Methods of identifying combinations of antibodies with an improved anti-tumor activity and compositions and methods using the antibodies   

This application is a divisional of U.S. patent application Ser. No. 12/320,207, filed on Jan. 21, 2009 which is a divisional of U.S. patent application Ser. No. 11/342,615, filed on Jan. 31, 2006, now U.S. Pat. No. 7,498,142 issued on Mar. 3, 2009. The contents of all of the above applications are incorporated by reference as if fully set forth herein.

This invention was made in part by the government support under contract No. R01 CA072981 awarded by the National Cancer Institute. The United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to methods of identifying combinations of antibodies with a combined improved anti tumor activity. The present invention further relates to pharmaceutical compositions comprising such antibodies which can be used for therapy, such as for cancer therapy.

Receptor tyrosine kinases (RTK) constitute a large family of cell surface proteins that act as scaffolding mediators and molecular switches in many signal transduction pathways, affecting cell growth, proliferation, differentiation, survival and migration. Receptors with tyrosine kinase activity share a similar molecular topology, essentially an extracellular ligand binding domain, a membrane spanning hydrophobic domain, and a cytoplasmic domain that comprises a highly conserved tyrosine kinase catalytic domain. RTKs comprise an array of extracellular domains that bind a variety of growth factors. Characteristically, the extracellular domains are comprised of one or more identifiable structural motifs, including cysteine-rich regions, fibronectin III-like domains, immunoglobulin-like domains, EGF-like domains, cadherin-like domains, kringle-like domains, Factor VIII-like domains, glycine-rich regions, leucine-rich regions, acidic regions and discoidin-like domains. Although diverse, RTK activation, initiation of signal transduction, and signal termination follow the same universal model (Yarden, Y., et al., Ann. Rev. Biochem 57:443-478, 1988; Ullrich, A. and Schlessinger, J., Cell 81, 203-212, 1990).

RTKs play a central role in the onset and progression of human disease, particularly cancer, such as breast, colon, lung and prostate cancers. As such, RTKs are preferred targets for the design and configuration of various therapeutic modalities.

Following ligand binding and onset of signaling, ligand-bound receptors are down regulated by removal from the cell surface. This universal mode of down regulation involves ligand-induced internalization by means of endocytosis, primarily via clathrin-coated pits, followed by receptor degradation. RTKs can also be endocytosed from invaginations other than clathrin-coated pits (e.g., caveoli), but the significance of this alternative internalization in RTK downregulation is not yet known. Deregulation of RTK endocytosis is a recurring factor in cancer progenesis (Peschard and Park, Cancer Cell 3: 519-523, 2003; Bache, K. G. et al., EMBO 23: 2707-2712, 2004). Indeed, more than 30 RTKs were found to be associated with cancer, through defective down regulation alone (Blume-Jensen, P. and Hunter, T. Nature 411: 355-365, 2001). Following this and other lines of research, a broad range of RTK inhibitors are being developed for use as anticancer agents or therapeutic agents against other RTK related diseases such as epidermal hyperplasia. RTK inhibitors e.g., epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), Fms-like tyrosine kinase (Flt-3) are constantly being developed [for examples see U.S. Pat. Nos. 6,900,186 (e.g., RTK down regulation for the treatment of skin disorders); 6,399,063 (e.g., down regulation of ErbB-2 with mAb); 5,837,523 (kinase deficient Neu mutants); 5,705,157 (e.g., mAb against EGFR increase receptor down regulation).

While the invention will be described herein in more detail with respect to the ErbB family of RTKs, it is to be understood that the invention is applicable for all RTKs.

The ErbB family of receptor tyrosine kinases, which includes the prototype, epidermal growth factor receptor (EGFR), also termed ErbB-1, and the related proteins ErbB-2, ErbB-3 and ErbB-4 is widely known and researched. The four known members of the ErbB family and their multiple ligand molecules form a layered signaling network, which is implicated in human cancer. Thus, overexpression of ErbB-1/EGF receptor (EGFR) as well as ErbB-2 has been correlated with poor prognosis in various human malignancies. Specifically, deletion mutants of EGFR exist in brain tumors and point mutations have recently been reported in lung cancer. By contrast, ErbB-2/HER2 is rarely mutated in solid tumors. Instead, the ErbB-2 gene is frequently amplified in breast, ovarian, and lung cancer.

