Biomarkers for predicting outcomes of cancer therapy with ErbB3 inhibitors

Abstract

Methods for predicting a therapeutic response in a patient (e.g., a cancer patient) to ErbB3 inhibitors, and methods of treating a cancer patient with targeted therapies.

Claims

1. A method of treating a patient having a heregulin positive (HRG+) cancer with a solid tumor having a ratio of expressed ErbB4 to ErbB3 of less than 1.3, wherein the expressed ErbB4 and ErbB3 are each detected by RNA in situ hybridization (RNA-ISH) or by RT-PCR, the method comprising: administering a therapeutically effective amount of an anti-ErbB3 antibody to the patient, wherein the anti-ErbB3 antibody comprises CDRH1, CDRH2, and CDRH3 sequences comprising the amino acid sequences set forth in SEQ ID NO:1 (CDRH1), SEQ ID NO:2 (CDRH2), and SEQ ID NO:3 (CDRH3); and CDRL1, CDRL2, and CDRL3 sequences comprising the amino acid sequences set forth in SEQ ID NO:4 (CDRL1), SEQ ID NO:5 (CDRL2), and SEQ ID NO:6 (CDRL3).

2. The method of claim 1, wherein the tumor is a NSCLC tumor.

3. The method of claim 1, wherein the tumor is a melanoma tumor.

4. The method of claim 1, wherein the tumor is a breast tumor.

5. The method of claim 1, wherein the tumor is an ovarian tumor.

6. The method of claim 1, wherein the tumor is platinum-resistant or refractory.

7. The method of claim 1, wherein the anti-ErbB3 antibody is seribantumab.

8. A method of treating a patient diagnosed with a heregulin positive (HRG+) solid tumor having a ratio of expressed ErbB4 to ErbB3 of less than 1.3, wherein the expressed ErbB4 to ErbB3 are each detected by RNA in situ hybridization (RNA-ISH) or by RT-PCR, the method comprising: administering a therapeutically effective amount of an anti-ErbB3 antibody to the patient, wherein the anti-ErbB3 antibody comprises CDRH1, CDRH2, and CDRH3 sequences comprising the amino acid sequences set forth in SEQ ID NO:1 (CDRH1), SEQ ID NO:2 (CDRH2), and SEQ ID NO:3 (CDRH3); and CDRL1, CDRL2, and CDRL3 sequences comprising the amino acid sequences set forth in SEQ ID NO:4 (CDRL1), SEQ ID NO:5 (CDRL2), and SEQ ID NO:6 (CDRL3).

9. The method of claim 8, wherein the cancer is NSCLC.

10. The method of claim 8, wherein the cancer is melanoma.

11. The method of claim 8, wherein the cancer is breast cancer.

12. The method of claim 8, wherein the cancer is an ovarian cancer.

13. The method of claim 8, wherein the anti-ErbB3 antibody is seribantumab.

14. A method of treating a patient diagnosed with a cancer selected from the group consisting of: non-small cell lung cancer (NSCLC), melanoma, breast cancer, and ovarian cancer, the cancer having a heregulin positive (HRG+) solid tumor having a ratio of expressed ErbB4 to ErbB3 of less than 1.3, wherein the expressed ErbB4 and ErbB3 are each detected by RNA in situ hybridization (RNA-ISH) or by RT-PCR, and the method comprises administering a therapeutically effective amount of seribantumab to the patient.

15. The method of claim 14, wherein the breast cancer is a HRG+, ER+, PR+ and HER2 negative breast cancer.

16. The method of claim 14, wherein the cancer is a NSCLC cancer.

17. The method of claim 14, wherein the cancer is an ovarian cancer.

18. The method of claim 14, wherein the cancer is a melanoma cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic of the signaling hypothesis disclosed herein. Seribantumab is a human monoclonal ErbB3 antibody that competes with HRG for binding to ErbB3. Seribantumab inhibits the effect of HRG only in tumors with low ErbB4/ErbB3 ratio, indicating that a high ErbB4/ErbB3 ratio might interfere with seribantumab efficacy.

(2) FIGS. 2A-E show the effect of seribantumab on HRG-stimulated cell proliferation in a panel of NCI-60 cell lines with different ErbB4/ErbB3 ratios. FIG. 2A is a list of cell lines with the ErbB4/ErbB3 ratio and percent inhibition by seribantumab indicated. Twenty percent growth inhibition by seribantumab (as compared to control) was used as the cutoff to separate responder cell lines from non-responder cell lines. The results are expressed as mean+/SD of triplicate wells. FIG. 2B shows examples of two responder cell lines, OVCAR-8 (ovarian) and A549 (lung) with low ErbB4/ErbB3 ratios. FIG. 2C shows examples of two non-responder cell lines with high ErbB4/ErbB3 ratios, IGR-OV1 (ovarian) and H522 (lung). FIG. 2D is a graph showing the correlation of HRG-stimulated cell growth with the ErbB4/ErbB3 ratio, and FIG. 2E shows the correlation of seribantumab-induced inhibition with the ErbB4/ErbB3 ratio.

