WO2009106321A1 - Treatment of resistant tumors with trifunctional antibodies - Google Patents

Abstract

The invention relates to the field of cancer treatment using therapeutic antibodies. More specifically, the invention relates to a trifunctional bispecific monoclonal antibody with specificities against HER-2 and a T-cell specific cell surface protein for use in a method of treatment of HER-2 expressing tumors that exhibit a resistance against tyrosine kinase inhibitors.

Description

Fresenius Biotech GmbH February 26, 2009

F65107PC1

Treatment of resistant tumors with trifunctional antibodies

Field of the invention

The present invention relates to the field of cancer treatment using therapeutic antibodies. More specifically, the invention relates to a trifunctional bispecific monoclonal antibody with specificities against HER-2 and a T-cell specific cell surface protein for use in a method of treatment of HER-2 expressing tumors that exhibit a resistance against tyrosine kinase inhibitors.

Background of the invention

Her2/neu, also known as ErbB2, belongs to the human epidermal growth factor receptor (EGFR) gene family or HER family. Her2/neu encodes a tyrosine kinase receptor (HER-2), which is over-expressed in approximately 25% of invasive breast cancers. HER-2 over- expression has consistently been found to confer resistance to cytotoxic and endocrine therapy and to account for an aggressive biological behaviour, thereby resulting in shorter disease-free and overall survival in both, patients with early and advanced breast cancer. Upon binding of a growth factor, receptors of the EGFR family dimerize using HER-2 as their preferred binding partner. Heterodimerization induces intrinsic receptor tyrosine kinase mediated autophosphorylation and subsequent activation of downstream signalling components via the MAPK and PI3K pathways, resulting in unresisted growth and enduring survival of the tumour cells. Therapy with the anti-HER-2 humanized monoclonal antibody trastuzumab (Herceptin^®, Genentech, South San Francisco, CA, US) has become a standard therapy in patients with HER-2 positive tumours, e.g. breast cancer and other types of tumors. There are several modes of action described for Herceptin^®. One is the effect of activating the immune system by binding the antibody to the tumor cell surface, referred to as antibody dependent cellular cytotoxicity (ADCC). Another important effect is the blocking of the surface receptor, thereby disturbing signal transduction. It is commonly accepted that the blocking of the dimerization and the signal transduction plays a more significant role for the potency of Herceptin^® than ADCC. For this reason, Herceptin^® also acts apoptotic in the absence of the immune system (see e.g. EP 865448). However, therapy with Herceptin^® is only suitable for patients showing a high expression rate of HER-2 on the tumor cell surface. HER-2 over-expression is generally due to gene amplification and has been defined by immunohistochemistry as being highest (3+) when receptor levels approach 2 million, or medium intensity (2+) when receptor levels are approximately 500,000, whereas normal levels of HER-2 are reported to be 20,000 per cell. The level of HER-2 gene amplification in human cancer cells can be classified by fluorescence in situ hybridization (FISH). A therapy with Herceptin^® is only believed to be promising in tumors of patients, which tumors can be classified 2+/3+ and FISH+, respectively. It has been thought that the HER-2 expression in the other remaining patient groups is not sufficiently high to achieve a satisfactory effect when using monospecific antibodies such as Herceptin^®. However, it was found that trifunctional bispecific antibodies also produce an anti-tumor effect in 1+ classified tumor cells (EP 1820513). The objective response rates to trastuzumab monotherapy are quite low, ranging from 12% to 34% for a median duration of nine months, i.e. up to 88% of the patients show a de novo resistance against trastuzumab. Of the patients showing an initial response to trastuzumab- based therapy, however, a majority acquires a secondary resistance within one year of treatment initiation. Identification of novel agents that inhibit the growth of trastuzumab-resistant cells is important for improving the survival of metastatic breast cancer patients whose tumors over-express HER-2 (2+/3+/FISH+). It is proposed that resistance against Herceptin^® mainly develops, if either the binding of the monospecific antibody to HER-2 is blocked, or the interruption of the signal transduction is not blocked although the binding occurred, or other tumor growth and survival promoting signal pathways have replaced the HER-2 signal pathway. Binding of the antibody to the surface receptor can be blocked due to a change of the surface epitope, or by masking the epitopes through other proximal surface proteins, e.g. the membrane-associated glycoprotein mucin-4 (MUC4). It is thought that several reasons may explain the absence of an effect on signal transduction despite of the binding of the antibody. Among others, the intracellular part may be permanently activated, independently from a stimulation of the extracellular part. For these reasons, Herceptin^® resistant cells may also be resistant against other monospecific antibodies, e.g. pertuzumab, which bind another epitope than Herceptin^® thereby inhibiting dimerization of HER-2 and blocking signal transduction. To tackle this kind of resistance, tyrosine kinase inhibitors have been developed which target the tyrosine binding site in the wtracellular part of HER-2. An example of such an intracellular tyrosine kinase inhibitor is lapatinib (Tykerb^®) (GlaxoSmithKline, Research Triangle Park, NC, US).

However, there may be patient groups which exhibit a general resistance against HER-2 tyrosine kinase inhibitors, administered alone or in combination with Herceptin^®. Results from early-phase trials indicate that clinical responses to lapatinib monotherapy in patients with ErbB2-overexpressing breast cancers are generally short-lived (Burris et al., J. Clin. Oncol. 2005). In general, the development of acquired resistance to ErbB2 tyrosine kinase inhibitors limits the clinical efficacy of this class of cancer therapeutics. In these patients, signal transduction initiates growth, even though the function of the transmembrane receptor, e.g. HER-2, is blocked intra- as well as extracellularly. Resistance against tyrosine kinase inhibitors may originate from the high expression of molecular transporter proteins, such as P-glycoprotein (Pgp) and Breast Cancer Resistance Protein (BCRP) (Polli et al., Drug Metab. Dispos. 2008). In cell based assays Lapatinib inhibits the growth of BT- 474 and SK-BR-3 tumor cells at a low concentration (Rusnak et al, MoI. Cancer Ther. 2001). The long term culture of Lapatinib susceptible tumor cells can lead to the selection of a resistant variant (Konecny et al., Cancer Res. 2006; Xia et al., Proc Natl Acad Sci USA 2006). Thus, there still remains the need to provide a treatment for patients having tumors being resistant against monospecific anti-HER-2 antibodies and/or tyrosine kinase inhibitors.

It is therefore an object of the invention to provide a trifunctional, bispecific antibody that is effective against tumor cells which are resistant against tyrosine kinase inhibitors, and/or monospecific anti-HER-2 antibodies, in particular Herceptin .

Summary of the invention

It has now surprisingly been found that a trifunctional, bispecific antibody can be used in a method of treatment of tumors exhibiting a de novo or secondary resistance against a tyrosine kinase inhibitor, such as lapatinib, as concluded from the Examples disclosed herein.