Because of their oncogenic potential and accessibility, ErbB proteins have emerged as attractive targets for pharmaceutical interventions. One major strategy involves the use of mAbs. Early studies uncovered the tumor-inhibitory potential of mAbs directed at ErbB-1 and ErbB-2, and later studies indicated that anti-ErbB mAbs are effective when combined with various chemotherapeutic agents. Indeed, the clinical benefit of combining mAbs with certain chemotherapeutic agents was notable, and led to the approval of mAbs to ErbB-2 (Herceptin) and EGFR(C225/Cetuximab) for the treatment of breast and colorectal cancer, respectively. Other antibodies now in clinical trials include MDX-210 (phase II, Medares), tgDCC-Eia (phase II, Targeted Genetics) and 2C4 (phase I, Genentech) (Zhang H. et al., Cancer Biol. Therap., 2: S122-S126, 2003). Unfortunately, the therapeutic efficacy of these and other RTK inhibitors is limited and varies dramatically between patients. There is thus a need to elucidate the mechanism underlying antibody mediated therapy.

Two types of mechanisms have been implicated in ErbB-directed immunotherapy. The first involves mAb-mediated recruitment of natural killer cells through the Fc-γ activation receptors of these immune effector cells to the tumor site. The second type of mechanism relates to intrinsic mAb activities, which include blockade of ligand binding or receptor heterodimerization, inhibition of downstream signaling to Akt, and acceleration of receptor internalization. The latter mechanism is particularly attractive because ligand-induced endocytosis and degradation of active receptor tyrosine kinases (RTKs) is considered a major physiological process underlying attenuation of growth-promoting signals.

In order to improve the efficacy of antibody therapy, the use of mAb combinations had been attempted. Indeed a number of studies have been effected using at least two antibody combinations directed at distinct epitopes of ErbB-2 [Drebin J. A. et al., Oncogene 2(3):273-277, 1988; Kasprzyk et al., Cancer Res. 52(10):2771-2776, 1992; Harwerth et al., Br. J. Cancer 68(6):1140-1145, 1993; Spiridon et al. Clin. Cancer Res. 8:1720-1730, 2002]. However, while some were successful in improving tumor inhibition [e.g. Drebin et al., Oncogene 2(3):273-277] others reported only marginal or additive effect of such combinations when compared to a single antibody treatment [e.g. Harwerth et al., Br. J. Cancer 68(6):1140-1145, 1993].

Thus for example, Spiridon et al. (Clin. Cancer Res. 8:1720-1730, 2002), generated a panel of murine anti-ErbB-2 mAbs directed against nine different epitopes on the extracellular domain of ErbB-2. A combination of three of those mAbs was used to address the effect of an antibody combination (directed to distinct epitopes) against the breast cancer cell line BTB474 in vivo and in vitro. However, only a slightly better therapeutic efficacy was observed in these studies as evidenced by tumor cell killing, Fc-mediated effector function and VEGF secretion from the tumor cell.

These results suggest that selection of antibody combination only upon structural characteristics (i.e., binding to distinct epitopes) cannot be used as a sole criterion for selecting winning antibody combinations for therapy.

There is thus a widely recognized need for and it would be highly desirable to have a method for identifying RTK directed antibody combinations which display high therapeutic efficacy.

SUMMARY

According to one aspect of the present invention there is provided a method of identifying a combination of antibodies with a combined improved anti tumor activity the method comprising identifying at least two anti RTK antibodies capable of inducing synergistic endocytosis of the RTK in a cell expressing the RTK, thereby identifying the combination of antibodies with the combined improved anti-tumor activity.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising as an active ingredient at least two anti RTK antibodies capable of inducing synergistic endocytosis of the RTK in a cell expressing the RTK and a pharmaceutically acceptable carrier of diluent.

According to yet another aspect of the present invention there is provided an article-of-manufacturing comprising a packaging material and a pharmaceutical composition identified for treating cancer being contained within the packaging material, the pharmaceutical composition comprising as an active ingredient at least two anti RTK antibodies capable of inducing synergistic endocytosis of the RTK in a cell expressing the RTK.

According to still another aspect of the present invention there is provided a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of at least two anti RTK antibodies capable of inducing synergistic endocytosis of the RTK in a cell expressing the RTK, thereby treating cancer in the subject.

According to further features in preferred embodiments of the invention described below, the at least two antibodies are directed to distinct extracellular epitopes of the RTK.

According to still further features in the described preferred embodiments each of the at least two antibodies bind the RTK with an affinity of at least 200 nM.

