblots, with an anti-EGFR antibody, of conditioned media collected from parental cells and from MDA-MB-231 cells stably expressing the TRAP-Fc protein.

Figure 7C shows average tumor sizes and standard deviations (bars) of tumors obtained by inoculating parental MDA-MB-231 and TRAP-Fc-expressing cells (2.5xl0 ⁶ ) into the mammary fat pad of SCID mice (6 and 7 mice, respectively; lower panel) and representative blots obtained from tumors removed at the end of the experiment (day 40) from 4 mice of each group, homogenized, electrophoresed and immunoblotted with an anti- EGFR antibody. Figure 7D shows nodules (by number analysis) in the lungs of SCID mice at day 60 after receiving (by injection of 2.5xl0 ⁶ cells into their tail vein) parental MDA-MB-231 cells (control; n=13) or TRAP-Fc-expressing cells (n=9).

Figure 7E shows nodules (by number analysis) in the lungs of SCID mice treated with TRAP-Fc (100 μg per intraperitoneal injection on days 1, 3, 6 and 9; n=8).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to combination therapy comprising, as the active ingredients, a binding molecule and an additional therapeutic modality, such as, therapeutic agents, radiation therapy, or surgery , wherein the binding molecule is having binding affinity for one or more ErbB ligands. In certain embodiments, the binding molecule is a bivalent binding molecule having affinity for a first and second ErbB ligand at separate binding sites on a single amino acid chain.

The present invention is further directed to a composition comprising the binding molecule of the invention and a therapeutic agent. In addition, the present invention is directed to a kit comprising the binding molecule and one or more therapeutic agents.

Without being bound by any hypothesis or mechanism, it is assumed that by combining the ligand-binding specificities of ErbB-l/EGFR and either ErbB-3 or ErbB-4, the bivalent proteins of the invention sequester the majority, or all EGF-like ligands, thereby sensitizing a cell or a tumor to anti cancer therapeutic agent(s). The bivalent binding molecule of the invention is capable of binding ligands to multiple receptors, such as, ErbB receptors. Preferred binding molecules, also referred herein as "bivalent binding molecules", "Trap-Fc" or "double traps" are capable of binding ligands for at least two distinct receptors. In some embodiments, the binding molecules have substantial affinity for all ErbB ligands. Exemplary embodiments of binding molecules are illustrated diagrammatically in FIG. 1A or 2A. The full length ectodomain for ErbB receptors contains four sub-domains, referred to as LI, CI, L2 and C2 (also known as subdomains 1, 2, 3 and 4; or subdomain LI, CRl, L2 and CR2; or subdomains LI, SI, L2 and S2), where L and CR are acronyms for large and cys-rich respectively. Amino acid sequence alignments of the ectodomains of ErbB 1, ErbB2, ErbB3 and ErbB4 have been determined (see US 7,449,559, FIGS. 1A and IB).

According to some embodiments, the binding molecule comprises at least LI, CI and L2 subdomain of each ErbB receptor and further comprises a portion of the C2 subdomain of said receptor, on a single amino acid chain. .

According to some embodiments, the binding molecule comprises the extracellular subdomains LI, CI, L2 and a portion of C2 of ErbBl receptor and the extracellular subdomains LI, CI, L2 and a portion of C2 of ErbB3 receptor on a single amino acid chain.

According to some embodiments, the binding molecule comprises the extracellular subdomains LI, CI, L2 and a portion of C2 of ErbBl receptor having the amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 18 and the extracellular subdomains LI, CI, L2 and a portion of C2 of ErbB3 receptor having the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 11 and SEQ ID NO: 12.

According to further embodiments, the binding molecule comprises the extracellular subdomains LI, CI, L2 and a portion of C2 of ErbBl receptor and extracellular subdomains LI, CI, L2 and a portion of C2 of ErbB4 receptor on a single amino acid chain.

According to some embodiments, the binding molecule comprises the extracellular subdomains LI, CI, L2 and a portion of C2 of ErbBl receptor having the amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 18 and the extracellular subdomains LI, CI, L2 and a portion of C2 of ErbB4 receptor having the amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14. The aforementioned sub-domains of ErbB receptors are composed of smaller domains known as subregions. For example, the CI sub-domain contains 8 disulfide bonded subregions sometimes known as modules which are numbered 1-8 as they extend from the amino to carboxy terminal direction. C2 contains 7 modules numbered 1 -7. However, the inventors of the present invention found that by maintaining the native LI, CI and L2 subdomains of each ErbB receptor the affinity of the bivalent molecule and the therapeutic activity of said molecule are significantly higher compared to molecules devoid of the native CI subdomain as a whole, or the native L2 subdomain as a whole. As exemplified hereinbelow, including the LI subdomain and merely portions (certain modules) of the CI subdomain, or without including CI, results with none or poor affinity. Accordingly, each ErbB binding arm of the bivalent molecule of the invention comprise at least the native LI, CI and L2 subdomains of the corresponding native ErbB receptor and a portion of the C2 subdomain or the complete C2 subdomain.

As the bivalent binding molecules have substantial binding affinity for ligands that bind distinct receptors, these molecules can include portions of the ectodomains of receptors preferably covalently joined in a single amino acid sequence. In instances where the spectrum of ligands bound by two receptors overlap, each binding moiety of the bivalent binding molecule made from portions of those receptors may bind similar or identical ligands.

The bivalent binding molecule may be soluble in aqueous solutions. As such, each binding moiety of the bivalent binding molecule can be a soluble portion containing extracellular domain of a receptor.

The binding molecule may encompass any suitable receptor according to the principles of the invention. Thus, the terms "suitable receptors" as used herein generally refers to the various family members of ErbB receptors. Thus, the bivalent binding molecule may encompass a combination of the extracellular ligand binding domains of ErbB receptors or portions thereof, for example ErbBl and ErbB3, ErbBl and ErbB4 or other combinations. The binding domains can exist in any order on the amino acid chain so long as suitable binding affinity for receptor ligands is maintained.

The terms "an extracellular ligand binding domain of an ErbB receptor that binds a ligand to the ErbB receptor" and "a portion of an extracellular domain of an ErbB receptor that binds a ligand to the ErbB receptor" as used herein, are interchangeable, and refer to the extracellular ligand binding domains of ErbB receptors, wherein each extracellular ligand binding domain includes at least the LI, CI, L2 and a portion of C2 subdomains of ErbB receptor. For example, SEQ ID NOs: 1, 5, 10 and 18 among others with respect to ErbBl receptor; SEQ ID NO: 2, 11 and 12 among others with respect to ErbB3 receptor ; and SEQ ID NOs: 3, 4, 13 and 14 among others with respect to ErbB4 receptor; .