Accordingly, the present invention relates to a trifunctional, bispecific antibody for use in the treatment of a HER-2 expressing tumor, wherein the tumor is or becomes resistant against one or more tyrosine kinase inhibitor, as defined in the claims. Furthermore, a pharmaceutical composition comprising one or more trifunctional, bispecific antibody and one or more anti-HER-2 antibody is provided as it is defined in the claims. Finally, a kit comprising one or more trifunctional, bispecific antibody and one or more anti-HER-2 antibody as defined in the claims is also provided. The various aspects of the invention as defined in the independent claims and the preferred embodiments contained in the dependent claims are herewith incorporated by reference.

Detailed description of the preferred embodiments

According to a first aspect, the present invention relates to a trifunctional, bispecific antibody having the following properties: (a) binding to a T-cell specific cell surface protein, (b) binding to the tumor-associated antigen HER-2 on a tumor cell, and (c) binding to Fc-gamma-receptor type I and/or type III positive cells, for use in the treatment of a HER-2 expressing tumor, wherein the tumor is or becomes resistant against one or more tyrosine kinase inhibitor. A trifunctional, bispecific antibody for use in the present invention may be prepared in accordance with the procedures described in EP 763128, EP 826696 and EP 1820513. Preferably, the trifunctional, bispecific antibody binds to the T- cell specific cell surface protein CD3. In a particular advantageous embodiment, the trifunctional, bispecific antibody is an anti-HER-2 x anti-CD3 antibody binding to Fc- gamma-receptors type I and/or III. In particular, the Fc portion comprises the isotype combination rat-IgG2b/mouse-IgG2a. A particularly preferred antibody is ertumaxomab. Ertumaxomab is an intact bispecific antibody targeting HER-2 and CD3 with selective binding of activatory Fcγ type I/III receptors.

The trifunctional, bispecific antibody described above is used in the treatment of a HER-2 expressing tumor, wherein the tumor is or becomes resistant against one or more tyrosine kinase inhibitor. In a preferred embodiment, the tumor additionally is or becomes resistant against one or more monospecific anti-HER-2 antibody. As used herein, the term "is or becomes resistant" means that the tumor does not respond to the respective agent, i.e. shows a de novo resistance, or, respectively, the initial responders demonstrate disease progression within a certain period after initiation of the treatment, i.e. develop a secondary resistance (see also Bartsch et al., 2007). The treatment of the HER-2 expressing tumor preferably comprises administration of one or more tyrosine kinase inhibitors and/or one or more monospecific anti-HER-2 antibodies. The term "tyrosine kinase inhibitor" refers to a molecule, in particular a small molecule, which can pass the cell membrane and target the intracellular domain of HER-2 and/or the intracellular domain of any binding partner of HER-2 (e.g. Nahta et al., 2007). Preferred examples of such tyrosine kinase inhibitors are lapatinib (Tykerb)/GW572016, GlaxoSmithKline, NC, US), gefitinib (Iressa^®, ZDl 839), imatinib (Gleevec^®, STI-571), erlotinib (Tarceva^®), lanafamib (Sarasar^®), sorafinib and/or sunitimib (see also Bartsch et al., 2007). The one or more monospecific anti-HER-2 antibody may be selected from, e.g. trastuzumab (Herceptin^®) and/or pertuzumab (Omnitarg^®, 2C4), or any other monospecific anti-HER-2 antibody suitable for the described treatment. The antibodies trastuzumab and pertuzumab are both commercially available (Genentech, US). It is believed that, based on the present disclosure, one of average skill in the art can define a protocol for use of the one or more tyrosine kinase inhibitor and/or one or more monospecific anti-HER-2 antibody.

The HER-2 expressing tumor to be treated may be a breast tumor, ovarian tumor, prostate tumor, colon tumor, pancreas tumor, stomach tumor, esophagus tumor, endometrium tumor, skin tumor, oropharynx tumor, larynx tumor, cervix tumor, bladder tumor, preferably a carcinoma, more preferably an adenocarcinoma and/or a squamous cell carcinoma. HER-2 is reported to be usually present on a cell at a level of 20,000 receptors per cell. Accordingly, a "HER-2 expressing tumor", as used herein, refers to a tumor expressing HER-2 at a level of at least about 20,000, preferably at least about 25,000, more preferably at least about 30,000, even more preferably at least about 35,000, or most preferably at least about 40,000 to about 10,000,000 HER-2 receptors per cell. Due to HER-2 gene amplification or chromosome 17 polysomy in human cancer cells HER-2 will typically be over-expressed in tumor cells from a number of primary as well as secondary tumors. Thus, in a further preferred embodiment, the HER-2 expressing tumor is a HER-2 over-expressing tumor. The term "HER-2 over-expressing tumor", as used herein, refers to a tumor expressing HER-2 at a level of about 50,000 to about 10,000,000 receptors/tumor cell, preferably at least about 75,000, 100,000, 125,000, 150,000, 200,000, 300,000, 400,000, 500,000, 1,000,000, 2,000,000 receptors/tumor cell to 10,000,000 receptors/tumor cell. The level of expression of HER-2 on the tumor cells can be determined in accordance with standard procedures known in the art. Preferably, the expression level of HER-2 is quantified by flow cytometry, as described in the experimental section below (see, e.g. Example 1). In a preferred embodiment, the status of HER-2 over-expression is evaluated by histochemical analysis using the HercepTest (DAKO, CA, US). The HercepTest is approved by the US Food and Drug Administration (FDA) for determining the suitability for trastuzumab treatment. The test provides an evaluation system for HER-2 comprising four steps, 0, 1+, 2+, 3+, referring to an approximate number of expressed target antigens on the surface of a tumor cell. In accordance with this system, cells expressing less than 20,000 HER-2 molecules on the target cell are classified as negative; cells with an expression of more than about 20,000 and up to about 100,000-1 10,000 molecules are classified as 1+; cells with an expression of up to 500,000 molecules as 2+, and cells with an expression of between about 2,000,000 and about 10,000,000 molecules are classified as 3+. In accordance with a preferred embodiment, the HER-2 over-expressing tumor is classified by a value in the HercepTest of 2+ and/or 3+. Alternatively, or in addition to the above procedures for determining the level of expression of HER-2 receptors per tumor cell described above, the status of HER- 2 expression can be determined by fluorescence in-situ hybridization (FISH). The FISH assay was initially approved by the FDA for assessing prognosis and predicting response to standard chemotherapy and has now also been approved for determining the eligibility for trastuzumab treatment. The assay is commercially available (PathVysion test; Vysis, IL, US). It is particularly preferred that the HER-2 over-expressing tumor is a FISH positive (FISH+) tumor. The treatment described herein may be particular advantageous in case of tumors classified as FISH+ and 2+ and/or FISH+ and 3+.