(3) FIG. 3 is a set of images showing the effect of seribantumab on HRG signaling in cells with different ErbB4/ErbB3 ratios. Five cell lines spanning a range of ErbB4/ErbB3 ratios (cells used were A549 (ratio 0.016, lung), HCC-1428 (ratio 0.26, breast), CAMA-1 (ratio 1.0, breast), IGR-OV1 (ratio 5.3, ovarian), and H522 (ratio 36.5, lung)) were pretreated with 250 nM of seribantumab for 1 hour, followed by stimulation with 10 nM of HRG for 10 min. Cells were lysed, and levels of phosopho-ErbB3 (pErbB3), phospho-Akt (pAkt), and phospho-ERK (pERK) were measured by western blotting. GAPDH was used as a loading control.

(4) FIGS. 4A-C demonstrate that ErbB4 depletion renders IGR-OV1 cells sensitive to seribantumab. IGR-OV1 cells were infected with lentivirus containing either ErbB4 short hairpin RNA (shErbB4) or scrambled sequence shRNA (shSCR) as a negative control. The parental cell line refers to the wild-type cells. Puromycin resistant cells were collected and analyzed by western blot. FIG. 4A shows western blot analysis of extracts from transduced IGR-OV1 cells to confirm knockdown of ErbB4 by shErbB4, and shows the other ErbB members to demonstrate specificity. FIG. 4B shows the effect of seribantumab activity on HRG-induced signaling in cells transduced with shSCR or shErbB4, as analyzed by western blot. FIG. 4C shows the relative cell viability of wild type IGR-OV1 cells (parental, left panel), cells transduced with either shSCR control (middle panel), or shErbB4 (right panel), and shows the effect of seribantumab on HRG-induced growth as tested by the CellTiter-Glo (CTG) assay.

(5) FIGS. 4D-G demonstrate that ErbB4 depletion also renders CAMA-1 cells sensitive to seribantumab. CAMA-1 cells were transduced with lentivirus containing ErbB4 short interfering RNA (siRNA; siErbB4), ErbB4 short hairpin RNA (shErbB4), scrambled sequence siRNA (siSCR) or scrambled sequence shRNA (shSCR). Both siErbB4 and shErbB4 significantly down-regulated ErbB4 (western blot analysis, FIGS. 4D and 4F) and these cells, when treated with HRG, subsequently had a greater response to treatment with seribantumab (CTG assay, FIGS. 4E and 4G).

(6) FIGS. 5A-G show that overexpression of ErbB4 renders OVCAR-8 cells resistant to seribantumab. OVCAR-8 cells were infected with lentivirus encoding ErbB4-JMaCYT1 (the ErbB4 isoform comprising JMa and CYT1 splice variants), ErbB4-JMaCYT2 (the ErbB4 isoform comprising JMa and CYT2 splice variants), or GFP as a control. Puromycin resistant cells were selected and tested. FIG. 5A is an image of a western blot showing ErbB4 and ErbB3 levels to confirm ErbB4 overexpression (and GADPH levels as a control). FIG. 5B is an image of a western blot of cells transduced with GFP control, ErbB4 JMaCYT1 or ErbB4 JMaCYT2 in order to test the effect of seribantumab activity on HRG-induced signaling in cells overexpressing these isoforms of ErbB4. FIG. 5C shows the relative cell viability of OVCAR-8 cells transduced with the GFP control (GFP, left panel), ErbB4-JMaCYT1 (middle panel), or ErbB4-JMaCYT2 (right panel), and shows the effect of seribantumab on HRG-induced growth in cells overexpressing ErbB4, as tested by the CTG assay. FIG. 5D is an image of a western blot showing ErbB4 expression in individual selected clones of OVCAR-8 cells infected with lentivirus encoding ErbB4-JMaCYT1. FIG. 5E is an image of a western blot showing ErbB4 and ErbB3 levels for select clones. FIG. 5F is an image of a western blot showing the effect of seribantumab activity on HRG-induced cell signaling in select clones. FIG. 5G shows the effect of seribantumab on HRG-induced cell growth as tested by CellTiter-Glo (CTG) assay.

(7) FIGS. 6A-C show that ErbB4 depletion re-sensitizes ErbB4-overexpressing OVCAR-8 cells to seribantumab.

(8) In ErbB4 JMaCYT2 over-expressing OVCAR-8 cells, ErbB4 was knocked down with ErbB4 siRNA to test the sufficiency of ErbB4 to cause resistance to seribantumab. Scrambled sequence siRNA was used as control. FIG. 6A is a western blot showing ErbB4 and ErbB3 levels in ErbB4 siRNA and scrambled sequence siRNA transfected cells. FIG. 6B is a western blot showing the effect of seribantumab on HRG signaling in OVCAR-8-GFP control cells and ErbB4 over-expressing OVCAR-8 cells transfected with scrambled sequence siRNA or ErbB4siRNA. FIG. 6C shows the effect of seribantumab on HRG-induced cell growth tested by the CTG viability assay (HRG: 10 nM, seribantumab: 1250 nM).

(9) FIGS. 7A-B show the effect of seribantumab on the in vivo tumor growth of Erbb4 engineered cell lines. IGR-OV1 (FIG. 7A) or OVCAR8 (FIG. 7B) cells were subcutaneously injected into nude mice to establish tumor xenografts. Once tumors reached approximately 200 mm.sup.3 in volume, the mice were randomized into treatment groups to receive either 600 g seribantumab every 3 days (Q3D) or PBS (Q3D) as a control. Tumor dimensions were measured twice a week and the tumor volumes were calculated using the formula: /6LW.sup.2.