The term "binding affinities" as used herein refers to affinities that are sufficient to trap, otherwise bind, ErbB ligands in a physiological matrix. Preferably, dissociation constants will be no higher than about 100-fold to about 1,000-fold above the dissociation constants of the native receptors. More preferably, dissociation constants in the nanomolar range or lower are preferred. Nevertheless, any affinity that is sufficient to bind and trap ErbB ligands thereby preventing or interfering with their binding to ErbB receptors are suitable for use in the disclosed compositions and can find use in the disclosed methods.

Ligands and receptor binding specificity for the ErbB receptors is listed below in Table

1.

Table 1 - Ligands and receptor binding specificity for the ErbB receptors

The complete nucleotide sequences and amino acid sequences of the ErbBl, ErbB2, ErbB3 and ErbB4 are known in the art and can be found in Genbank as accession #: NM_005228 for ErbBl, accession # NM_004448 for ErbB2, accession #: M29366 or NM 001982 for ErbB3, and accession #: NM 005235 for ErbB4. For purposes of this specification a full length EGFR ectodomain refers to the ectodomain consisting of amino acid residues 1-621 of ErbB 1 (SEQ NID NO: 19) or equivalent residues of other members of the EGF receptor family. The amino acid sequence of the full length ectodomains for the ErbB receptor family is also known, portions of these sequences include but are not limited to the sequences listed below:

SEQ ID NO. 1 for ErbBl amino acid residues 24-524,

SEQ ID NO. 2 for ErbB3 amino acid residues 19-518,

SEQ ID NO. 3 for ErbB4 amino acid residues 25-521,

SEQ ID NO. 4 for ErbB4 amino acid residues 26-553,

SEQ ID NO. 5 for ErbBl amino acid residues 25-556,

SEQ ID NO. 10 for ErbBl amino acid residues 1-532,

SEQ ID NO. 11 for ErbB3 amino acid residues 1-499,

SEQ ID NO. 12 for ErbB3 amino acid residues 1-531,

SEQ ID NO. 13 for ErbB4 amino acid 1-496,

SEQ ID NO. 14 for ErbB4 amino acid residues 1-528, and

SEQ ID NO. 18 for ErbBl amino acid residues 1-500, among others. Corresponding nucleotide sequences that encode these amino acids are also known. The full length ectodomain for ErbB receptors contains four sub-domains, referred to as LI, CR1, L2 and CR2, where L and CR are acronyms for large and cys-rich respectively. Amino acid sequence alignments of the ectodomains of ErbBl, ErbB2, ErbB3 and ErbB4 have been determined (see US 7,449,559, FIGS. 1A and IB).

The CR2 sub-domain of ErbB receptors is thought to link the ligand binding domain (LI, CR1 and L2) with the membrane spanning region and consists of seven additional modules which are joined by linkers of 2 or 3 amino acid residues and bounded by cysteine residues. For ErbBl these modules extend from amino acid positions 482-499, 502-511, 515- 531, 534-555, 558-567, 571-593, and 596-612 for modules 1-7, respectively. For ErbB2 these modules extend from 490-507, 510-519, 523-539, 542-563, 566-575, 579-602 and 605-621 for modules 1-7, respectively. For ErbB3 481-498, 501-510, 514-530, 533-554, 557-566, 570-591, and 594-610 for modules 1-7, respectively. For ErbB4 these modules extend from 478-495, 498-507, 511-527, 530-552, 555-564, 568-589, and 592-608 for modules 1-7, respectively.

In some embodiments the amino acid sequence of one or both of the binding moieties may be modified provided that the modification does not adversely affect the binding affinity of the binding moiety for its ligand(s). For example, modified binding moieties may be constructed by making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for binding activity. Generally, substitutions should be made conservatively; for example, the most preferred substitute amino acids are those having physiochemical characteristics resembling those of the residue to be replaced. Similarly, when a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity should be considered. In order to preserve the biological activity of the binding moieties, deletions and substitutions will preferably result in homologous or conservatively substituted sequences, meaning that a given residue is replaced by a biologically similar residue. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, Met or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Moreover, particular amino acid differences between human, murine or other mammalian EGFRs is suggestive of additional conservative substitutions that may be made in ErbB binding moieties without altering the essential biological characteristics of the binding moiety.

In some embodiments the bivalent binding molecules is arranged according to one of the following motifs: B-L-B-F; B-L-rB-F and B-F-B where B represents a binding moiety which can originate from a receptor. The binding moieties can be the same or different. rB represents a binding moiety in which the amino acid sequence is reversed such that the amino-terminal amino acids become the carboxy-terminal residues. An exemplary sequence for ErbBl is SEQ ID NO.: 15 which is a nucleotide sequence encoding one such reverse sequence to provide an amino sequence which is the reverse of the sequence in SEQ. ID NO.: 10. Similar inversions can be constructed for ErbB3 and ErbB4, as desired. Such reverse sequences can be positioned as the carboxy-terminal binding moiety to mimic the structure of receptors as they are found in the membrane. With respect to ErbBl, amino acids 24-524, 25-556, 1-500 and 1-532 [SEQ ID NOS. 1, 5, 18 and 10, respectively] can be used to form an active binding molecule; with respect to ErbB3, amino acids 19-518, 1-499 and 1-5312 [SEQ ID NOS. 2, 11 and 12, respectively] can be used to form an active binding molecule; and with respect to ErbB4 amino acids 25-521, 26-553, 1-496 and 1-528 [SEQ ID NOS. 3, 4, 13 and 14, respectively] can be used such that when ErbBl and ERbB3 or ErbB4 are joined in a single amino acid chain they form a bivalent binding molecule having a substantial affinity for both ErbBl and ErbB3 or ErbB4 ligands regardless of whether ErbB 1 is positioned on the amino or carboxy-terminal side of ErbB3 or ErbB4. "L" is an optional linker moiety which can be used to join binding moieties. Many suitable linker molecules are known and can be used. Preferably, the linker will be non- immunogenic. For linkers and methods of identifying desirable linkers, see, for example, George et al. (2003) Protein Engineering 15:871-879, herein specifically incorporated by reference. A linker sequence may include one or more amino acids naturally connected to a binding moiety and can be added to provide specifically desired sites of interest, allow component domains to form optimal tertiary structures and/or to enhance the interaction of a component with its target molecule. One simple linker is (Gly ₄ Ser)x wherein "X" can be any number from 1 to about 10 or more in certain embodiment linkers wherein "X" is three [SEQ ID NO. 6]. "F" is an optional fusion partner and can be any component that enhances the functionality of the bivalent binding molecule. Suitable fusion partners may enhance the biological activity of the bivalent binding molecule, aid in its production and/or recovery, or enhance a pharmacological property or the pharmacokinetic profile of the fusion polypeptide by, for example, enhancing its serum half-life, tissue penetrability, lack of immungenicity, or stability.