In another preferred embodiment, the treatment of HER-2 (over-)expressing tumors additionally comprises the administration of one or more monospecific antibody against an antigen other than HER-2, preferably a member of the HER family such as epidermal growth factor receptor (EGFR), HER-3, and/or HER-4. Particularly preferred are one or more anti-EGFR antibodies, in particular the monospecific anti-EGFR antibody cetuximab. Cetuximab is a chimeric monoclonal antibody targeting the extracellular domain of EGFR. In another aspect, the invention relates to a pharmaceutical composition comprising (i) one or more trifunctional, bispecific antibody; and (ii) one or more anti-HER-2 antibody; optionally further comprising (iii) one or more monospecific antibody against an antigen other than HER-2, preferably one or more anti-EGFR antibodies, and/or (iv) one or more tyrosine kinase inhibitor. The components (i) to (iv) may take any form as defined above and any combination thereof.

According to the still further aspect of the invention, there is provided a kit comprising (i) one or more trifunctional, bispecific antibody, and (ii) one or more anti-HER-2 antibody; optionally further comprising (iii) one or more monospecific antibody against an antigen other than HER-2, preferably one or more anti-EGFR antibodies; and/or (iv) one or more tyrosine kinase inhibitor; wherein component (i) to (iv) are the above-described embodiments taken either alone or in combination with other embodiments described herein.

In a still further aspect, it is provided a bispecific antibody or a fragment thereof, having the following properties: (a) binding to a tumor-associated antigen HER-2 on a tumor cell, and (b) binding to immunocompetent cells, preferably Fc-gamma-receptor type I and/or type III positive cells, in particular binding to an epitope on the Fc-gamma-receptor or to CD3, for use in the treatment of a HER-2 over-expressing tumor, wherein the tumor is or becomes resistant against one or more tyrosine kinase inhibitor. Preferably, the antibody fragment is a single chain antibody (scFv).

Description of the Figures

Figure 1. Schematic antibody structure of ertumaxomab.

A hybrid-hybridoma derived intact bispecific antibody with specificities against HER-2 and CD3 combining the two subclasses mouse IgG2a and rat IgG2b, which are evolutionary related and highly homologous Ig-subclasses.

Figure 2. Analysis of proliferation of JIMT-I and BT-474 cells incubated with trastuzumab; cell proliferation analyzed at cell density of 2.5x10⁴ cells/mL.

Figure 3. Analysis of proliferation of JIMT-I and BT-474 cells incubated with trastuzumab; cell proliferation analyzed at cell density of 5x10⁴ cells/mL.

Figure 4. Analysis of ertumaxomab mediated cellular cytotoxicity towards JIMT-I cells. Data show mean % residual tumor cells (plus standard deviation). Figure 5. Analysis of ertumaxomab mediated cellular cytotoxicity towards SK-BR-3 cells. Data show mean % residual tumor cells (plus standard deviation).

Figure 6. Analysis of ertumaxomab mediated cellular cytotoxicity towards SK-OV-3 cells. Data show mean % residual tumor cells (plus standard deviation).

Figure 7. Analysis of ertumaxomab mediated cellular cytotoxicity towards JIMT-I cells. Data show mean % residual tumor cells (plus standard deviation). Figure 8. Analysis of proliferation of BT-474HR and BT-474 cells incubated with trastuzumab; cell proliferation analyzed at cell density of 2.5x10⁴ cells/mL.

Figure 9. Analysis of proliferation of BT-474HR and BT-474 cells incubated with trastuzumab; cell proliferation analyzed at cell density of 5x10⁴ cells/mL.

Figure 10. Analysis of ertumaxomab mediated cellular cytotoxicity towards BT-474HR cells.

Figure 11. Analysis of ertumaxomab mediated cellular cytotoxicity towards BT-474 cells.

Figure 12. Analysis of proliferation of MKN-7 and BT-474 cells incubated with trastuzumab; % cell proliferation (±SD) was analyzed at a cell density of

2.5xl 0⁴ cells/mL.

Figure 13. Analysis of proliferation of MKN-7 and BT-474 cells incubated with trastuzumab; % cell proliferation (±SD) was analyzed at a cell density of 5x10⁴ cells/mL.

Figure 14. Analysis of ertumaxomab mediated cellular cytotoxicity against MKN-7 cells. Data show mean % residual tumor cells (± SD).

Figure 15. Analysis of ertumaxomab mediated cellular cytotoxicity against BT-474 cells. Data show mean % residual tumor cells (±SD).

Figure 16. Dose-response of Lapatinib on SK-BR-3 and SK-BR-3Lap cells. Shown are the mean values of fa (Effect) in correlation to the μM dose of Lapatinib (Dose).

Figure 17. Dose-response of Lapatinib on BT-474 and BT-474Lap cells. Shown are the mean values of fa (Effect) in correlation to the μM dose of Lapatinib (Dose).

Figure 18. Anti-tumoral activity of ertumaxomab against SK-BR-3 cells. Analysis of % residual tumor cells after 3 days of co-cultivation of SK-BR-3 cells with MNC in the presence of ertumaxomab. Shown are the mean values of % residual tumor cells ± SD of control without any antibody versus antibody concentration.

Figure 19. Anti-tumoral activity of ertumaxomab against SK-BR-3Lap cells. Analysis of % residual tumor cells after 3 days of co-cultivation of SK-BR-3Lap cells with MNC in the presence of ertumaxomab. Shown are the mean values of % residual tumor cells ± SD of control without any antibody versus antibody concentration.

Figure 20. Anti-tumoral activity of ertumaxomab against BT-474 cells. Analysis of % residual tumor cells after 3 days of co-cultivation of BT-474 cells with MNC in the presence of ertumaxomab. Shown are the mean values of % residual tumor cells ± SD of control without any antibody versus antibody concentration.

Figure 21. Anti-tumoral activity of ertumaxomab against BT-474Lap cells. Analysis of % residual tumor cells after 3 days of co-cultivation of BT-474Lap cells with

MNC in the presence of ertumaxomab. Shown are the mean values of % residual tumor cells ± SD of control without any antibody versus antibody concentration.

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Example 1: Characterization of the tumor target cells

The functionality and cytotoxic activity of the test item ertumaxomab (anti-HER-2 x anti- CD3) was investigated by an in vitro test system using the trastuzumab resistant HER-2 positive human tumor cell line JIMT-I (breast). As control test systems for the activity of trastuzumab the HER-2 positive human tumor cell lines SK-OV-3 (ovary), BT-474 (breast) and SK-BR-3 (breast) were used. The features of the cell lines used in this study are summarized in Table 1.

The expression level of HER-2 in these cell lines was quantified by flow cytometry using a murine antibody directed against HER-2 (clone 9G6.10, Alexis) and fluorescence calibration beads (Quifikit, DAKO). 5xl0⁵-10⁶ tumor cells were incubated with 20 μg/mL anti-HER-2 antibody followed by staining with saturating concentrations of FITC- conjugated anti-murine IgG antibodies. All antibody incubations were conducted for 30 min at 2-8°C. In parallel beads which have known numbers of binding sites for secondary anti-mouse IgG antibodies were stained with fluorescein isothiocyanate conjugated anti- mouse antibodies (Dianova). This allows correlating the fluorescence intensity signals with HER-2 binding sites per cell.