(10) FIG. 8 is a table showing collected data from the Examples. In the two right-hand columns, stimulation and inhibition refer to effects on cell proliferation. In the second column, parental/engineered refers to the nature of the cell line named in the same row in the first column.

DETAILED DESCRIPTION

(11) Provided herein are methods for selecting and/or optimizing therapy for patients having cancer (e.g., non-hematological cancers) by determining whether the patient will benefit from treatment with an ErbB3 inhibitor (e.g., an antibody, such as seribantumab), based on particular biomarker scores obtained from a biological sample of the patient (i.e., ErbB3 and ErbB4, and the ratio of ErbB4:ErbB3 (also denoted herein as ErbB4/ErbB3).

(12) ErbB3 and HER3 both refer to human ErbB3 protein, as described in U.S. Pat. No. 5,480,968.

(13) ErbB3 inhibitor indicates a therapeutic agent that inhibits, downmodulates, suppresses or downregulates activity or expression of ErbB3, e.g., an agent that does one or more of the following: reduces cellular ErbB3 levels, reduces ligand binding to ErbB3, and reduces ErbB3-mediated intracellular signal transduction. The term is intended to include small molecule kinase inhibitors, antibodies, interfering RNAs (shRNA, siRNA), soluble receptors, and the like. Exemplary ErbB3 inhibitors are an anti-ErbB3 antibody, an anti-heregulin antibody, a heregulin-binding ErbB3 receptor fragment, or an ErbB3 anti-sense nucleic acid molecule. Representative inhibitors of ErbB3 and HRG binding and methods of their use are disclosed, e.g., in U.S. Pat. Nos. 7,125,680 and 7,314,916.

(14) An anti-ErbB3 antibody is an antibody that immunospecifically binds to the ectodomain of ErbB3. The antibody may be an isolated antibody. Exemplary anti-ErbB3 antibodies inhibit ligand-mediated phosphorylation of ErbB3 by HRG, and some (such as seribantumab) also inhibit phosphorylation of ErbB3 mediated by one or more of the EGF-like ligands EGF, TGF, betacellulin, heparin-binding epidermal growth factor, biregulin, epigen, epiregulin, and amphiregulin.

(15) An antibody, is a protein consisting of one or more polypeptides comprising binding domains substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes, wherein the protein immunospecifically binds to an antigen. One type of naturally occurring immunoglobulin structural unit (e.g., an IgG) comprises a tetramer that is composed of two identical pairs of polypeptide chains, each pair having one light (about 25 kD) and one heavy chain (about 50-70 kD). VL and VH refer to the variable regions of these light and heavy chains respectively. Antibodies include intact proteins as well as antigen-binding fragments, which may be produced by digestion of intact proteins, e.g., with various peptidases, or may be synthesized de novo either chemically or using recombinant DNA expression technology. Such fragments include, for example, F(ab)2 dimers and Fab monomers, and single chain antibodies. Single chain antibodies exist, generally due to genetic engineering, as a single polypeptide chain, e.g., single chain Fv antibodies (scFv) in which a VH fragment and a VL fragment are joined together (directly or through a peptide linker) to form a continuous polypeptide that retains immunospecific binding activity. Inhibitors can inhibit growth of such tumors.

(16) The terms suppress, suppression, inhibit and inhibition as used herein, refer to any statistically significant decrease in biological activity (e.g., tumor cell growth), including full blocking of the activity. For example, inhibition can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity.

(17) The term patient indicates a human subject receiving either prophylactic or therapeutic treatment.

(18) The terms treat, treating, and treatment, as used herein, refer to therapeutic or preventative (prophylactic) measures such as those described herein. The methods of treatment employ administration to a patient of an ErbB3 inhibitor as provided herein, for example, a patient having cancer, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the cancer, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.

(19) The term effective amount, as used herein, refers to that amount of an agent, such as an anti-ErbB3 antibody, which is sufficient to product a therapeutic benefit when administered to a patient.

(20) The terms anti-cancer agent and antineoplastic agent refer to drugs used to treat malignancies, such as cancerous growths.

(21) ErbB3 Inhibitors

(22) Methods provided herein can be used to predict efficacy of therapeutic treatment using any suitable ErbB3 inhibitor or combination of inhibitors.

(23) In one embodiment, the ErbB3 inhibitor is an anti-ErbB3 antibody, e.g., a monoclonal antibody. In an exemplary embodiment, the ErbB3 inhibitor is seribantumab. Alternately, the anti-ErbB3 monoclonal antibody is an antibody that competes with seribantumab for binding to ErbB3. In another embodiment, the anti-ErbB3 antibody is an antibody comprising the V.sub.H and V.sub.L CDR sequences of seribantumab in the same relative order as they are present in seribantumab, and which are disclosed herein as SEQ ID NOs: 1-3 (V.sub.H CDR1, 2, 3) and 4-6 (V.sub.L CDR1, 2, 3), respectively. Other examples of anti-ErbB3 antibodies include Ab #3, Ab #14, Ab #17 and Ab #19, also described further in WO 2008/100624 and U.S. Pat. No. 7,846,440, and having V.sub.H and V.sub.L sequences as disclosed in the patent as SEQ ID NOs: 9 and 10, 17 and 18, 25 and 26, and 33 and 34, respectively. In another embodiment, the anti-ErbB3 antibody is an antibody comprising the V.sub.H and V.sub.L CDR sequences of Ab #3 (shown in the patent as SEQ ID NOs: 11-13 and 14-18, respectively) or antibody comprising the V.sub.H and V.sub.L CDR sequences of Ab #14 (shown in SEQ ID NOs: 19-21 and 22-24, respectively) or an antibody comprising the V.sub.H and V.sub.L CDR sequences of Ab #17 (shown in the patent as SEQ ID NOs: 27-29 and 30-32, respectively) or an antibody comprising the V.sub.H and V.sub.L CDR sequences of Ab #19 (shown in the patent as SEQ ID NOs: 35-37 and 38-40, respectively), each of said CDRs being present in the same relative order as they are present in the corresponding Ab # antibody.