For a fusion partner which is a serum protein or fragment thereof, the fusion partner may be can be a- 1 -microglobulin, AGP-1, orosomuciod, a-acid glycoprotein, vitamin D binding protein (DBP), hemopexin, human serum albumin (hSA), transferrin, ferritin, afamin, haptoglobin, a-fetoprotein thyroglobulin, a-2-HS-glycoprotein, β-2-glycoprotein, hyaluronan-binding protein, syntaxin, C1R, Clq a chain, galectin3-Mac2 binding protein, fibrinogen, polymeric Ig receptor (PIGR), α-2-macroglobulin, urea transport protein, haptoglobin, IGFBPs, macrophage scavenger receptors, fibronectin, giantin, Fc (especially including an IgG Fc domain), α-1-antichyromotrypsin, a- 1 -antitrypsin, antithrombin III, apolipoprotein A-I, apolipoprotein B, β-2-microglobulin, ceruloplasmin, complement component C3 or C4, CI esterase inhibitor, C-reactive protein, cystatin C, and protein C. The inclusion of a fusion partner component may extend the serum half-life of the fusion polypeptide of the invention when desired.

Based on the ligands and receptor binding specificity for the ErbB receptors shown in Table 1, combination of an ErbBl and ErbB3 binding moiety can be used to create a bivalent binding molecule with specificity for EGF, TGFa, HB-EGF, Betacellulin, Amphiregulin, Epiregulin, Epigen, Neuregulin la, Neuregulin 1 β, Neuregulin 2a and Neuregulin 2β. The combination of binding domains for ErbBl and ErbB4 have binding affinity for EGF, TGFa, HB-EGF, Betacellulin, Amphiregulin, Epiregulin, Epigen, Neuregulin la, Neuregulin 1β, Neuregulin 2a, Neuregulin 2β, Neuregulin 3 and Neuregulin 4, which includes all of the known ErbB ligands. Bivalent binding molecules may generally include signal sequences at their amino terminal ends. Any suitable signal sequence, of which many are known, can be used. For example, the ErbB ectodomain in the first position of the bivalent binding molecule can contain its own native signal peptide. Alternatively, that signal peptide can be modified to consist the amino acid sequence CTC GAG ATGG (SEQ ID NO. 17). Additional signal sequences include, but are not limited to, a signal peptide consisting of the amino acid sequence as set forth in SEQ ID NO: 7 and encoded by the nucleotide sequence as set forth in SEQ ID NO:20 or any other signal sequences disclosed in US 2009/0318346.

According to a further embodiment, the binding molecule comprises the signal peptide as set forth in SEQ ID NO: 7, followed by a first binding site comprising a portion of the ErbB4 receptor, consisting of SEQ ID NO: 4, a second binding site comprising a portion of the ErbBl receptor, consisting of SEQ ID NO: 5 and an Fc domain of immunoglobulin G2 consisting of SEQ ID NO: 8, wherein the first and second binding sites are linked with a (Gly4Ser)3 linker consisting of SEQ ID NO: 6. According to a certain embodiment, the binding molecule consists of the amino acid sequence as set forth in SEQ ID NO: 9. The bivalent binding molecules is prepared from amino acid sequences expressed from recombinant DNA molecules. As indicated above, the recombinant DNA molecule can include a first nucleotide sequence encoding a portion of a first receptor protein and a second nucleotide sequence encoding a portion of a second receptor protein. The receptor proteins can be the same or different. It is noted that a bivalent binding molecule encompassing different receptor proteins will bind a broader spectrum of binding molecules. In such cases the first and second receptor proteins are generally encoded from different genes.

Nucleotide sequences that encode the bivalent binding moieties, optional linker and an optional fusion partner can be cloned into a recombinant DNA construct in an arrangement with transcription and translation sequences such that the bivalent binding molecule can be expressed as a single polypeptide chain in a suitable host. Any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding the fusion polypeptides of the invention under control of transcriptional/translational control signals. It is well within the skills of one having skill in the art to select transcription and translation sequences that can be used to express genes in suitable hosts. Any host cell that can produce the disclosed molecules from their recombinant genes can be used. Suitable host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. In many circumstances receptors are glycosylated and glycosylation can influence ligand binding. Thus, the selection of a host can depend on the glycosylation pattern generated by the host cell. Any host cell that can produce ligand binding molecules with suitable binding affinities can be used. In the case of an ErbB -containing binding molecule a mammalian host cell can be used for example and, more specifically CHO cells, for example.

Many suitable promoter and enhancer elements are known in the art. Promoters that may be used to control expression of the chimeric polypeptide molecules include, but are not limited to, a long terminal repeats; SV40 early promoter region, CMV, M-MuLV, thymidine kinase promoter, the regulatory sequences of the metallothionine gene; prokaryotic expression vectors such as the β-lactamase promoter, or the tac promoter; promoter elements from yeast or other fungi such as Gal 4 promoter, ADH, PGK, alkaline phosphatase, and tissue-specific transcriptional control regions derived from genes such as elastase I.

The bivalent binding molecules may be purified by any technique which allows for stable bivalent binding of the resulting double trap molecules. For example, the bivalent binding molecules may be recovered from cells either as soluble proteins or as inclusion bodies, from which they may be extracted quantitatively by 8M guanidinium hydrochloride and dialysis, as is known. Alternatively, the bivalent binding molecules, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration may be used. Affinity techniques that utilize immobilized ligands or ligand mimetics can also be used.

Binding affinity and inhibitor potency of the bivalent binding molecules can be measured for candidate truncated ectodomains using biosensor technology or by classic binding assays such as ELISA which are well known in the art.

The bivalent binding molecules of the invention are used in combination therapies. Advantageously, combining the bivalent molecule with additional therapeutic agents, specifically, anti cancer drugs, bolsters the therapeutic effect synergistically. Particularly, as exemplified herein for the first time, using the bivalent binding molecule in combination with various anticancer agents prolongs survival in vivo of animal models for cancer. Combination therapy according to the present invention includes administering a single pharmaceutical dosage formulation containing all active components, namely, the bivalent binding molecule and the at least one therapeutic agent. Combination therapy according to the present invention also includes administering the bivalent binding molecule and the at least one therapeutic agent, each in its own separate pharmaceutical dosage formulation. The bivalent binding molecule and the therapeutic agent may be administered to a patient together, substantially concomitantly. For example, a dosage form with the bivalent trap is administered immediately before a dosage form with the anti-cancer therapeutic agent is similarly administered. Alternatively, according to another embodiment of the present invention, the bivalent binding molecule and the therapeutic agent may be administered sequentially. For example, a dosage form with the bivalent protein is administered in the morning and another dosage form with one or more the at least one therapeutic agent is administered in the evening or vise versus.