The expression levels of the tumor target cells used in these examples are shown in Table 2. The number of binding sites per cell was related to the clinical HER-2 score according to Ross et al. MoI Cell Proteomics 2004; 3(4): 379-398. All cell lines are known to have an amplification of the Her-2/neu gene but show different levels of expression. Whereas JIMT-I cells are classified for the HER-2 status 2+ BT-474, SK-BR-3 and SK-OV-3 cells are classified as 3+ according to Ross et al., supra.

Table 2: Expression level of tumor target cells

To verify the resistance of JIMT-I cells to trastuzumab, JIMT-I tumor cells were incubated with trastuzumab and cell proliferation was determined by thymidine incorporation into the cellular DNA. As a control, BT-474 tumor cells that are sensitive to trastuzumab were used.

In detail, tumor cells were incubated for 9 days following the instructions of the manufacturer in medium supplemented with 10 and 100 μg/mL trastuzumab. As a control, cells were incubated without trastuzumab. Cells were then seeded into 96-well plates at 2.5xlO⁴ cells or 5xlO⁴ cells/well and pulsed with thymidine for 18 h at 37°C, 5%CO₂ and 95 relative humidity (rH). Each concentration was tested in 32 well of a 96-well plate. The whole plates were frozen and stored at -20°C until further use. For determination of the thymidine incorporation, cells were thawed for 1 h at 37°C. The medium was decanted and cells were detached with trypsin for 20 min at 37°C. Using a cell harvester, cell suspensions were collected onto a filter membrane and counted using a beta-counter. Results are determined as counts per minute (cpm). For further analysis proliferation was calculated as % of control (without trastuzumab):

„, ,. _ ,. [cpm sample - cpm control] X 100 % proliferation = — ±- ±- - cpm control

Mean values and standard deviations are derived from 32 wells per antibody concentration. Proliferation of the control cell line BT-474 was inhibited by trastuzumab at a concentration of 10 μg/mL and 100 μg/mL, whereas JIMT-I cells were not inhibited by trastuzumab even at 100 μg/mL (Table 3, Table 4, Figure 2, Figure 3).

Table 3 : Analysis of proliferation of JIMT-I and BT-474 cells incubated with trastuzumab at 5xlO⁴/mL.

Table 4: Analysis of proliferation ol JIMT-I and BT-474 cells incubated with trastuzumab at 2.5x10⁴AnL.

In this example, the cytotoxic in vitro activity of ertumaxomab towards the HER-2 over- expressing human breast cancer cell line JIMT-I (FISH+/2+) was investigated. The JIMT- 1 cell line was isolated from a trastuzumab resistant patient with breast cancer and represents a model for trastuzumab (Herceptin^®) resistance in vitro and in vivo (Tanner et al., MoI. Cancer Ther. 2004; 3(12): 1585-1592). JIMT-I cells are characterized by an amplification of the Her-2/neu oncogene without any mutations in the coding sequence, a low shedding of HER-2 and a lack of growth inhibition by transtuzumab in vitro. Although JIMT-I cells express HER-2 only at a level of 100,000 molecules per cell (Mocanu et al., Cancer Lett. 2005; 227(2): 201-212) the expression levels of ER, PR, HER-I, HER-3 and HER-4 are similar to that of the trastuzumab-sensitive cell line SK-BR-3 (Mocanu et al, supra; Szollosi et al., Cancer Res. 1995; 55: 5400-5407). Trastuzumab resistance of JIMT- 1 cells is supposed to be based on reduced availability and a lack of activation of HER-2 (Nagy et al., Cancer Res. 2005; 65(2): 473-482). They express MUC4, a membrane- associated mucin that contributes to the masking of membrane proteins. It was concluded that masking of HER-2 in JIMT-I may lead to diminished trastuzumab binding. This study shows low binding of trastuzumab to JIMT-I cells when compared to SK-BR-3 cells that also over-express HER-2 but have no MUC4 expression.

In this example, the trastuzumab resistant phenotype of the JIMT-I cell line has been verified in vitro. Cell proliferation of JIMT-I cells was not decreased after incubation in the presence of 100 μg/mL trastuzumab for 9 days. In contrast, the trastuzumab sensitive cell line BT-474 showed a strong reduction of proliferation after incubation even with 10 μg/mL for 9 days. Example 2: Binding of ertumaxomab and trastuzumab to tumor target cells

To confirm binding of ertumaxomab and trastuzumab to tumor target cells, 5xl0⁵-lxl0 JIMT-I cells were incubated with 4 μg/mL ertumaxomab or trastuzumab for 30 min at 2°-

8°C. Binding of antibodies was detected by using FITC-conjugated secondary antibodies directed against rat IgG (for analysis of ertumaxomab binding) or antibodies directed against human IgG (for detection of trastuzumab binding) in saturating concentrations.

This second incubation was conducted for 30 min at 2-8°C. After a washing step, mean fluorescence intensity (MFI) was measured by flow cytometry. Cells were gated to exclude cell debris and dead cells. As a control, binding analysis was performed with SK-OV-3

(ovary), BT-474 and SK-BR-3 (breast) tumor cells.

Binding of ertumaxomab and trastuzumab to JIMT-I cells was demonstrated (Table 5, Table 6), but weak compared to the binding to SK-OV-3, BT-474 and SK-BR-3 cells.

Table 5: Binding of ertumaxomab to tumor cells.

Example 3: Analysis of ertumaxomab mediated killing of JIMT-I cells

The test item ertumaxomab is supposed to induce a specific cell-mediated elimination of tumor cells in the presence of peripheral blood mononuclear cells (PBMC). To assess the cytotoxic in vitro activity, the test item was added in varying concentrations to co-cultures of tumor target cells and mononuclear cells at an effector to target ratio of 10:1. In detail, SK-BR-3, SK-OV-3, BT-474 and JIMT-I tumor cells were seeded with 10⁴ cells per well in 96- well plates and incubated at 37⁰C and 5% CO2 for 24 h to assure adherence of the cells. The tumor cells were incubated for 3 days with medium containing mononuclear cells in the presence of varying concentrations of ertumaxomab or trastuzumab. Each concentration was tested in 8 wells of a 96-well plate. After 3 days incubation at 37°C and 5% CO2 the residual surviving tumor cells were quantified using the XTT-method:

Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple formazan. The absorbance of the resulting purple solution was measured spectrophotometrically at a wavelength of 450-500 nm. This method determines the enzymatic activity of viable cells which is directly correlated to the number of viable cells per sample. Data acquisition was performed with the software Magellan (Tecan). For further analysis the activity of residual tumor cells was calculated as percentage of residual tumor cells using the following formula:

. . . ., [mean absorbance sample - mean absorbance medium controllx 100

% residual tumor cells = - -

[mean absorbance control without antibody - mean absorbance medium control]

Mean values / SD are derived from 8 wells per antibody concentration.