(24) Yet other anti-ErbB3 binding sites (or portions thereof, such as CDRs, V domains or chains) that may be used are those from the anti-ErbB3 antibodies 1B4C3 (cat # sc-23865, Santa Cruz Biotechnology) and 2D1D12 (U3 Pharma AG), both of which are described in, e.g., U.S. Pat. No. 9,011,851, and are produced by hybridoma cell lines DSM ACC 2527 or DSM ACC 2517 (deposited at DSMZ); AV-203 (SEQ ID NO:190 (heavy chain) and SEQ ID NO:206 (light chain) in U.S. Pat. No. 8,481,687 (Aveo Pharmaceuticals); 8B8 (produced by ATCC hybridoma #HB-12070 and described in WO 1997/035885, Genentech); the monoclonal antibody mAb 205.10.2 (SEQ ID NO:8 (heavy chain) and SEQ ID NO:10 (light chain) in U.S. Pat. No. 8,859,737, Roche Glycart); the murine anti-ErbB3 antibody described in U.S. Pat. No. 8,362,215 (Trellis Biosciences) or the bispecific anti-ErbB3/anti-EGFR antibody MEHD7945a, Genentech).

(25) Patient Populations

(26) Provided herein are effective methods for treating cancer in a human patient and for selecting patients to be so treated. In one embodiment, the human patient suffers from a cancer selected from the group consisting of non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), melanoma (e.g., cutaneous or intraocular malignant melanoma), colorectal cancer, serous ovarian carcinoma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, breast cancer, lung cancer, uterine cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), spinal axis tumor, glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, and mesothelioma. The disclosed methods are also applicable to treatment of metastatic cancers. In a particular embodiment, the cancer is ovarian cancer. In another particular embodiment, the cancer is breast cancer. The breast cancer may be either or both of ER+ and PR+ breast cancer (ER+ and/or PR+). The breast cancer may be HER2 negative. The breast cancer may be either or both of 1) ER+ and/or PR+ and 2) HER2 negative. Methods for testing ER and PR status are used as a matter of clinical routine in the treatment of gynecological tumors. Such methods may be carried out in accordance with the well-established guidelines of Hammond, M E et al., American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunological Testing of Estrogen and Progesterone Receptors in Breast Cancer Arch Pathol Lab Med. 2010; 134:E1-E16. HER2 status may be determined using HCT, with a score of 3+ being considered HER2 positive and a score of 2+ or 1+ or 0 being considered HER2 negative.

(27) In one embodiment, a human patient for treatment using the subject methods and compositions has evidence of recurrent or persistent disease following primary chemotherapy.

(28) In another embodiment, a human patient for treatment using the subject methods and compositions has had at least one prior platinum based chemotherapy regimen for management of primary or recurrent disease.

(29) In another embodiment, the patient has a cancer that is platinum-resistant or refractory. In one example, the platinum-resistant cancer is ovarian cancer.

(30) In another embodiment, a human patient for treatment using the subject methods and compositions has evidence of recurrent or persistent disease following a) primary treatment, e.g., with an anti-estrogen therapy or b) an adjuvant treatment with a non-steroidal aromatase inhibitor and/or tamoxifen.

(31) In another embodiment, the cancer undergoing treatment is advanced. In one aspect, the term advanced cancer denotes a cancer above Stage II. In another, advanced refers to a stage of disease where chemotherapy is typically recommended, which is any one of the following: 1) in the setting of recurrent disease: any stage or grade; 2) stage IC or higher, any grade; 3) stage IA or IB, grade 2 or 3; or 4) in the setting of incomplete surgery or suspected residual disease after surgery (where further surgery cannot be performed): any stage or grade.

(32) Outcomes

(33) The efficacy of the treatment methods provided herein can be assessed using any suitable means. In one embodiment, the treatment produces at least one therapeutic effect selected from the group consisting of reduction in growth rate of tumor, reduction in size of tumor, reduction in number of metastatic lesions over time, increase in duration of progression-free survival, and increase in overall response rate.

(34) With respect to target lesions, responses to therapy may include:

(35) Complete Response (CR):

(36) Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm;

(37) Partial Response (PR):

(38) At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters;

(39) Progressive Disease (PD):

(40) At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression); and

(41) Stable Disease (SD):

(42) Neither sufficient shrinkage to qualify for PR, nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study. (Note: a change of 20% or less that does not increase the sum of the diameters by 5 mm or more is coded as stable disease). To be assigned a status of stable disease, measurements must have met the stable disease criteria at least once after study entry at a minimum interval of 6 weeks.