Use of the binding molecule of the invention for treating a disease associated with one or more ErbB ligands, includes combining the binding molecule with therapy, such as, a therapeutic agent. The therapeutic agent may be an anticancer drug, including, a chemotherapeutic agent, tyrosine kinase inhibitor and targeted therapy agent among other types of anticancer drugs. Chemotherapy refers to the treatment of cancer with antineoplastic drugs which affect cell division or DNA synthesis and/or function in some way. Since chemotherapy acts by killing cells that divide rapidly it also harms cells that divide rapidly under normal circumstances, such as, cells in the bone marrow, digestive tract and hair follicles. Most chemotherapeutic drugs fall under one of the following categories: alkylating agents, antimetabolites, anthracyclines, hormone treatments, plant alkaloids and topoisomerase inhibitors. The group of alkylating agents is named so because of the ability to alkylate many nucleophilic functional groups in cells. This group includes, but is not limited to, cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide.

Anti-metabolites prevent purines and pyrimidines from becoming incorporated in to DNA during the "S" phase of the cell cycle, thereby stopping cell development and division. This group of drugs also affects RNA synthesis.

The group of plant alkaloids and terpenoids refers to alkaloids derived from plants that block cell division by preventing microtubule function. Examples of plant alkaloids and terpenoids are vinca alkaloids and taxanes, such as, paclitaxel (Taxol®) and docetaxel.

Topoisomerase inhibitors refer to inhibitors of the topoisomerase enzyme, which are essential for maintaining the topology of DNA. Inhibition of type I or type II topoisomerases interferes with both transcription and replication of DNA by upsetting proper DNA supercoiling. Type I topoisomerase inhibitors include camptothecins, e.g. irinotecan and topotecan. Examples of type II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.

Cytotoxic antibiotics include, but are not limited to, the following antibiotics: actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin and mitomycin.

The therapeutic agent according to the principles of the present invention may be an agent suitable for targeted therapy. Targeted therapy refers to therapy that blocks the growth of cancer cells by interfering with specific targeted molecules needed for carcinogenesis and tumor growth, rather than interfering with rapidly dividing cells. Therefore, targeted cancer therapies may be more effective than chemotherapy and less harmful to normal cells. Typically, agents for targeted therapy are monoclonal antibodies, such as, rituximab (MabThera® or Rituxan®), which targets CD20 on B cells and used in non Hodgkin lymphoma; trastuzumab (Herceptin®) that targets the Her2/neu (ErbB2) receptor expressed in some types of breast cancer; cetuximab (Erbitux®) which targets the epidermal growth factor receptor and is used in the treatment of colon cancer and non-small cell lung cancer; bevacizumab (Avastin®) that targets circulating VEGF ligand and is used for the treatment of colon cancer, breast cancer, non- small cell lung cancer, sarcoma and brain tumors.

The tyrosine-kinase inhibitors (TKIs) are agents that inhibit tyrosine kinases, the enzymes responsible for the activation of signal transduction cascades through phosphorylation of various proteins. This family of compounds includes imatinib mesylate (Gleevec®, or STI-571®) that is approved for chronic myelogenous leukemia, gastrointestinal stromal tumor and some other types of cancer; gefitinib (Iressa® or ZD 1839®), that targets the epidermal growth factor receptor (EGFR) tyrosine kinase and is used in non small cell lung cancer; erlotinib (Tarceva®) that was shown to increase survival in metastatic non small cell lung cancer when used as second line therapy; bortezomib (Velcade®) that is approved for treating multiple myeloma that has not responded to other treatments; and apatinib, a selective VEGF Receptor 2 inhibitor among others.

According to some embodiments, the at least one therapeutic agent in the combined therapy and pharmaceutical composition of the invention is selected from the group consisting of: erlotinib, imatinib mesylate, gefitinib, lapatinib, CI-1033, AG1478, gemcitabine, cetuximab, trastuzumab (Herceptin ^® ), anti-ErbB3 monoclonal antibody, bevacizumab, docetaxel and panitumumab. Each possibility represents a separate embodiment of the present invention.

Where separate dosage formulations are used, the binding molecules of the invention and one or more additional therapeutic agents can be administered concurrently, or at separately staggered times, i.e., sequentially.

Therapy may also include other therapeutic modalities, such as, radiation therapy, organ transplantation, or surgery intended for removal of the cells, tissue, part of an organ or an organ, in order to treat the disease. The present invention also provides pharmaceutical compositions comprising a bivalent binding molecule and at least one therapeutic agent. Such compositions comprise a therapeutically effective amount of a bivalent binding molecule, a therapeutically effective amount of the at least one therapeutic agent and a pharmaceutically acceptable carrier.

The terms "pharmaceutical composition," "formulation" and "dosage form" are used herein interchangeably to encompass formulated preparations comprising the pharmacologically active ingredients, and one or more pharmaceutically acceptable excipients, diluents or carriers. Compositions, formulations and dosage forms can be designed for administration by all possible administration routes to achieve the desired therapeutic response. The terms used may refer to the physical format of the product which is dispensed and administered to the patient, for example, a capsule or a patch. Alternately or in addition, the terms used may refer to any of: the mode of administration, the mode of delivery or the mode of release of the drug, for example a transdermal delayed release formulation.

The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations and the like. Pharmaceutically acceptable carriers include other ingredients for use in formulations such as DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing the protein or analog), dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation. Pharmaceutically acceptable diluents include buffers having various contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., TWEED™80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations. Liposome, microcapsule or microsphere, inclusion complexes, or other types of carriers are also contemplated. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof. Therapeutic formulations suitable for oral administration, e.g. tablets and pills, may be obtained by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by mixing the constituent(s), and compressing this mixture in a suitable apparatus into tablets having a suitable size. A tablet may be coated or uncoated. An uncoated tablet may be scored. A coated tablet may be coated with sugar, shellac, film or other enteric coating agents. Each possibility represents a separate embodiment of the present invention.

Therapeutic formulations suitable for parenteral administration include sterile solutions or suspensions of the active constituents. An aqueous or oily carrier may be used. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soy bean oil, mineral oil, sesame oil and the like. Formulations for parenteral administration also include a lyophilized powder comprising all or part of the active ingredients and, optionally, further active constituents, that is to be reconstituted by dissolving in a pharmaceutically acceptable carrier that dissolves the active constituents, e.g. an aqueous solution of carboxymethylcellulose and lauryl sulphate. Each possibility represents a separate embodiment of the present invention.