According to still further features in the described preferred embodiments the at least two antibodies bind the RTK with a similar affinity.

According to still further features in the described preferred embodiments the RTK is an ErbB protein.

According to still further features in the described preferred embodiments the ErbB protein is selected from the group consisting of ErbB-1, ErbB-2, ErbB-3 and ErbB-4.

According to still further features in the described preferred embodiments the RTK is selected from the group consisting of c-met, PDGFR, ErbB, VEGFR, EphR, FGFR, INSR and AXL.

According to still further features in the described preferred embodiments the at least two antibodies comprise a recombinant antibody.

According to still further features in the described preferred embodiments the recombinant antibody is a bispecific antibody or a single chain antibody.

According to still further features in the described preferred embodiments the at least two antibodies comprise a monoclonal antibody.

According to still further features in the described preferred embodiments the at least two antibodies comprise a polyclonal antibody.

According to still further features in the described preferred embodiments the at least two antibodies comprise a humanized antibody.

According to still further features in the described preferred embodiments the at least two antibodies comprise an antibody fragment.

According to still further features in the described preferred embodiments the at least two antibodies comprise at least a bivalent antibody.

According to still further features in the described preferred embodiments the endocytosis is c-Cbl independent.

According to still further features in the described preferred embodiments the endocytosis is ubiquitylation independent.

According to still further features in the described preferred embodiments the method further comprising subjecting the subject to a therapy selected from the group consisting of a radiotherapy and a chemotherapy.

The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of identifying combinations of antibodies with a combined improved anti tumor activity.

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

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

FIGS. 1a-d depict distinct endocytic behavior of EGFR treated with EGF and mAbs. FIG. 1a is a graph depicting cell-surface EGFR following treatment with EGF and the indicated antibodies for various time periods. KB cells were treated with EGF (100 ng/ml) or mAbs (20 μg/ml) at 37° C. for various time intervals. The cells were then washed and acid-stripped, and levels of surface receptor were determined (average ±SD) using radiolabeled EGF; FIG. 1b is an immunoblot showing surface expression of EGFR in cells treated for 4 or 32 hours with anti EGFR antibodies, EGF or no treatment, as determined by surface biotinylation. KB cells were treated as indicated and washed as in FIG. 1a. Cells were either subjected to surface biotinylation followed by lysis and immunoprecipitation and blotting with streptavidin-horseradish peroxidase (Upper panel) or directly lysed and immunoblotted with anti-EGFR Ab (Lower panel); FIG. 1c is an immunoblot showing that down-regulation of EGFR by mAbs is independent of receptor ubiquitilation. CHO cells transiently expressing EGFR, c-Cbl and hemagglutinin (HA)-ubiquitin were incubated with EGF (100 ng/ml) or the indicated mAbs (20 μg/ml) for various intervals. Cells were lysed and subjected to immunoprecipitation with anti EGFR and immunoblotting with anti HA. FIG. 1d is an immunoblot showing that antibody-mediated EGFR endocytosis is c-Cbl independent. CHO cells were co-transfected with WT- or Y1045F-EGFR (which cannot directly recruit c-Cbl), along with c-Cbl. Transfected cells were incubated with EGF (100 ng/ml; 1 h), saline, or the indicated mAbs (10 μg/ml; 18 h). Cells were washed for removal of bound ligands, lysed, and subjected to immunoprecipitation and immunoblotting with anti EGFR;