In a first set of experiments ertumaxomab and trastuzumab were used at 0.33 ng/mL to 125 ng/mL (Table 7, Table 8, Table 9). In this setting the HER-2 over-expressing (3+) tumor cell lines SK-BR-3 (breast) and SK-OV-3 (ovary) were used as positive control for trastuzumab mediated cellular cytotoxicity.

Table 7: Analysis of ertumaxomab and trastuzumab mediated cellular cytotoxicity towards JIMT-I tumor cells.

Table 8: Analysis of ertumaxomab and trastuzumab mediated cellular cytotoxicity towards SK-BR-3 tumor cells.

Table 9: Analysis of ertumaxomab and trastuzumab mediated cellular cytotoxicity towards SK-OV-3 tumor cells.

In a second set of experiments ertumaxomab and trastuzumab were used at different, overlapping concentrations (ertumaxomab: 0.069 to 35.2 ng/mL; trastuzumab: 11.7 to 230.9 μg/mL). For this experiment BT-474 tumor cells were used as a positive control for trastuzumab mediated cellular cytotoxicity (Table 10, Table 1 1).

Table 10: Analysis of ertumaxomab and trastuzumab mediated cellular cytotoxicity towards JIMT-I tumor cells, n.d. = not determined.

Table 11 : Analysis of ertumaxomab and trastuzumab mediated cellular cytotoxicity towards BT-474 tumor cells, n.d. = not determined.

Ertumaxomab mediated a concentration-dependent decrease of SK-BR-3, SK-OV-3 and JIMT-I tumor cells. Ertumaxomab and trastuzumab are both efficient in killing BT-474 (see also Fig. 1 1), SKBR-3 (Figure 5) and SK-OV-3 tumor cells (Figure 6) in vitro. The efficiency of ertumaxomab was higher than that of trastuzumab for these cell lines. Trastuzumab showed no dose-dependent cytotoxicity towards JIMT-I tumor cells (Figure 4, Figure 7).

Ertumaxomab is able to mediate a cellular cytotoxicity towards JIMT-I cells in vitro. This is in accordance with the finding that in vivo JIMT-I cells are prone to cellular cytotoxicity

(Barok et al., MoI. Cancer Ther. 2007; 6(7): 2065-2072). Ertumaxomab and trastuzumab are both efficient in killing BT-474, SK-OV-3 and SK-BR-3 cells in vitro. The efficiency of ertumaxomab was higher than that of trastuzumab for these cell lines. Trastuzumab showed no cytotoxicity against JIMT-I cells. The observed different cytotoxic activities may be explained by the different mode of action.

In summary, this example shows that ertumaxomab efficiently kills the human breast cancer cell line JIMT-I in the presence of PBMC in a dose-dependent manner. No cytotoxic effect on JIMT-I cells could be observed with trastuzumab under the same experimental conditions. These in vitro data are believed to be predictable for use of the invention in a patient, because the efficient killing activity of ertumaxomab was shown for an accepted model of trastuzumab resistance.

Without being bound to a particular scientific theory, based on the above findings, it is contemplated that there is a different mode of action that is responsible for the different functionality of trastuzumab vs. ertumaxumab. Even though ertumaxomab binds to a different epitope of HER-2 than trastuzumab, it is believed that epitope binding is not a major contributor to resistance, as cross-resistance of Herceptin-resistant tumor cells to alternative monospecific antibodies (pertuzumab) does occur (Nahta et al., 2006). This functionality may be independent from the level of binding to the tumor cells (cf. Example 2). The cytotoxicity of trastuzumab is believed to be mainly based on the interruption of signal transduction, whereas that of ertumaxumab results from an enhanced ADCC (cf. e.g. Table 7).

Example 4: Analysis of the effect of ertumaxomab on a trastuzumab resistant variant of the cell line BT-474

The aim of this study was to analyze the biological activity of the trifunctional, bispecific antibody ertumaxomab (anti-HER-2/neu x anti-CD3) towards a trastuzumab (Herceptin^®) resistant variant of the tumor cell line BT-474.

The human tumor cell line BT-474 was cultivated in DMEM medium supplemented with 10% FCS, 10% NCTC- 135, 10 mM HEPES, 100 mM Sodium pyruvate 1 mM, oxaloacetic acid 1.2 mM, bovine insuline 0.01 mg/ml plus 50 μg/mL trastuzumab. Medium was exchanged every 2 to 3 days and when cells were grown to near confluency cell were detached and split split at a ratio of 1 :4 and 1 :8 into new culture dishes to keep a subconfluent cell layer. For each split the cells were incubated with PBS/1 mM EDTA for 10 min at 37°C. The BT-474 cells were cultivated for a period of 5 weeks in the presence of 50 μg/mL trastuzumab for the selection of trastuzumab resistant cell clones. A cell bank was established by expansion of cells in the presence of 50 μg/mL trastuzumab for further 4 weeks.

Following an decrease of cell numbers during the first 2 weeks down to very few cells the cell number increased again in the last 2 weeks of the selection culture phase. Following the 5 week long selection phase a cell bank of the selected cell line (now named BT- 474HR) was generated by further expansion of these cells in the presence of 50 μg/mL trastuzumab. For each further experiment using BT-474HR cells were taken from the cell bank and expanded in the presence of 50 μg/mL trastuzumab. 3 days prior to each experiment using this cell line the cell culture medium was changed to medium without trastuzumab to prevent interference of residual trastuzumab bound to the cells with the experimental assays.

Table 12: Overview of cell lines used in this example

The expression level of HER-2/neu was quantified as described in Example 1. The number of binding sites per cell was related to the clinical HER-2/neu score according to Ross et al., supra. Both cell lines have an amplification of the HER-2/neu gene but show different levels of expression. BT-474 and BT-474HR cells are classified as 3+ according to Ross et.al., supra.

Table 13: Expression level of BT-474HR and BT-474 cells

Analysis of binding of ertumaxomab was performed as described in Example 2. Cells were gated to exclude cell debris and dead cells. Both BT-474 and BT-474HR show a high binding of trastuzumab and ertumaxomab.

Table 14: Binding of ertumaxomab to BT-474HR and BT-474 cells

Table 15: Binding of Trastuzumab to BT-474HR and BT-474 cells

BT-474 and BT-474HR cells were analyzed for amplification of the HER-2/neu gene by FISH (fluorescence in situ hybridization). This analysis was performed by an external laboratory (Blandfort & Lahr Institut fur Chromosomendiagnostik, Kaiserslautern,

Germany). Cells were fixed on glass slides and stained for DNA/nuclei by DAPI, the centromer region of the HER-2/neu gene locus (chromosome 17) by a centromer specific probe and the HER-2/neu gene by a HER-2/neu gene specific probe. Analysis was performed in 10 metaphase nuclei and 100 intact non-overlapping interphase nuclei. The overall gene-to-chromosome (17) ratio is calculated. A tumor is designated as "positive" for gene amplification if gene-to-chromosome (17) ratio is >2.0.