The compounds of the present invention may be formulated into injections by dissolving, suspending or emulsifying the active ingredients in water-soluble solvent such as saline and 5% dextrose, or in water-insoluble solvents such as vegetable oils, synthetic fatty acid glyceride, higher fatty acid esters and propylene glycol. The formulations of the invention may include any of conventional additives such as dissolving agents, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

When the pharmaceutical composition is a capsule, it may contain a liquid carrier, such as fatty oil, e.g. cacao butter.

Thus, the pharmaceutical composition may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Each possibility represents a separate embodiment of the present invention.

The formulation of the invention optionally further comprises a flavoring agent. The flavoring agent may be selected from the group consisting of synthetic or natural oil of peppermint, oil of spearmint, citrus oil, fruit flavors, sweeteners (sugars, aspartame, saccharin, Estevia, etc.), and mixtures thereof. Menthol can also act as a flavoring agent.

The therapeutic ingredients may be delivered in a controlled release system. The term "controlled release" is used herein to refer to a pharmaceutical dosage form in which release of the active ingredient is timed or modified to a rate sufficient to maintain the desired therapeutic level over an extended period of time. The release may be a "sustained release" or a "delayed release" such that release of the active ingredient from the pharmaceutical dosage form is other than promptly after administration of the dosage form, but rather is withheld or delayed following administration.

In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the central nervous system, thus requiring only a fraction of the systemic dose. Other controlled release systems are discussed, for example, in U.S. Patent No. 5,120,548 which is directed a controlled-release drug delivery device comprised of swellable polymers. U.S. Patent No. 5,073,543 also describes controlled-release formulations containing a trophic factor entrapped by a ganglioside-liposome vehicle. U.S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer. Biodegradable microparticles are also known for use in controlled-release formulations. U.S. Patent No. 5,733,566 describes the use of polymeric microparticles that release antiparasitic compositions.

The controlled-release of the active ingredient may be stimulated by various inducers, for example, pH, temperature, enzymes, water, or other physiological conditions or compounds.

The amount of the active bivalent binding molecule and the therapeutic agent that will be effective for its intended therapeutic use can be determined by standard clinical techniques based on the present description. Preferably, due to the synergistic effect rendered by combining the bivalent molecule and the therapy agent, the effective amounts of each of these active agents are lower than the effective amounts of each agent when administered alone. In vitro assays may be employed to help identify optimal dosage ranges. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the active per kilogram of body weight, preferably 0.1-150 micrograms per kilogram. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Dosage amount and interval may be adjusted individually to provide plasma levels of the compounds that are sufficient to maintain therapeutic effect. In cases of local administration or selective uptake, the effective local concentration of the compounds may not be related to plasma concentration. The dosage regimen involved in a method for treatment will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of disease, time of administration and other clinical factors.

The amount of each active compound administered will be also dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician. The combined therapy may be repeated intermittently while symptoms are detectable or even when they are not detectable.

The methods of the invention are directed to treating a disease associated with ErbB ligands in a patient in need thereof and inhibiting cell proliferation in a subject in need thereof comprising administering to said subject a binding molecule having binding affinity for at least one ErbB ligand, or for a first and a second ErbB ligand at separate binding sites on a single amino acid chain, in combination with therapy. The method of the invention are also directed to preventing a disease associated with ErbB ligands in a patient in need thereof comprising administering to said subject a binding molecule having binding affinity for at least one ErbB ligand, or for a first and a second ErbB ligand at separate binding sites on a single amino acid chain. A disease associated with ErbB ligand(s) refers to any disease that may be cured or prevented by inhibiting the activity, or the activity induced by, one or more ErbB ligands. Diseases associated with ErbB ligand(s) include, but are not limited to, atherosclrosis, malignant diseases, cardiovascular diseases or disorders, skin disorders, coronary diseases and disorders, psoriasis and neurodegenerative diseases or disorders. The method of the invention are also directed to preventing or treating cosmetic disorders, comprising administering to a subject a binding molecule having binding affinity for at least one ErbB ligand, or for a first and a second ErbB ligand at separate binding sites on a single amino acid chain, alone, or in combination with therapy.

As used herein, the term "treating" encompasses substantially ameliorating, relieving, alleviating and preventing symptoms of the disease in a patient in need thereof.

The term "preventing" encompasses delaying, or inhibiting the onset of a disease. This term also refers to preventing the recurrence of a disease, following treatment, in a patient in need thereof.

A patient in need of preventing the disease may be a healthy individual with a risk of having the disease. The risk is usually a statistical risk based on hereditary, familial, environmental and/or genetic information.

A patient in need of preventing the recurrence of the disease is typically a patient in the stage of remission post treatment for said disease. For this type of patient, there remains a statistical risk of relapse. As used herein, a "malignant disease" or "malignancy" refer a severe and progressively worsening disease, typically, cancer, and also describes the tendency of a medical condition, especially tumors, malignant rather than benign, to become progressively worse and to potentially result in death. Malignancy in cancers is characterized by anaplasia, invasiveness, and metastasis. A malignant tumor is not self-limited in its growth but is capable of invading into adjacent tissues, and may be capable of spreading to distant tissues (metastasizing). Examples of malignant disease to be treated according to the principles of the present invention, include breast cancer, non Hodgkin lymphoma, non-small cell lung cancer, colon cancer, sarcoma, brain tumors, leukemia, gastrointestinal stromal tumor, multiple myeloma, bladder cancer, prostate cancer, lung cancer, colorectal cancer, head and neck cancer, ovarian cancer, cervical cancer and pancreatic cancer.

As used herein, the term "administering" refers to delivery of a pharmaceutical compound to a subject by any means that does not affect the ability of the compound to perform its intended function. Specifically, the methods of the invention are directed to coadministering, concomitantly or in sequence, the binding molecule of the invention together with an additional therapeutic agent.

According to some embodiments, the route of administration is selected from the group consisting of: oral, buccal, sublingual, transdermal, transmucosal, intranasal, intratracheal, intravenous ( .v.), intraperitoneal (i.p.), intramuscular (i.m.), subcutaneous (s.c.) or intrathecal (i.t). According to a particular embodiment, the intratracheal administration refers to introduction of aerosols comprising the binding molecule of the invention and/or the additional therapeutic agent directly through the endotracheal tube.