FIGS. 2a-d depict better down regulation of EGFR following treatment with combinations of anti-receptor mAbs as compared to treatment with individual mAbs. FIG. 2a is an immunoblot showing surface expression of EGFR in KB cells treated for 13 hours with individual anti EGFR mAbs, a combination of antibodies (total: 20 μg/ml), EGF (100 ng/ml) or no treatment. Following incubation, cells were stripped of bound ligands, lysed and extracts were subjected to immunoprecipitation and immunoblotting with anti EGFR; FIG. 2b is an immunoblot showing surface expression of EGFR in KB cells after incubation for various time intervals with anti EGFR antibodies, a combination of antibodies (total: 20 μg/ml), EGF (100 ng/ml) or no treatment. Extracts were analyzed as in FIG. 2a. FIG. 2c shows three graphs depicting cross competition analyses between three mAb. KB cells were treated for 1 h at 4° C. with mAbs 111 (closed circle), 143 (closed triangle), 565 (open circle), or EGF (closed square). The indicated radiolabeled mAbs (8 nM) were then added, and the cells were incubated for an additional 15 min before determination of radioactivity. The graphs depict the percent of bound radiolabled antibody as it is affected by the incubation with other antibodies, or EGF; FIG. 2d is an immunoblot showing rate of receptor down-regulation is proportional to the size of Ab-receptor lattice. The upper panel is an immunoblot showing surface expression of EGFR in cells treated for 18 h with saline (Cont) or EGF (100 ng/ml), or co-treated with mAbs 111 (10 μg/ml) and increasing amounts (2.5-20 μg/ml) of 565. The lower panel is an immunoblot showing surface expression of EGFR in KB cells treated with saline, EGF (100 ng/ml), or the indicated mAbs (total: 10 μg/ml) for 3 h at 4° C., followed by a change of medium of all cells (except lane 3) to a fresh medium containing a goat anti-mouse IgG (second Ab: either 40 or 10, 20, and 40 μg/ml). Cells were incubated for additional 18 h at 37° C., washed, lysed, and subjected to immunoprecipitation followed by immunoblotting with anti-EGFR;

FIGS. 3a-d depict better down regulation of ErbB-2 treated with combinations of antibodies as compared to treatment with individual mAbs. FIG. 3a is a graph depicting cross competition analyses between 2 mAbs. SKBR-3 cells were treated with various concentrations of the mAb L26 (circles) or Herceptin™ (triangles) at 4° C. Radiolabeled mAb L26 (8 nM; closed symbols) or Herceptin™ (open symbols) were then added, and the cells were incubated for 15 min. After washing, radioactivity was measured and expressed as average percent of bound antibody ±SD. FIG. 3b an immunoblot showing surface expression of ErbB-2 is lower in cells treated with a combination of mAb. HEK-293T cells (Upper panel) ectopically expressing ErbB-2 or T47D cells (Lower panel) were treated with L26 and/or Herceptin™ (Her; total: 20 μg/ml) or preimmune Abs (PI) or pAb, at 37° C. for the indicated time intervals. Cells were then washed lyzed and subjected to immunoblotting with anti-ErbB-2. FIG. 3c is a graph showing the percent of internalized 4D5 (a parental form of Herceptin) is higher when it is used in treatment together with L26. SKBR-3 cells were treated with fluorescein-labeled 4D5-mAb (10 μg/ml) at 37° C. for the indicated time intervals in the absence (closed rectangle) or presence (closed square) of L26. Thereafter, cells were washed and acid-stripped, and internalized 4D5 was determined by using a cell sorter. FIG. 3d is a confocal microscopy photograph of CB2 cells that were incubated with a mixture of L26 and Herceptin™ (20 μg/ml each) or L26 alone (40 μg/ml) at 37° C. for the indicated time periods. Thereafter, cells were washed, fixed, and permeabilized, and ErbB-2 was detected by using confocal microscopy with a Cy3-conjugated anti-mouse IgG;

FIGS. 4a-c depict down-regulation of ErbB-2 by combinations of mAbs is dynamin-dependent but requires no cytoplasmic or transmembrane portions of ErbB-2. FIG. 4a is an immunoblot showing that down regulation of EGFR and ErbB-2 is dynamin dependent. HEK-293T cells were cotransfected either with plasmids encoding EGFR (Upper panel) or ErbB-2 (Lower panel), along with plasmids encoding c-Cbl and dynamin (WT or K44A—a dominant negative form of dynamin which blocks EGF endocytosis). After 48 h, cells were treated with EGF (100 ng/ml) or a combination of L26 and Herceptin™ (Her; total: 20 μg/ml). Cells were then lyzed immunoblotted with anti-EGFR (Upper panel) or anti-ErbB-2 (Lower panel), and anti-Dynamin Ab; FIG. 4b is a diagram showing the ErbB-2 molecules analyzed. The diagram shows either WT, a mutant lacking the cytoplasmic domain (ECD-TM), or the full ectodomain fused to a GPI-attachment signal (ECD-GPI). FIG. 4c is an immunoblot showing that no cytoplasmic or transmembrane motifs of ErbB-2 are necessary for mAb-induced internalization and degradation. CHO cells were trasfected with either WT, ECD-GPI or ECD-TM mutant forms of ErbB-2. Cells were treated for 0 or 3 h with a mixture of mAbs L26 and Herceptin™ (5 μg/ml each), and washed to remove bound ligands. Cells were then either subjected to lysis, immunoprecipitation and blotting with anti-ErbB-2 Ab (Upper panel), or were directly lysed and immunoblotted with anti-ErbB-2 Ab. The lower panel shows that when electrophorased, the two mutants display two bands corresponding to 120 and 135 kDa, but experimental data has shown that only the 135 kDa species had reached the plasma membrane and underwent down-regulation upon treatment with mAb (data not shown). In accordance, only the 135 kDa surface-localized forms of ECD-GPI and ECD-TM (arrows) were affected by mAbs;