(43) With respect to non-target lesions, responses to therapy may include:

(44) Complete Response (CR):

(45) Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis). If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response;

(46) Non-CR/Non-PD:

(47) Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits; and

(48) Progressive Disease (PD):

(49) Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase.

(50) In exemplary outcomes, patients treated according to the methods disclosed herein may experience improvement in at least one sign of a cancer, such as platinum resistant/refractory advanced ovarian cancer.

(51) In one embodiment, the patient so treated exhibits CR, PR, or SD.

(52) In another embodiment, the patient so treated experiences tumor shrinkage and/or decrease in growth rate, i.e., suppression of tumor growth. In yet another embodiment, one or more of the following can occur: the number of cancer cells is reduced; tumor size is reduced; cancer cell infiltration into peripheral organs is inhibited, retarded, slowed, or stopped; tumor metastasis is slowed or inhibited; tumor growth is inhibited; recurrence of tumor is prevented or delayed; or one or more of the symptoms associated with cancer is relieved to some extent.

(53) In other embodiments, such improvement is measured by a reduction in the quantity and/or size of measurable tumor lesions. Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter is to be recorded) as >10 mm by either or both of CT scan (CT scan slice thickness no greater than 5 mm) and caliper measurement via clinical exam, or as >20 mm by chest X-ray. The size of non-target lesions, e.g., pathological lymph nodes, can also be measured for improvement. In one embodiment, lesions can be measured on chest x-rays or CT or MRI outputs.

(54) In other embodiments, cytology or histology can be used to evaluate responsiveness to a therapy. The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease can be considered to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease.

(55) The following Examples are illustrative and should not be construed as limiting the scope of this disclosure in any way, as many variations and equivalents will become apparent to those skilled in the art upon reading the present disclosure.

EXAMPLES

Materials and Methods

Materials

(56) The ErbB4 shRNA lentivirus is from Sigma-Aldrich (St. Louis, Mo., USA; cat # TRCN00000014011). The ErbB4 JMaCYT1 and ErbB4 JMaCYT2 isoform cDNA lentivirus (Catalog Nos. LP-A0212-Lv105 and LP-Z4265-Lv105, respectively) are from GeneCopoeia (Rockville, Md., USA). The 3D NanoCulture plates are from SCIVAX Life Sciences, Inc. (Woburn, Mass., USA). All materials for western blot assays are from Bio-Rad Laboratories (Hercules, Calif., USA). Antibodies against pErbB3, pAkt, pERK, total EGFR and ErbB4 are from Cell Signaling Technology (Beverly, Mass., USA; cat #s 4561, 9271, 4370, 2232, and 4795, respectively). The antibody against total ErbB3 is from Abcam (Cambridge, Mass., USA; cat #1186-1). The antibodies against GAPDH (cat#MAB374) and total ErbB2 (cat#04-291) are from EMD Millipore (Temecula, Calif., USA). The secondary antibodies are from Li-Cor Biosciences (Lincoln, Nebr., USA; IRDye 680 anti-mouse IgG: #926-32222 and IRDye 800 anti-rabbit IgG: #926-32211). HRG1-beta 1 is from R&D Systems (Minneapolis, Minn., USA; cat#396-HB). CellTiter-Glo Luminescent Cell Viability Assay kit is from Promega (Madison, Wis., USA). ErbB4 siRNA (cat# L-003128-00), scrambled sequence siRNA (cat# D-001310-01) and transfection reagent (Dharmafect, cat# T-2001-01) are purchased from Dharmacon, Inc. (Lafayette, Colo., USA). Cell culture medium and all other reagents are from Gibco/Life Technologies unless otherwise specified.

(57) Cell Culture

(58) Cell lines from NCI-60 panel (NCI, Rockville, Md., USA) are cultured in RPMI 1640 (ATCC 30-2001) supplemented with 10% fatal calf serum (FCS), 2 mM of L-glutamine, and 100 units/ml penicillin/streptomycin. ErbB4-engineered cell lines are cultured in above medium plus puromycin to maintain their genotype. All cells are grown in a humidified atmosphere at 37 C. with 5% CO.sub.2, unless otherwise indicated. Cells are sub-cultured every 3-4 days to maintain a logarithmic growth phase. Cell lines from the NCI-60 anti-cancer cell lines used in the assays disclosed herein include, e.g., those in FIG. 2A. FIG. 8 is a summary of all parental and engineered cell lines used in the Examples below.

(59) Cell Viability/Proliferation Assay

(60) Cells are sub-cultured one day before to make sure they are in log growing phase. Cells are then seeded either into 96-well 3D NanoCulture plate at a density of 5000 cells/well or into 96-well 2D culture plate at a density of 800-2000 cells/well depending on cell growth rate and cultured in RPMI containing 2% fetal calf serum for 24 hr. The cells are then treated with different doses of seribantumab in the presence or absence of 10 nM HRG, each dose is in triplicates. At day 5, cell viability is measured using the CTG assay according to the manufacturer's instructions. Results are expressed as mean+/SEMD of triplicate wells.