For buccal administration, buccal tablets or sublingual tablets may be used. These tablets are typically small, flat and soft, designed to be placed in the side of the cheek (i.e. buccal cavity) or designed to be placed under the tongue, to be directly absorbed through the buccal mucosa for a systemic effect. Other dosage forms suitable for buccal administration are, for example, oral films administered on the gyngiva or tongue.

Sublingual spray is also a buccal formulation for delivery to the sublingual mucosa in the form of a spray for a systemic effect, typically provided in spray actuators, designed to access the mucosal surfaces under the tongue or the lips. For transdermal delivery of the composition of the invention, the composition may be provided in the form of a patch. The major approaches for transdermal delivery include use of chemical penetration enhancers; physical enhancers, such as ultrasound, iontophoresis, electroporation, magnetophoresis, and microneedles; vesicles; particulate systems, such as those incorporating liposomes, niosomes, transfersomes, microemulsions, or solid lipid nanoparticles, as described for example in Rizwan et al., Recent Pat Drug Deliv Formul., 2009, 3(2): 105-24. A method for treating a patient in need of treatment may include pretreating the patient's blood ex vivo by contacting the patient's blood or serum with the binding molecule thereby removing a portion of the ErbB ligands from the serum. For this purpose the binding molecule may be immobilized onto a solid support, such as, an apheresis or biocore support by standard methods. When the binding molecule is immobilized to a solid support the serum or blood of the patient can be placed in contact with the solid support in the apheresis column to remove a portion of the ErbB ligand from the blood. Thereafter, the pretreated blood or serum is transfused back to the patient. Following transfusion, the patient receives anti cancer therapy. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with the active ingredients of the pharmaceutical compositions of the invention, specifically, the binding molecule and the therapeutic agent(s). Optionally associated with such container(s) can be a notice in the form described by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

According to some embodiments, the kit for treating a disease associated with an ErbB ligand in a subject in need thereof, comprises a pharmaceutical composition comprising a binding molecule having binding affinity for at least one ErbB ligand and at least one therapeutic agent.

The kit may further comprise at least two containers, as follows: (a) a first container comprising the pharmaceutical composition; and (b) a second container comprising the at least one therapeutic agent. The kit may further comprise instructions for using said kit.

The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention. EXAMPLES

Example 1. Construction, Expression and Function of TRAP-His Fusion Proteins

Four recombinant constructs, named TRAP-His, were designed. The resulting constructs combined a signal peptide (SP; SEQ ID NO: 7) flanked by the ligand-binding domains of EGFR/ErbB-1 called domains I, II, III and a part of domain IV, a linker (Gly ₄ Ser) ₃ (SEQ ID NO: 6) followed by the respective domains of ErbB-3 or ErbB-4, and a six-histidine tag (HisX6) (Fig. 1A). The resulting constructs were stably expressed in HEK- 293 cells. Immunoblot analyses detected three of the four fusion proteins in the medium of the transfected cells (Fig. IB), and similar analysis of cell lysates revealed that TRAP-His 1-4 that was not detected in the medium, was instead detected in the cytoplasm.

Covalent cross-linking of radioactive EGF or NRGl-β to the TRAP-His molecules validated retention of ligand binding specificities. All TRAP-His molecules, as well as IgB-1, a molecule comprising the full-length extracellular domain of ErbB-l/EGFR fused to the Fc portion of human immunoglobulin (Chen et al, J Biol Chem., 271 :7620-7629, 1996), could bind EGF, and unlabeled EGF prevented this binding. Likewise, TRAP-His proteins 1-3 and 3-1 underwent specific cross-linking to NRG1 (Fig. 1C).

Binding of EGF to the TRAP-His molecules was supported by another experiment that assayed cellular uptake of the radioactive ligand. HeLa cells were incubated with ¹²⁵ I- EGF, washed, lysed and the amount of ligand that underwent uptake by the cells was determined. The results obtained clearly indicated that all secreted TRAP-His molecules decreased uptake of ¹²⁵ I-EGF, in line with an ability to sequester ligands (Fig. ID).

TRAP-Fc also inhibits ligand-induced phosphorylation of EGFR and ErbB-3 by all ligands. As shown in Figure IE, HeLa cells (derived from human cervical cancer) and T47D cells (derived from human breast cancer) were seeded in 24-well plates and allowed to adhere overnight. Twenty-four hours later cells were washed and incubated with the ligands indicated in the figure (5 ng/ml), in the absence or presence of the indicated fusion proteins (each at 60 μg/ml). Following a 10-min long incubation, the cells were lysed, and cleared extracts immunoblotted (IB) with an anti-phosphotyrosine antibody. All ligands are from human origin, except for two selected ligands that are from rodents origin: mTGFa and mNRGl . Example 2. Construction, Expression and Functional Tests of TRAP-Fc protein

The His tag was replaced with the Fc domain of human immunoglobulin 2 (Fig.2A). In addition, as ErbB-4 binds more ligands than ErbB-3 and with higher affinity, the successfully secreted ErbB-4-ErbB-l configuration was used in subsequent studies. The recombinant TRAP-Fc structure combined a signal peptide having the amino acid sequence as set forth in SEQ ID NO: 7 which is encoded by the nucleotide sequence as set forth in SEQ ID NO: 20, flanked by domains I-III, along with a portion of domain IV of ErbB-4, followed by a (Gly ₄ Ser) ₃ linker domain, and the corresponding part of EGFR (Fig. 2A). The resulting molecule consist of the amino acid sequence as set forth in SEQ ID NO: 9. First, secretion of TRAP-Fc from transfected HEK-293 human kidney cells was verified. Under reducing conditions, a single immunoreactive species was detectable in the medium (Fig. 2B; R). By contrast, under non-reducing conditions (Fig. 2B; NR) a larger, major immunoreactive species was detected, confirming that the secreted TRAP-Fc represents a disulfide linked dimer molecule. The binding capability of TRAP-Fc to TGFa, HB-EGF and NRG1 ligands was evaluated using an Enzyme Linked Immunosorbent Assay (ELISA). Briefly, Ninety- six-well plates were coated with solutions of the recombinant proteins (8 μg/ml) by incubating overnight at room temperature. Plates were washed, blocked for lh with saline containing 1% albumin and 0.05% Tween-20, followed by 2-h incubation with increasing concentrations of the indicated ligands. After washing, the plates were incubated for 2h with avidin-bound antibody that targeted a ligand epitope, followed by 20 min incubation with biotin- horseradish peroxidase. Subsequently, the plates were incubated with ATBS (Sigma). Signals were determined using an ELISA reader (420 nm). It was confirmed that the IgBs (namely IgB-1, IgB-3 and IgB-4) that have binding affinity to the TGFa, HB-EGF and NRG1 ligands can bind the cognate ligands with affinities similar to those of the intact receptors (Chen et al., ibid), but TRAP-Fc was able to bind all three ligands with apparent affinities similar to or better than the respective IgB molecules (Fig. 2C; TRAP-Fc, full squares; IgB-1, diamonds; IgB-3, empty squares and IgB-4, crosses).