FIGS. 5a-e depict inhibition of growth factor signaling, promotion of differentiation, and reduction of tumor growth by combinations of anti-ErbB-2 Abs. FIG. 5a is an immunoblot showing that NDF mediated ErbB-2 phosphorylation on tyrosine residues, is inhibited by treatment with a combination of mAb. T47D cells were incubated for 12 h with either L26, Herceptin™ (Her), a mixture of both (mix) or polyclonal antibodies (pAb) (total: 10 μg/ml). Cells were then washed and stimulated with NDF (50 ng/ml) for 15 min, lysed and immunoblotted with antiphosphotyrosin Abs, anti p-Erk Ab, and anti Erk2 Abs. FIG. 5b is a bar graph showing that NDF stimulation of transcription from the serum response element (as shown by the activity of a c-fos promoter-luciferase reporter gene) is inhibited by treatment with a combination of mAb. MCF-7 cells were transfected with a reporter plasmid, and, 24 h later, cells were split and incubated for 12 h with the indicated Abs, including a control human IgG. Later (47 h), cells were washed and stimulated for 1 h with NDF (50 ng/ml), followed by analysis using a luminator. FIG. 5c is a photograph showing that differentiation of tumor cells is higher in cells treated with a combination of mAb. AU-565 cells were treated for 3 d with mAbs (30 μg/ml) and then stained for neutral lipids (which are the product of differentiated tumor cells). FIG. 5d is a bar graph showing tumor volume of tumors from CD-1/nude mice that were injected s.c. with 3×106 N87 cells. mAbs (600 μg per animal) were injected i.p. 3, 7, and 10 d later. Saline-injected mice were used for control. Tumor volumes were measured after 18 d, and the mean volume of each group of four mice was plotted. The difference between treatments with each mAb alone and their combination is statistically significant (P<0.05). FIG. 5e is a diagram which shows the size of ErbB-Ab complexes formed at the cell surface is larger when the lattice is formed by more than one mAb. According to the model, the rate of internalization is proportional to the size of surface-associated antigen-Ab lattices; and

FIGS. 6a-b is a schematic presentation of teravalent bi-epitopic antibodies to ErbB-2. The design of each bi-epitopic arm of the antibody is shown in FIG. 6a, along with the sequence of linkers. FIG. 6b shows an assembled disulfide-held tetravalent antibody.

The present invention relates to methods of identifying combinations of antibodies with a combined improved anti tumor activity. The present invention further relates to pharmaceutical compositions comprising such antibodies and methods of using same. Specifically, the present invention can be used for the treatment of receptor-tyrosine kinases associated diseases, such as cancer.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Overexpression and aberrant function of receptor tyrosine kinases (RTKs) and their ligands have been reported in many human cancers, providing a rationale for targeting this signaling network with novel approaches. The ErbB family of receptor tyrosine kinases is a selective target for inhibiting cancers because their activation often confers a proliferative advantage. For example, activation of the ErbB-1 tyrosine kinase provides signals that drive deregulated proliferation, invasion, metastasis, angiogenesis, and cell survival, and its inhibition has potential in both the treatment and prevention of these malignancies. Based on the structure and function of RTKs, therapeutic strategies have been developed. These include the use of human monoclonal antibodies (mAbs) generated against the receptor\'s ligand-binding extracellular domain. These mAbs block binding of receptor-activating ligands, and, in some cases, can induce receptor endocytosis and downregulation. Early clinical studies suggest only limited therapeutic activity in many common malignancies.

In order to improve the efficacy of antibody therapy, the use of mAb combinations had been attempted. Indeed a number of studies have been effected using at least two antibody combinations directed at distinct epitopes of ErbB-2 However, while some were successful in improving tumor inhibition, others reported only marginal or additive effect of such combinations when compared to a single antibody treatment.