(61) In Vivo Efficacy Study

(62) Female nu/nu nude mice (NU-Foxn1nu, Charles River Laboratories, Wilmington, Mass., USA), 4-5 weeks of age, weighing 160.5 g, are inoculated with 0.2 ml of cell suspension in phosphate buffered saline (PBS): Growth Factor Reduced Matrigel (BD Biosciences) of IGR-OV1 (8*10.sup.6/mouse) or A549 (5*10.sup.6/mouse), or OVCAR-8 (8*10.sup.6/mouse). Each tumor model is represented by a parental control, green fluorescent protein, or scrambled sequence control and ErbB4 modified versions. Once tumors reached approximately 200 mm.sup.3 in volume, the mice are randomized into treatment groups (10 mice/group) to receive either 600 g seribantumab or PBS (control) every 3 days (Q3D). Tumor dimensions are measured twice a week and the tumor volumes calculated using the formula: /6LW2, where L and W, respectively, represented the larger and smaller tumor diameter. At the end of the four week treatment, mice are sacrificed and tumor bulk collected and frozen.

(63) Stable transduction of target cells with lentivirusesStable ErbB4 knockdown or over-expression engineered cell lines are established by transducing either ErbB4 shRNA lentivirus or ErbB4 JMaCYT1 and JMaCYT2 isoform lentivirus into proper cell lines. Cells are seeded into 24-well plates and cultured in complete medium overnight. At 70-80% confluence, transduce the cells by removing the old medium and replacing it with 0.5 ml of complete medium (without antibiotics) containing 8 g/ml of Polybrene and diluted lentivirus. Culture the transduced cells in a standard 37C cell culture incubator overnight. Replace the virus containing old medium with fresh complete culture medium and culture it overnight. Two days after the transduction, subculture the cells into 10-cm petri-dish with complete medium containing the different concentrations of selection antibiotics determined by kill curve of each target cell lines. The selection medium is changed every 3-4 days. The ErbB4 knockdown or over-expression in the selection drug resistant pooled cells are validated by western blotting.

(64) Western Blotting

(65) Cells are seeded into 6-well culture plates at 70% confluence and cultured in RPMI containing 10% FBS overnight. The medium is changed to 2% FBS medium and cells are pre-incubated with 250 nM of seribantumab, or buffer (control), for 1 hr. The cells are then stimulated with HRG-1-beta-1 EGF domain at a final concentration of 10 nM for 10 minutes. The reaction is stopped by removing the supernatants and washing with ice-cold PBS. The cells are harvested in 2 protein sample buffer by scraping the cells. The genomic DNA is sheared by passing it through a 21-gauge syringe needle several times. The homogenized cell lysates are boiled for 5 min and centrifuged at 12,000 rpm for 5 min. Protein from 5*10.sup.4 cells (25 g) is subjected to electrophoresis on 4-12% gradient gels and electro-transferred to nitrocellulose membrane (BioRad). Nonspecific binding is blocked by incubating with blocking buffer (#927-40000, LI-COR). Western blots of the gels are probed with different primary antibodies (Cell Signaling, see materials above) followed by incubating with secondary antibody (LI-COR). The blots are then imaged on a LI-COR Odyssey infra-red imaging system. GAPDH is detected in each blot as an internal control.

Example 1: Effect of ErbB4/ErbB3 Ratio on Seribantumab Activity in Various Cell Lines

(66) To test the inhibitory effect of seribantumab, a panel of human cell lines with different ErbB4/ErbB3 ratios was selected from the NCI-60 panel (FIG. 2A). A CTG assay was used as the readout to test the effect of ErbB4/ErbB3 ratio on the ability of seribantumab to inhibit HRG-induced cell proliferation. Inhibition was defined as a 20% reduction in cell proliferation (via CTG) by 1.25 M of seribantumab as compared to control, wherein a cell line with 20% or more reduction in cell proliferation was identified as a responder, and a cell line with less than 20% inhibition of proliferation was identified as a non-responder. The A549 and OVCAR-8 cell lines (see FIG. 2B) are an example of responder cell lines. Both cell lines have an ErbB4/ErbB3 ratio of less than 0.1, and 10 nM of HRG induced cell proliferation by 4.1-fold and 1.5-fold, respectively. Seribantumab significantly inhibited the HRG stimulation by 70% and 41%, respectively. The IGR-OV1 and H522 cell lines shown in FIG. 2C are an illustrative example of non-responder cells. In both cell lines, each with an ErbB4/ErbB3 ratio of more than 5, 10 nM of HRG induced cell proliferation by 3-fold (IGR-OV1) and 2-fold (H522). However, seribantumab did not block the effect of HRG in either cell line. It was observed that seribantumab non-responder cells all had high ErbB4 levels, and there was a clear correlation between the ErbB4/ErbB3 ratio and the responsiveness of the cells to seribantumab inhibition (FIG. 2C). For all the cell lines analyzed, the ErbB4/ErbB3 ratio didn't affect the sensitivity of cells to HRG stimulation (FIG. 2D), but there was an inverse correlation between the ErbB4/ErbB3 ratio and seribantumab activity (FIG. 2E).