The binding affinities of TRAP-Fc to TGFa, HB-EGF and NRG1 ligands were evaluated using plasmon resonance measurements. Solutions containing the TRAP-Fc (1-100 nM) were passed over surfaces coated with TGFa, HB-EGF or NRG1 ligands to derive dissociation constants. Direct determination of binding affinities by surface plasmon resonance (SPR) demonstrated high affinity interactions of the TRAP-Fc with ligands that bind EGFR/ErbB-1 and ErbB-3/4 (Fig. 2D). The affinities were in agreement with previous reports. Next, the ability of TRAP-Fc to inhibit ligand-induced receptor phosphorylation was examined using either HeLa cells (and TGFa) or, T47D mammary cells (and NRG1). As shown in Figures 2E and 2F, increasing concentrations of TRAP-Fc gradually reduced ligand-induced phosphorylation levels. This observation was extended to additional EGF-like ligands from human and rodent origins (Fig. IE). Altogether, the results confirmed the ability of TRAP-Fc to effectively sequester multiple growth factors with efficiencies and specificities similar to those displayed by the full-length ectodomains of ErbB-l/EGFR and ErbB-4.

Example 3. TRAP-Fc Inhibits Cancer Cell Proliferation Dose-dependent inhibition of proliferation, up to 40%, was observed when BxPC3 human pancreatic cells were incubated with increasing concentrations of TRAP-Fc (Fig. 3 A). Similar or smaller inhibitory effects were observed with six additional cancer cell lines of mammary, prostate, lung and pancreatic origins (Fig. 3B). Inhibition was determined by comparing the control/non-treated cells (Fig. 3B; black bars) to cells treated (incubated) with TRAP-Fc (Fig. 3B; white bars).

In addition, TRAP-Fc inhibited proliferation of lung (H1437), pancreatic (BxPC3) and PC3 cells, which were exposed to exogenously added ligands (i.e., NRG1, HB-EGF, TGFa and EGF; Fig. 3C). Similarly, anchorage-independent growth of H1437 lung cancer cells was dramatically decreased in the presence of TRAP-Fc (Figs. 3D and 3E). In conclusion, the recombinant TRAP-Fc molecule can inhibit growth of carcinoma cells under both autocrine and paracrine settings.

Example 4. Anti-tumorigenic activities of TRAP-Fc in animal models

The ability of TRAP-Fc to inhibit growth of a wide spectrum of cultured carcinoma cells predicted an anti-tumorigenic activity in animals. This was assessed using human pancreatic (BxPC3), lung (H1437) and prostate (PC3) xenograft models (Figure 4). Female nude mice (6 week old) were inoculated subcutaneously with the human tumor cells BxPC3 (upper panel), H1437 (middle panel) and PC3 (lower panel), 2X10 ⁶ cells per animal. Once tumors became palpable, mice were randomized into three groups and injected intraperitoneally with vehicle (open circle), TRAP-Fc (100 μg; Fig. 4, closed circles), or with a combination of anti- TGFa and anti HB-EGF mAbs (each at 250 μg/mouse; Fig. 4, open diamond in BxPC3 graph and open triangle in H1437 graph). Mice bearing BxPC3 xenografts were injected on days: 9, 16, 20, 23, 26, 30, 33, 37, 40 and 44. Both the control and the treatment group included 8 mice. Tumors of two mice of the treatment group completely disappeared but these cases of total regression were excluded from the statistical analyses. The mAb group included 3 mice. Mice bearing H1437 xerografts were injected on days 6, 10, 14, 18 and 21. The control group included 15 mice, the TRAP-Fc-treated group included 7 mice and the mAb group included 11 mice. Mice bearing PC3 xenografts were injected on days 1, 3, 6, 9, 13, 17, 20, 23, 27 and 30. The control group included 11 mice and the TRAP-Fc-treated group included 10 mice (two of them lost their tumors and are not represented). As can be seen in Figure 4, treatment with the combination of antibodies partially inhibited tumorigenic growth of BxPC3 and H1437 cells, while the inhibitory effect obtained for treatment with the TRAP-Fc recombinant (Fig. 4; black circles) protein was significantly greater. Moreover, two of the eight BxPC3 -treated mice and two of the ten treated PC3 mice completely lost their tumors, but these cases of total regression were excluded from the statistical analyses. Advantageously, no significant weight loss was observed in these experiments. In conclusion, the TRAP-Fc protein is endowed with an anti-tumorigenic activity towards different human xenograft models.

Example 5. TRAP-Fc Sensitizes Tumor Cells to Therapeutic Agents

Self-produced growth factors may play essential roles in acquired resistance to chemotherapeutics and targeted therapies. Hence, blocking such autocrine loops may re- sensitize tumors to specific drugs. As an initial test of this scenario the combined effect of TRAP-Fc and each of the following agents on pancreatic tumor cells, was examined in vitro: mAbs specific to EGFR or to HER2, ErbB-specific TKIs, and gemcitabine. Cell proliferation assay of BxPC3 pancreatic tumor cells using the MTT assay was conducted. The results presented in Figure 5A verified the ability of the TRAP-Fc molecule to augment the inhibitory effects of the EGFR- and HER2- targeting mAbs, namely, cetuximab, panitumumab and trastuzumab, respectively. Combining TRAP-Fc with the TKIs lapatinib, erlotinib, CI-1033, AG1478 and gefitinib, also resulted in significantly enhanced inhibitory effects.