The FISH analysis of interphase and metaphase nuclei from BT-474 and BT-474HR cells showed a high HER-2/neu gene amplification that was not quantifiable.

For BT-474 cells 60% of interphase nuclei had 6 signals for centromer of chromosome 17, about 30% showed 5 signals and some showed 4 signals for centromer of chromosome 17. 6 metaphase nuclei showed 6 signals for centromer of chromosome 17, 4 metaphase nuclei showed 5 signals for centromer of chromosome 17. In metaphase nuclei 7 to 9 multiple signals in form of heterogeneous signals were detected additional to 5 to 7 single signals. The gene-to chromosome (17) ratio for HER-2/neu in BT-474 cells is >2. For BT-474HR cells 75% of interphase nuclei had 6 signals for centromer of chromosome 17, about 20% 5 signals for centromer of chromosome 17. 6 metaphase nuclei showed 6 signals for centromer of chromosome 17, one metaphase nucleus showed 5 signals, 3 metaphase nuclei showed 4 signals for centromer of chromosome 17. In metaphase nuclei 3 to 8 multiple signals in form of heterogeneous signals were detected additional to 1 to 7 single signals. The gene-to chromosome (17) ratio for HER-2/neu in BT-474HR cells is >2. The functionality and antitumoral activity of the test item ertumaxomab (anti-HER-2/neu x anti-CD3) was investigated by an in vitro test system using the trastuzumab resistant cell variant BT-474HR and BT-474 cells. To verify the resistance of BT-474HR cells to trastuzumab, this cell line was incubated with trastuzumab and subsequently cell proliferation was measured using the ³H-thymidine incorporating method, as described in Example 1, but wherein the tumor cells were incubated for a total of 9 days in cell culture medium supplemented with 5 and 50 μg/mL trastuzumab. As a control the parental BT- 474 tumor cells that are sensitive to trastuzumab were used. Cells were seeded at two densities (2.5 x 10⁴ /mL and 5x 10⁴/mL) into 96-well plates prior to ³H-thymidine incorporation. Proliferation of the control cell line BT-474 was inhibited by trastuzumab at a concentration of 10 μg/mL whereas BT-474HR cells were not inhibited even at 50 μg/mL trastuzumab (see Fig. 8 and Fig. 9). This confirms the resistant phenotype of the selected BT-474 cell variant.

To assess the cytotoxic in vitro activity, the test item was added in varying concentrations to co-cultures of tumor target cells and mononuclear cells at an effector to target ratio of 10:1. The residual tumor cells were quantified using the XTT-method, as described in Example 3. Ertumaxomab and trastuzumab were used at different but overlapping concentrations (ertumaxomab: 0.069 to 35.1 ng/niL; trastuzumab 5.85 ng/mL to 1 15450 ng/mL). BT-474 tumor cells were used as positive control for trastuzumab mediated cellular cytotoxicity.

Ertumaxomab mediated a concentration dependent decrease of residual BT-474HR and BT-474 tumor cells in two independent experiments. Trastuzumab mediated less cellular cytotoxicity towards BT-474HR and BT-474 tumor cells than ertumaxomab (see Fig. 10 for BT-474HR; Fig. 1 1 for BT-474).

Ertumaxomab is able to mediate a cellular cytotoxicity towards both BT474 cells and cell of the trastuzumab resistant variant in vitro.

In summary this example shows efficacy of ertumaxomab against the trastuzumab resistant variant of the BT-474 cell line in vitro. It is also expected that ertumaxomab is able to efficiently kill cells with acquired resistance to both trastuzumab and tyrosin kinase inhibitors, such as Lapatinib. Example 5: Analysis of the effect of ertumaxomab on the trastuzumab resistant cell line MKN-7

The aim of this example was to analyze the biological activity of the tri functional, bispecific antibody ertumaxomab (anti-HER-2/neu x anti-CD3) against the HER-2/neu positive tumor cell line MKN-7 that is known to be resistant to trastuzumab (Herceptin^®). The functionality and cytotoxic activity of the test item ertumaxomab (anti-HER- 2/neu x anti-CD3) was investigated by an in vitro test system using the trastuzumab resistant HER-2/neu positive cell line MKN-7. The MKN-7 cell line was isolated from a patient with gastric cancer and is known to be insensitive to trastuzumab growth inhibition in vitro. This is suggested to be due to a certain association of HER-2/neu and integrins in lipid rafts. As control test system for the activity of trastuzumab the cell line BT-474 (breast) was used. The features of the cell lines used in this example are summarized in Table 16.

Table 16: Overview of cell lines used in this study

The expression level of HER-2/neu was quantified by flow cytometry as described in Example 1. Cells were gated to exclude cell debris and dead cells. The number of binding sites per cell was related to the clinical HER-2/neu score according to Ross et al., supra. Both cell lines have an amplification of the HER-2/neu gene but show different levels of expression. MKN-7 cells are classified for the HER-2/neu status 2+, whereas BT-474 cells are classified as 3+ according to Ross et. al., supra.

Table 17: Expression level of MKN-7 cells and BT-474 cells

Ertumaxomab binding to MKN-7 cells was also tested. As a control, binding analysis was performed with BT-474 tumor cells. Due to different secondary antibodies used for the detection of ertumaxomab and trastuzumab mean fluorescence intensity can not be compared directly. Binding of ertumaxomab and trastuzumab to MKN-7 cells was lower than binding to BT-474 cells (Table 18, Table 19).

Table 18: Binding of ertumaxomab to MKN-7 cells and BT-474 cells

Table 19: Binding of trastuzumab to MKN-7 cells and BT-474 cells

MKN-7 cells were analyzed for amplification of the HER-2/neu gene by FISH (fluorescence in situ hybridization), as described in Example 4 above, but that analysis was performed in 4 metaphase nuclei and 100 intact non-overlapping interphase nuclei. The FISH analysis of interphase and metaphase nuclei from MKN-7 cells showed a high HER-2/neu gene amplification that was not quantifiable. 93% of interphase nuclei had 2 signals for centromer of chromosome 17, 4% 3 signals and 3% 4 signals for centromer of chromosome 17. All 4 metaphase nuclei showed 2 signals for centromer of chromosome 17, at least 3 single signals for HER-2/neu and at least 3 multiple signals in form of heterogeneous signals. The gene-to chromosome (17) ratio for HER-2/neu in MKN-7 cells is >2.

To verify resistance of MKN-7 tumor cells to trastuzumab, MKN-7 cells were incubated for a total of 9 days with 5 μg/mL and 50 μg/mL trastuzumab and proliferation was determined by thymidin incorporation, as described in Example 4. As a control BT-474 tumor cells that are sensitive to trastuzumab were used. Cells were seeded at two densities (2.5x10⁴/mL and 5xlO⁴/mL) into 96-well plates prior to thymidine incorporation. Mean values / SD are derived from 32 wells per antibody concentration. Proliferation of the control cell line BT-474 was inhibited by trastuzumab at a concentration of 5 μg/mL and 50 μg/mL whereas MKN-7 cells were not inhibited even by 100 μg/mL trastuzumab (Fig. 12 and Fig. 13).