(67) To examine the mechanism by which the high ErbB4/ErbB3 ratio attenuated seribantumab activity, the effect of the ErbB4/ErbB3 ratio on seribantumab inhibition of HRG-induced signaling was evaluated by western blot. As shown in FIG. 3, seribantumab potently inhibited HRG-induced ErbB3 phosphorylation in all the cell lines. However, in cell lines with low ErbB4/ErbB3 ratios, such as A549 and HCC1428, seribantumab treatment only inhibited events downstream of HRG induced signaling, such as production of pAkt and/or pERK. In contrast, seribantumab treatment was ineffective in cell lines with high ErbB4/ErbB3 ratios such as IGR-OV1 and H522. Seribantumab itself did not show any ErbB3 agonist effect (FIG. 3). Both the phenotypic data and signaling data support the hypothesis that the high ErbB4/ErbB3 ratios attenuated seribantumab activity.

Example 2: Effect of Knocking Down ErbB4 in High ErbB4/ErbB3 Ratio Cells on Seribantumab ActivityIn Vitro Study

(68) The effect of ErbB4/ErbB3 ratio on seribantumab activity was tested using ErbB4-engineered cells. To determine whether seribantumab non-responders with a high ErbB4/ErbB3 ratio could become responders by reducing expression of (knocking down) ErbB4 protein, ErbB4 knockdown cells were created by treating IGR-OV1 and CAMA-1 cells with sh/siRNA. As shown in the western blot image in FIG. 4A, the expression of ErbB4 sequence-specific shRNA dramatically knocked down ErbB4 expression in IGR-OV1 cells by more than 80% without interfering with other ErbB family members. Cells transfected with scrambled sequence shRNA were used as a control. The ErbB4/ErbB3 ratio of IGR-OV1-parental, scrambled sequence and ErbB4 knockdown were 5.3, 4.4, and 0.93, respectively. These data show correlation between shRNA-induced ErbB4 down-regulation and the ability of seribantumab to inhibit HRG induced downstream production of pAkt and pERK (FIG. 4B). Next the, effect of seribantumab on HRG induced proliferation among these cell lines was investigated. Consistent with the data shown in FIGS. 2A-E, while seribantumab was ineffective at inhibiting HRG-induced cell growth in parental and scrambled sequence IGR-OV1 cells with high ErbB4/ErbB3 ratios, it dose-dependently inhibited HRG-induced cell growth in ErbB4 knockdown IGR-OV1 cells (FIG. 4C).

(69) Similar results were obtained from CAMA-1 cells as well (FIG. 4D-G). Both ErbB4 siRNA and shRNA significantly down-regulated ErbB4 (FIGS. 4D and 4F) and these cells subsequently had a greater response to treatment with seribantumab (FIGS. 4E and 4G). These data show that decreasing the ErbB4/ErbB3 ratio in seribantumab non-responder cells by knocking down ErbB4 rescued the ability of cells respond to seribantumab.

Example 3: Effect of ErbB4 Overexpression on Seribantumab ActivityIn Vitro Study

(70) To further test the relationship of the ErbB4/ErbB3 ratio to seribantumab activity, ErbB4 was overexpressed in the responder cell line OVCAR-8 which has a low ErbB4/ErbB3 ratio. Cells were transduced with a lentivirus vector encoding either the JMaCYT1 isoform of ErbB4 or the JMaCYT2 isoform of ErbB4 and cultured in puromycin selection medium. Cells transduced with GFP-encoding lentivirus vector were used as control. The ErbB4 expression level in pooled cell lines was tested by western blot. Both ErbB4 isoforms were highly expressed compared to that in parental and GFP-expressing control cells, as shown in FIG. 5A. The ErbB4/ErbB3 ratio of OVCAR-8 parental, GFP-expressing control, ErbB4 JMaCYT1, and JMaCYT2-expressing cell lines were 0.00014, 0.00022, 1.8, and 2.6, respectively. Increasing the ErbB4/ErbB3 ratio significantly decreased the ability of seribantumab to inhibit HRG-induced pAkt and pERK (FIG. 5B). Seribantumab significantly inhibited HRG induced pAkt and pERK in GFP control cells, but not in ErbB4 JMaCYT1- and ErbB4 JMaCYT2-expressing cells with high ErbB4/ErbB3 ratios.