The large effect observed when TRAP-Fc was combined with gemcitabine, the mainstay single-agent drug of advanced pancreatic tumors was also tested in vivo. The effect of the combined treatment was addressed in an animal model of pancreatic cancer. BxPC3 cells were injected subcutaneously into female nude mice (6-week old) and allowed to grow until palpable tumors appeared. Thereafter, mice were randomized into four groups: a control group which received no treatment (9 mice), TRAP-Fc group (6 mice), injected intraperitoneally with TRAP-Fc (100 μg) for the first 3 weeks only, gemcitabine group (9 mice) treated with gemcitabine (intraperitoneal injection, 25 mg/kg), and the combination group (6 mice) treated with a combination of gemcitabine and TRAP-Fc. Mice were injected with TRAP-Fc on days 19, 28, 32, 35, 39, 42, and 45. Gemcitabine injections were given on the same days and repeated three more times on days 49, 52 and 55. Body weights were measured once a week, but no consistent differences were observed. Kaplan-Meier analysis of animal survival showed that the combined treatment led to a statistically significant prolongation of survival: the median (95% CI) survival of the control group was 52±1.5 days (Fig. 5B, pixeled line - large pixels). Treatment with TRAP-Fc or gemcitabine alone moderately prolonged survival to 61 (p=0.071; Fig. 5B, pixeled line - small pixels) and 64 (p=0.103; Fig. 5B, full line) days, respectively. Namely, each of TRAP-Fc or gemcitabine alone, extended the survival by 9 and 12 days, respectively, with respect to control. The expected additive effect was prolongation of median survival by 21 days relative to control. Surprisingly, the combination resulted in a prolongation of median survival by 80 days (p=0.006; Fig. 5B, broken line), extending survival by 28 days relative to control. In conclusion, the TRAP-Fc molecule exhibits anti-tumor activity when tested in animals, and this activity is enhanced, in a synergistic manner, when combined with an established anticancer agent. Example 6. TRAP-Fc inhibits invasive growth of mammary tumor cells

ErbB signaling has been implicated in invasion and metastasis of cancer cells. To test the TPvAP-Fc's effect on the ability of cancer cells to invade through tissue barriers, the highly metastatic MDA-MB-231 human breast cancer cell line was used. Eight-chambered plates were coated with an extracellular matrix (Matrigel). MDA-MB-231 cells (2000 cells/well) were mixed with medium containing Matrigel and then added to the chambers. Cells were incubated with or without TRAP-Fc (30 μg/ml), and phase contrast photomicrographs were captured six days later. When plated in a natural preparation of extracellular matrix (Matrigel™), MDA-MB-231 cells tend to invade the surrounding matrix by growing long and branched extensions (Fig. 6A, left panel). However, when the TRAP-Fc was added to their medium, cells exhibited a round, non-invasive morphology (Fig. 6A, right panel). The inhibitory effect of TRAP-Fc on cell migration was evaluated using a dual chamber Transwell tray. MDA-MB-231 cells (1.5 ^x 10 ⁵ ) were allowed to migrate through pores within a nitrocellulose filter, which separates the chambers (Transwell chambers) in the presence or absence of TRAP-Fc (30 μg/ml). Thereafter (following 19h of incubation) cells that adhered to the bottom of the filter were photographed, and the percentage of the area covered by the cells was quantified (Fig. 6B). Migration was normalized to the input number of cells. The results obtained in repeated experiments indicated that migration of TRAP-Fc- treated cells was inhibited by 27±7%. The "wound closure" (scratch) assay was used to further test the TRAP-Fc 's ability to inhibit TGFa-induced collective cell migration of untransformed MCF-10A. The cells were plated into two compartments separated by an insert, resulting within 24 hours in a confluent, but split layer. Thereafter, the inserts were removed and TGFa was added, either in the absence or in the presence of TRAP-Fc (100 μg/ml), and cells were allowed to migrate. Snapshots taken following 24 hours are presented in Figure 6C. By this time, the residual wound areas differed significantly: control cells covered approximately 60% of the gap area, while TRAP-Fc-treated cells covered only 33% of the gap area. Those results are in line with the ability of TRAP-Fc to inhibit collective cell's migration (Fig. 6C). Example 7. TRAP-Fc exhibits anti-metastasis activities in vivo

The effect of the TRAP-Fc on metastasis was examined on MDA-MB-231 cells, which were initially tested in vitro using a cell proliferation assay. These cells exhibited a partial growth inhibitory effect when incubated with TRAP-Fc (30 μg/ml) (Fig. 7A). The MDA- MB-231 cells were transfected with a TRAP-Fc-encoding plasmid and expression of the decoy was confirmed by immunoblotting (Fig. 7B). In the next step, these cells were implanted subcutaneously in the flanks of mice, and tumor size was monitored. High concentrations of tumor's TRAP-Fc showed a persistent correlation with effective growth inhibition (Fig. 7C). The ability of the ectopically expressed decoy to modulate metastasis in the lungs was examined. Parental and TRAP-Fc-expressing MDA-MB-231 cells were injected into the tail vein of SCID mice, and two months later lung metastases were assessed by counting the number of nodules. Compared to the parental cells, the TRAP-Fc-secreting MDA-MB-231 derivatives displayed an 80% reduction in lung metastasis (Fig. 7D). This anti-metastasis effect was verified by using a recombinant TRAP-Fc and the parental MDA-MB-231 cells. Cells were injected into the tail vein of SCID mice, and half of the mice were additionally injected intraperitoneally with the TRAP-Fc protein (100 μg per injection) on days 0, 3, 6 and 9. Two months later, we assessed metastasis by counting nodules in the lung. Consistent with the results obtained with the stably expressing clones of MDA-MB-231 cells, the injected TRAP-Fc reduced metastasis to the lung by 46% (Fig. 7E).

Example 8. Truncated TRAP-Fc exhibit poor binding

It has been suggested that truncation of ErbB receptor binding sites, by removal of subdomains or parts thereof, particularly, subdomain CI and L2, would produce smaller and more biocompatible molecules while maintain the binding affinities. The inventors of the present invention tested the aforementioned assertion by designing several truncated molecules and testing their ability to bind tagged ErbB ligands (NRG - 100 ng/ml; EGF - 0.15 nM), in an ELISA assay. The results are presented as percentage of binding compared to the binding of a control molecule (100% binding). As control served a bivalent molecule comprising the native binding domains of the ErbBl and ErbB4 receptors, where each binding domain includes all four subdomains (LI, CI, L2, C2; Dl, D2, D3 and D4) of the corresponding ErbB receptor.

As shown in Table 2, removal of domains (D), such as, CI (also termed domain 2, or D2) results with molecules having poor binding affinities to ErbB ligands compared to control. It is also shown that removal of domain 3 and 4 (D3-4, also known as L2, C2) results with a molecule that is capable of binding only one type of ErbB ligand. Stated otherwise, removal of domains 3 and 4 results with a monovalent molecule. It is further exemplified that removal of subdomains can result with molecules that cannot be expressed.

Table 2:

Domain of ErB4 Domain of ErBl Ligand Binding (%) Remarks Dl D2 D3-4 Dl D2 D3-4 NRG EGF

+ + + + + + 100 100 Control

+ + + + + 57.51 0.29

+ + + + + 18.19 25.53

+ + + + 6.41 0.15

+ + + - - + 62.34 0.06

+ + - - + 9.01 0.09

+ - - - + 0 0.23

+ + + + - - 2.54 0

+ + + + + - 88.30 0.24

+ + - - - + 9.61 1.24

+ - - + + No expression in cells

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.