In order to assure adherence, the tumor cells were seeded with 1x10⁴ cells per well in 96- well plates and incubated at 37°C and 5% CO₂ for 24 h before start of the assay. To assess the cytotoxic in vitro activity, ertumaxomab was added in varying concentrations to co- cultures of BT-474 and MKN-7 tumor cells and mononuclear cells at an effector to target ratio of 10:1. Each concentration was tested in 8 wells of a 96-well plate. After 3 days incubation at 37°C and 5%CO₂ the residual tumor cells were quantified using the XTT- method, as described in Example 3. Mean values / SD are derived from 8 wells per antibody concentration. Ertumaxomab and trastuzumab were used at different but overlapping concentrations (ertumaxomab: 0.034 to 17.6 ng/mL; trastuzumab: 1.1 to 281.6 μg/mL). Ertumaxomab mediated a concentration dependent decrease of residual MKN-7 and BT- 474 tumor cells in two independent experiments. Killing of MKN-7 cells > 82% was observed in the first experiment with 17.6 ng/mL. BT-474 cells were killed >97% with concentrations <17.6 ng/mL. The dose response curve for ertumaxomab with these cells was lower than compared to MKN-7 cells. In the second experiment killing of MKN-7 cells >73% was observed with 17.6 ng/mL. BT-474 were killed >45% at a concentration of 17.6 ng/mL.

Trastuzumab mediated its cellular cytotoxicity against BT-474 tumor cells (36% and 24% killing at 281.6 ng/mL) less efficiently than ertumaxomab and no cytotoxicity against MKN-7 tumor cells (Fig. 14, Fig. 15).

In this study the trastuzumab resistant phenotype of the MKN-7 cell line has been verified in vitro. Cell proliferation of MKN-7 cells was not decreased after incubation in the presence of 50 μg/mL trastuzumab for 9 days. In contrast, the trastuzumab sensitive cell line BT-474 showed a strong reduction of proliferation after incubation even with 5 μg/mL for 9 days. HER-2/neu gene amplification has been verified by heterogeneously stained regions not restricted to chromosome 17 by FISH.

Ertumaxomab is able to mediate a cellular cytotoxicity against MKN-7 cells in vitro. Whereas trastuzumab shows killing of BT-474 cells no killing of MKN-7 cells was observed. The efficiency of ertumaxomab was higher than that of trastuzumab for both cell lines.

In summary, this example shows that ertumaxomab efficiently kills the human gastric cancer cell line MKN-7 in the presence of PBMC in a dose dependent manner. No cytotoxic effect on MKN-7 cells could be observed with trastuzumab even at high concentrations. It is also expected that ertumaxomab is able to efficiently kill cells with acquired resistance to both trastuzumab and tyrosin kinase inhibitors, such as Lapatinib.

Example 6: Analysis of cytotoxicity of ertumaxomab against Lapatinib resistant cell lines in vitro

The aim of this study was the characterization of the biological activity of the trifunctional, bispecific antibody ertumaxomab (anti-HER-2/neu x anti-CD3) towards human tumor cells with acquired Lapatinib resistance. In this model of acquired resistance, chronic exposure to Lapatinib converts ErbB2-overexpressing breast cancer cells that are initially sensitive to Lapatinib-induced apoptosis to resistant cells. Resistance is mediated by enhanced estrogen receptor (ER) signalling, resulting in ER playing a more significant role in regulating cell survival and survivin rather than loss of ErbB2 expression or insensitivity of the ErbB2 pathway to Lapatinib.

BT-474 and SK-BR-3 tumor cells were cultivated for several weeks in the presence of Lapatinib. The cell culture medium containing Lapatinib was changed every 2-3 days. The initial concentration of Lapatinib was 0.058 μM for SK-BR-3 cells and 0.029 μM for BT- 474 cells. Each time when cells have adapted to the growth inhibiting effects of Lapatinib and started proliferating the concentration of Lapatinib was increased by a factor of 0.5 to 1.5 in the cell culture medium. This procedure was repeated for a total selection time span of 56 days for BT-474 cells and 74 days for SK-BR-3 cells. The final concentration of Lapatinib at which the tumor cells were still proliferating was 0.3 μM for BT-474 cells and 0.8 μM for SK-BR-3 cells.

To verify the effect of selection process the ED50 of Lapatinib was analyzed for the SK- BR-3 (SK-BR-3Lap) and BT-474 (BT-474Lap) cells that underwent the selection procedure in comparison to SK-BR-3 and BT-474 cells that were cultivated under standard conditions without Lapatinib. Prior to the evaluation of the ED50 those cells that underwent the selection process were cultivated for 3 days in the absence of Lapatinib to remove any residual Lapatinib.

To evaluate the ED50 of Lapatinib 1x10⁵ cell/mL BT-474 were incubated for 5 days in the presence of 0.00036 - 7.047 μM Lapatinib, BT-474 Lap cells were incubated for 5 days in the presence of 0.0001 1 - 24.3 μM Lapatinib. SK-BR-3 cells were incubated for 5 days in the presence of 0.0072 - 14.094 μM Lapatinib, SK-BR-3Lap cells were incubated for 5 days in the presence of 0.001 1 - 7.2 μM Lapatinib. The incubation was performed under cell culture conditions (37°C, 5% CO₂ in a humidified atmosphere). Subsequently, non adherent cells were removed from the cultures by washing using PBS. Residual viable tumor cells were quantified using the XTT reagent of cell proliferation kit II following the manufacturer instructions. Data acquisition was performed with the software Magellan (Tecan). For further analysis the activity of Lapatinib was calculated as fraction affected (fa):

Fraction affected [fa]= 1 - [mean absorbance sample - mean absorbance medium control]/[mean absorbance control without Lapatinib - mean absorbance medium control]. For calculation of the ED50 and the correlation coefficient r of the dose-response curve the Calcusyn software version 2.1 (Biosoft) was used. The ED50's evaluated for SK-BR-3Lap and SK-BR-3 cells are summarized in Table 20 and the dose response curves are shown in Fig. 16. The long term culture of SK-BR-3 cells with Lapatinib lead to a >38-fold increase of the ED50 of Lapatinib indicating the acquisition of Lapatinib resistance by the SK-BR-3Lap cell line. The ED50's evaluated for BT-474Lap and BT-474 cells are summarized in Table 20 and the dose response curves are shown in Fig. 17. The ED50 of Lapatinib on BT-474Lap cells was not quantifiable.