(71) Next, the impact of increasing the ErbB4/ErbB3 ratio on seribantumab ability to inhibit cell growth was examined. In parental and GFP-expressing control cells with a very low ErbB4/ErbB3 ratio (<0.01), seribantumab at 1.25 M inhibited HRG induced cell proliferation by 53.7% and 40.8%, respectively. But for both OVCAR-8-JMaCYT1 and OVCAR-8-JMaCYT2 cells, seribantumab inhibited less than 20% of HRG-induced cell growth (FIG. 5C), indicating that increasing the ErbB4/ErbB3 ratio by overexpressing ErbB4 made the OVCAR-8 cells less sensitive to seribantumab. It was observed that while OVCAR-8-JMaCYT2 cells became resistant to seribantumab, they also lost sensitivity to HRG stimulation. In order to determine whether this might be due to the heterogeneity of the pooled cell population (possibly containing some cells with unknown transfection-mediated changes that caused cells to be unresponsive to HRG), cells were collected from single clones of the pooled cells in order to produce a homogenous cell culture. The selected clones showed a high degree of variability in the amount of ErbB4, as analyzed by western blot (FIG. 5D), suggesting that the heterogeneity in the pooled cell lines may have been due to variations in the ErbB4 expression level. For ongoing experiments, clones #4, #5, and #9, representing ErbB4 low, high, and medium over-expressing levels, respectively, were tested for their response to HRG stimulation and sensitivity to seribantumab inhibition. The ErbB4 and ErbB3 levels were determined for each clone (FIG. 5E) and the ErbB4/ErbB3 ratio was measured (see FIG. 5G). First, a set of CTG assays was performed to determine the ability of seribantumab to inhibit cell proliferations in the different expression clones. As shown in FIG. 5G, 10 nM HRG stimulated cell growth in all cell lines but clone#5 (high ErbB4/ErbB3 ratio), which may have contributed to the HRG unresponsiveness of the pooled OVCAR-8-JMaCYT2 cells shown in FIG. 5C. Seribantumab inhibited HRG-induced cell growth by 49% in OVCAR8-GFP cells, 45% in low ErbB4 expression clone #4, 25% in medium ErbB4 expression clone #9, and 8% in high ErbB4 expression clone #5, indicating a clear correlation between the ErbB4/ErbB3 ratio and the ability of seribantumab to inhibit HRG-induced cell growth (FIG. 5G). Similar results were obtained when signaling was examined by western blot; HRG treatment resulted in phosphorylation of ErbB3, Akt, and ERK in all the cell lines. While seribantumab almost completely inhibited HRG-induced production of pErbB3 regardless of ErbB4 level, the ability of seribantumab to inhibit the production of pAkt and pERK was attenuated with increased ErbB4 level (FIG. 5F). Clone#9 was further tested in the in vivo xenograft model described below in Example 5.

Example 4: Rescue Effect of Knocking Down ErbB4 with siRNA in OVCAR8-ErbB4JMa CYT2 Cells

(72) As described above for FIG. 5A, overexpressing ErbB4 in OVCAR-8 cells also changed the level of ErbB3 and other signaling pathway members. In order to confirm that the observed effect was due to the presence of ErbB4, ErbB4 was then transiently knocked down by using ErbB4 sequence-specific siRNA in OVCAR-8-ErbB4JMaCYT2 cells to determine if seribantumab sensitivity could be rescued. Scrambled sequence siRNA was used as a control (FIG. 6A). As shown in FIG. 6B, in scrambled sequence cells with high ErbB4, seribantumab could not inhibit HRG-induced pAkt and pERK; whereas in ErbB4 cells expressing the ErbB4 siRNA, seribantumab inhibited HRG-induced pAkt and pERK to a similar extent compared with GFP control cells. The data detected by the CTG assay also showed that seribantumab inhibited HRG-induced cell growth in ErbB4 siRNA transfected cells, but not in scrambled sequence siRNA control (FIG. 6C). Both the signaling data and the CTG assay data showed that knockdown of ErbB4 in OVCAR-8-ErbB4 cells re-sensitized the cells to seribantumab, indicating that ErbB4 is sufficient to cause seribantumab resistance in OVCAR-8 cells.

Example 5: Effect of ErbB4/ErbB3 Ratio on Seribantumab Activity In Vivo

(73) In order to test if the observed effect of the ErbB4/ErbB3 ratio in vitro might also be observed in an in vivo setting, a xenograft study was performed using ErbB4 stable knockdown IGR-OV1 cells and ErbB4 stable overexpressing OVCAR-8 cells as described above. In each model, seribantumab activity was compared among parental cells, engineered control cells (either scrambled sequence or GFP overexpression), and ErbB4 engineered cells. The tumor-bearing mice were treated with either PBS as a control or 600 g/kg of seribantumab. For the IGR-OV1-ErbB4 knockdown model, consistent with in vitro data, treatment with seribantumab had no effect on parental IGR-OV1 or IGR-OV1-scrambled sequence tumor growth, whereas tumor cell proliferation from ErbB4 knockdown-IGR-OV1 was significantly inhibited by seribantumab (FIG. 7A). In the OVCAR8-ErbB4 over-expression model, tumor growth from both parental and GFP-expressing OVCAR-8 cells was significantly inhibited by seribantumab, whereas tumor growth from OVCAR-8-ErbB4-expressing clone #9 was resistant to seribantumab treatment (FIG. 7B).

(74) Endnotes

(75) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features set forth herein. The disclosure of each and every U.S., international, or other patent or patent application or publication referred to herein is hereby incorporated herein by reference in its entirety.

SEQUENCE SUMMARY

(76) TABLE-US-00001 SEQ IDNO: DESIGNATION SEQUENCE 1 HeavyChain HisTyrValMetAla CDR1(CDRH1)of Seribantumab Protein 2 HeavyChain SerIleSerSerSerGlyGly CDR2(CDRH2)of TrpThrLeuTyrAlaAspSer Seribantumab ValLysGly Protein 3 HeavyChain GlyLeuLysMetAlaThrIle CDR3(CDRH3)of PheAspTyr Seribantumab Protein 4 LightChain ThrGlyThrSerSerAspVal CDR1(CDRL1)of GlySerTyrAsnValValSer Seribantumab Protein 5 LightChain GluValSerGlnArgProSer CDR2(CDRL2)of Seribantumab Protein 6 LightChain CysSerTyrAlaGlySerSer CDR3(CDRL3)of IlePheValIle Seribantumab Protein