These results suggest that selection of antibody combination only upon structural characteristics (i.e., binding to distinct epitopes) cannot be used as a sole criterion for selecting winning antibody combinations for therapy.

While reducing the present invention to practice, the present inventors uncovered that a combination of at least two antibodies to a receptor tyrosine kinase (RTK) of interest, which is selected capable of inducing synergistic endocytosis of the RTK, mediate improved antitumorigenic activity when compared to the effect of each individual antibody.

As is illustrated in the Examples section which follows, the present inventors showed that combinations of anti-EGFR or ErbB-2 mAbs directed at distinct extracellular epitopes better down-regulate the receptor than treatment with each monoclonal antibody alone (see Examples 1 and 3 of the Examples section which follows). The present inventors further showed that antibody combinations with improved therapeutic efficacy are those which are capable of synergistic down-regulation of the cell-surface receptor (see Examples 5 of the Examples section which follows). Thus, for example, co-administration of L26 and Herceptin™ resulted in augmented reduction of tumor volume as compared to the effect of each antibody alone. Accordingly, it is expected that co-administration of 565+111 will exhibit a better therapeutic effect than of 143+565 although both combinations are directed at distinct epitopes of the EGFR. The hybridoma cell lines expressing antibodies 111 and 565 were deposited pursuant to the Budapest Treaty requirements at the Collection Nationale de Cultures de Microorganismes (CNCM) Institut Pasteur 25, Rue du Docteur Roux F-75724 Paris CEDEX 15, on Nov. 26, 2009 and are registered as CNCM I-4261 for anti-EGFR antibody 111 and CNCM I-4262 for anti-EGFR antibody 565.

FIG. 5e outlines the proposed model. Accordingly, because of their bivalence, mAbs are able to form receptor homodimers, but treatment with combinations of mAbs will generate much larger receptor-Ab complexes. For several reasons it is proposed that the rate of endocytosis of mAb-RTK complexes is proportional to their size, in analogy to the entry of viruses and other polyvalent ligands and pathogens. Firstly, both mAb combinations and a pAb (FIG. 3b) induce earlier (FIG. 2b) and more extensive receptor internalization. Secondly, the observed bell-shaped dose-response (FIG. 2d), as well as the ability of a secondary Ab to increase receptor degradation (FIG. 2d), may be interpreted in terms of a precipitin reaction occurring at the cell surface. Thirdly, the strict dependence of mAb cooperation on simultaneous engagement of more than one epitope (FIGS. 2c and 3a), in line with previous reports (Damke, H., et al., J. Cell Biol. 127:915-934, 1994), is compatible with the notion that large surface aggregates internalize faster than smaller complexes.

Based on the overall structural similarity and endocytic mechanism of RTKs it is suggested that the present findings can be extended to the targeting of any RTK.

Thus, the present invention offers strategies to enhance the therapeutic efficacy of clinically approved antibodies such as Avastin™, Herceptin™ and C225/Cetuximab™.

Thus, according to one aspect of the present invention there is provided a method of identifying a combination of antibodies with a combined improved anti tumor activity.

As used herein the phrase “anti tumor activity” refers to prevention of tumor formation and/or reduction of tumor size (e.g., volume) and/or metastasis potential.

The method comprising identifying at least two anti RTK antibodies capable of inducing synergistic endocytosis of said RTK in a cell expressing said RTK, thereby identifying the combination of antibodies with the combined improved anti-tumor activity.

As used herein the term “RTK” refers to the cell surface form of protein tyrosine kinase (E.C. 2.7.1.112) which cellular surface expression/activation is typically associated with the onset or progression of a disease, usually a malignant disease, such as cancer.

Examples of RTKs which can be used in accordance with this aspect of the present invention are listed in Table 1 below.

TABLE 1 Examples of Accession associated RTK Full name Reference number Pathologies RTK subfamily epidermal Silvestri G A and NP_958441 non-small cell EGFR/Erb ErbB growth Rivera M P, lung cancer B-1/HER1 subfamily factor Chest. receptor 128(6): 3975-84, 2005. Snyder L C, et colorectal cancer al., Clin head and neck Colorectal cancer Cancer. 1 2: S71-80, 2005. Slamon D J, et Sprot: breast ovarian and ErbB-2/ ErbB al,. Science 244: P04626 lung cancer HER2 subfamily 707-712, 1989. Visakorpi T, et transitional cell al., Clin. carcinoma of the Cancer Res. 9 bladder

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