Table 20: Analysis of ED50 and correlation coefficient r for Lapatinib

The acquisition of Lapatinib resistance by the selected variant BT-474Lap was shown by the fact that a dose response for Lapatinib was not found. Effectivity of Lapatinib against these cells was observed at a concentration of 24.3 μM only. These results suggest that both SK-BR-3Lap and BT-474Lap are variants of their parental cell lines with acquired resistance to Lapatinib.

For the analysis of ertumaxomab-mediated cellular cytotoxicity mononuclear cells/mL and tumor cells (SK-BR-3, SK-BR-3Lap, BT-474, BT-474Lap) were seeded in microtiter plates and allowed to adhere over night. After removing the supernatant on the next day co-cultures with MNC and several concentrations of ertumaxomab were initiated. 5x10⁵ MNC/mL were added to each of the tumor cell cultures to achieve an effector to target ratio of 10: 1. Ertumaxomab was analyzed in a concentration range between 150 ng/mL and 0.04 ng/mL. Co-cultures were incubated for three days at 37⁰C, 5% CO₂ in a humidified atmosphere. Non adherent cells were removed from the co-cultures by extensive washing using PBS. Residual viable tumor cells were quantified using the XTT reagent of cell proliferation kit II following the manufacturer instructions. Data acquisition was performed with the software Magellan (Tecan). For further analysis the activity of residual tumor cells was calculated as % residual tumor cells: residual tumor cells [%]=

[mean absorbance sample - mean absorbance medium control]/[mean absorbance control without antibody - mean absorbance medium control]xlOO.

Increase of residual tumor cells above 100% indicates tumor cell growth and absence of cytotoxic activity.

Ertumaxomab showed a similar high anti-tumoral activity against the Lapatinib resistant cell variants and the parental SK-BR-3 and BT-474 cell lines [Fig. 18, Fig, 19, Fig. 20, Fig. 21]. Killing of SK-BR-3Lap cells by >85% was achieved at 0.615 ng/mL ertumaxomab, of SK-BR-3 cells at 1.55 ng/mL, of BT-474Lap cells at 1.55 ng/mL and of BT-474 cells 9.6 ng/mL ertumaxomab.

It was found that ertumaxomab mediated an efficient, concentration dependent killing of SK-BR-3Lap, BT474Lap, SK-BR-3 and BT-474 tumor cells. Ertumaxomab mediated cellular cytotoxicity against Lapatinib resistant cell lines in vitro was observed at clinical concentrations. In conclusion, ertumaxomab is able to efficiently kill cells with acquired resistance to Lapatinib. It is also expected that ertumaxomab is able to efficiently kill cells with acquired resistance to both Lapatinib and trastuzumab.

References

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EP 1820513 Al Destruction of tumor cells expressing low to medium levels of tumor associated target antigens by trifunctional bispecific antibodies

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Claims

Fresenius Biotech GmbH February 26, 2009F65107PC1Claims

1. A trifunctional, bispecific antibody having the following properties: (a) binding to a T-cell specific cell surface protein,

(b) binding to the tumor-associated antigen HER-2 on a tumor cell, and

(c) binding to Fc-gamma-receptor type I and/or type III positive cells, for use in the treatment of a HER-2 expressing tumor, wherein the tumor is or becomes resistant against one or more tyrosine kinase inhibitor.

2. The use of claim 1 , wherein the tumor additionally is or becomes resistant against one or more monospecific anti-HER-2 antibody.

3. The use of claim 1 or 2, wherein the said treatment comprises administration of one or more tyrosine kinase inhibitor and/or one or more monospecific anti-HER-2 antibody.

4. The use of any of claims 1 to 3, wherein the one or more tyrosine kinase inhibitor (TKI) is lapatinib (Tykerb), gefitinib (Iressa^®, ZDl 839), imatinib (Gleevec^®, STI-

571), erlotinib (Tarceva^®), lanafamib (Sarasar^®), sorafinib and/or sunitimib.

5. The use of any of claims 1 to 4, wherein the one or more monospecific anti-HER-2 antibody is trastuzumab (Herceptin^®) and/or pertuzumab (Omnitarg^®, 2C4).

6. The use of any of the preceding claims, wherein said HER-2 expressing tumor is selected from the group consisting of breast tumor, ovarian tumor, prostate tumor, colon tumor, pancreas tumor, stomach tumor, esophagus tumor, endometrium tumor, skin tumor, oropharynx tumor, larynx tumor, cervix tumor, bladder tumor, preferably carcinoma, more preferably adenocarcinoma or squamous cell carcinoma.

7. The use of any of the preceding claims, wherein said HER-2 expressing tumor is a HER-2 over-expressing tumor, preferably wherein said HER-2 over-expressing tumor expresses HER-2 at a level of about 50,000, preferably 75,000, more preferably about 100,000 to about 10,000,000 receptors/tumor cell; preferably of greater than about 150,000 to about 10,000,000 receptors/tumor cell.

8. The use of claim 7, wherein the HER-2 over-expressing tumor is classified by a value in the HercepTest of 2+ or 3+.

9. The use of claim 7 or 8, wherein the HER-2 over-expressing tumor is a FISH positive (FISH+) tumor.

10. The use of any of the preceding claims, wherein the tumor is classified as (i) 2+ and FISH+ or (ii) 3+ and FISH+.

1 1. The use of any of the preceding claims, wherein the T-cell specific cell surface protein is CD3.

12. The use of claim 1 1, wherein said trifunctional bispecific antibody is an anti-HER-2 x anti-CD3 antibody binding to Fc-gamma-receptors type I and/or type III.

13. The use of claim 12, wherein the Fc portion comprises the isotype combination rat- IgG2b/mouse-IgG2a.

14. The use of any of the preceding claims, wherein said treatment further comprises administration of one or more monospecific antibody against an antigen other than HER-2, preferably one or more anti-EGFR antibodies, more preferably the monospecific anti-EGFR antibody cetuximab.

15. A pharmaceutical composition comprising (i) one or more trifunctional, bispecific antibody; and (ii) one or more anti-HER-2 antibody; optionally further comprising (iii) one or more monospecific antibody against an antigen other than HER-2, preferably one or more anti-EGFR antibodies, and/or (iv) one or more tyrosine kinase inhibitor; wherein component (i) to (iv) are as further defined in any of claims 1-14.

16. A kit comprising (i) one or more trifunctional, bispecific antibody, and (ii) one or more anti-HER-2 antibody; optionally further comprising (iii) one or more monospecific antibody against an antigen other than HER-2, preferably one or more anti-EGFR antibodies; and/or (iv) one or more tyrosine kinase inhibitor; wherein component (i) to (iv) are as further defined in any of claims 1-13.

17. A bispecific antibody or a fragment thereof, preferably wherein the antibody fragment is a single chain antibody (scFv), having the following properties: (a) binding to the tumor-associated antigen HER-2 on a tumor cell, and

(b) binding to immunocompetent cells, for use in the treatment of a HER-2 over-expressing tumor, wherein the tumor is or becomes resistant against one or more tyrosine kinase inhibitor.