WO2017161009A1 - Dosage and administration of combination therapies comprising targeted antibodies uses and methods of treatment - Google Patents

Abstract

Provided are methods of treating a patient with cancer with a targeted therapy in combination with chemotherapies. Also provided are methods of determining whether the patient is likely to respond to a treatment with the combinations.

Description

DOSAGE AND ADMINISTRATION OF COMBINATION THERAPIES COMPRISING TARGETED ANTIBODIES,

USES AND METHODS OF TREATMENT RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos. 62/309,254, filed March 16, 2016 and 62/323,411, filed April 15, 2016. The contents of the

aforementioned applications are hereby incorporated by reference. FIELD

Provided are methods of treating a patient with cancer with a targeted therapy in combination with chemotherapies. Additionally, methods of determining whether the patient is likely to respond to a treatment with the aforementioned combinations are described. BACKGROUND

Cancer therapy has advanced with the use of targeted agents that have significantly increased the utility of traditional chemotherapies as part of combination regimens. Most of the successes have been observed in those cancer subtypes in which a specific oncogenic protein is mutated, such as EGF receptor (EGFR), BRAF, or ALK, or the expression is amplified, such as ErbB2 in breast and gastric cancer. However, many patients never respond to these combination regimens or become refractory, suggesting the existence of uncharacterized tumor survival mechanisms, e.g., compensatory pathways. For example, although inhibition of IGF-1R was expected to eliminate a key resistance mechanism to anticancer therapies, clinical results to date have been disappointing. It has previously been established that adaptive v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3) signaling activated by its ligand heregulin (HRG) is a key factor limiting the utility of anti- IGF-1R agents. A series of biomolecules have been invented that co-inhibit IGF-1R and ErbB3, including a bispecific tetravalent antibody, MM-141, having the USAN "istiratumab". Istiratumab is a polyvalent bispecific antibody (PBA) that co-blocks IGF-1 and heregulin- induced signaling and induces degradation of receptor complexes containing IGF-1R and ErbB3, including their respective heterodimers with insulin receptor and with ErbB2. MM- 141 is disclosed in U.S. Patent No. 8,476,409, which also discloses a number of other novel PBAs that, like istiratumab, bind specifically to human IGF-1R and to human ErbB3 and are potent inhibitors of tumor cell proliferation and of signal transduction through their actions on either or (typically, as for istiratumab) both of IGF-1R and ErbB3. The invention of targeted biomolecules, such as MM-141, has resulted in a need for new approaches to combination therapies for cancer. The present invention addresses these needs and provides other benefits. SUMMARY

In one aspect, provided herein is a method of treating cancer in a patient, wherein the patient has a tumor that is HRG-positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-121, (b) MM-131, (c) MM-141, (d) MM-151, (e) MM-131 and MM-121, or (f) MM-131 and MM-141.

In another aspect, provided herein is a method of treating a cancer in a patient, wherein the patient has a tumor that is HGF-positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-131, (b) MM-141, (c) MM-151, or (d) MM- 13 land MM-151.

In another aspect, provided herein is a method of treating a cancer in a patient, wherein the patient has a tumor that is EGF-positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-151, (b) MM-131, (c) MM-131 and MM-121, (d) MM-131 and MM-141, or (e) MM-131 and MM-151.

In another aspect, provided herein is a method of treating a cancer in a patient, wherein the patient has a tumor that is IGF-2-positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-131, (b) MM-141, or (c) MM- 151.

In another aspect, provided herein is a method of treating a cancer in a patient, wherein the patient has a tumor that is both HRG- and IGF- 1 -positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-121, (b) MM-131, (c) MM-141, or (d) MM-151.

In some embodiments, the method further comprise administering an effective amount of at least one additional neoplastic agent.

In some embodiments, positivity for HRG, HGF, EGF, IGF2, or IGF-1 is determined using an FDA-approved test. In one embodiment, positivity for HRG, HGF, EGF, IGF2, or IGF-1 is determined by RNA in situ hybridization or RT-PCR.

In another aspect, provided herein is a method of treating a cancer in a patient, the method comprising co-administering to the subject a therapeutically effective amount of metformin and a bispecific anti-ErbB3/anti-IGF-lR antibody. In one embodiment, the metformin is metformin hydrochloride. In another embodiment, the bispecific anti- ErbB3/anti-IGF-lR antibody is istiratumab. In another embodiment, the istiratumab is administered at a dose of 2.8 g/ml, q2w. In one embodiment, the metformin hydrochloride is administered at a dose of 2000 mg daily. In another embodiment, the 2000 mg daily dose comprises two doses of 1000 mg each, administered about 12 hours apart. In another embodiment, the 2000 mg daily dose comprises three doses, one each of 500 mg metformin in the morning, 1000 mg at noon, and 500 mg in the evening. In another embodiment, the metformin is administered at a dose of 500 mg PO daily for the first week of treatment, 500 mg PO twice daily for the second and third week of treatment, and 850 mg twice daily for the duration of treatment of the patient. In another embodiment, the method further comprises administering a therapeutically effective amount of gemcitabine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic of the screening system for determining the effects of combinations of targeted therapies and ligands on the viability of cancer cell lines.

Figure 2 is a schematic showing the structures of MM-121 (seribantumab), MM-131, MM-141 (istiratumab), and MM-151. Figures 3A-3C show heat maps representing the ability of HRG, HGF, and EGF, respectively, to desensitize cancer cell lines (listed on x-axis) to various targeted inhibitors (listed on right axis).

Figures 4A-4F are plots showing the differential effects of MM- 121, MM- 131, MM- 141, and MM- 151 on cell viability in different ligand contexts. The data are normalized to DMSO in each cell line and ligand context. Dots represent different cell lines. The asterisks indicate significance of the effect compared to control based on a t-test (p<0.05). The grey boxes show 25% and 75% quartiles.

Figures 5A-5H show consensus and individual framework models for cancer cell lines from the screen described in Example 1. Figures 5A and 5B represent the consensus framework models showing average information flow and the heterogeneity of information flow, respectively. Figures 5C-5H show individual information flow models for H358 (NSCLC, KRAS^mut), H441 (NSCLC, KRAS^mut), LIM1215 (CRC), HCC827 (NSCLC, EGFR^mut), SNU5, and MKN45, respectively.

Figures 6A and 6B show the viability response of various cancer cell lines to MM-

131 combined with ligands and MM- 121, MM- 141, or MM- 151. The cell lines listed in the x-axis of Figure 6A are, from left to right, Hs746T, SNU16, OE19, AGS, KATO III, N87, KYSE-410, HGC27, MKN45, SNU5, and OE33; the combinations listed in the y-axis are, from top to bottom, EGF/MM-151, HRG/IGF1/MM-141, HGF/MM-131, HRG/MM-121, HRG, MM- 111, FGF2/control, EGF/control, HRG/IGF 1/control, IGF 1/control, HGF/control, HRG/control, and control/control.

Figure 7A shows a heat map demonstrating that HGF is a resistance ligand for MM- 151, and the combination with MM- 131 restored sensitivity. The cell lines listed in the x- axis are, from left to right, H23, H1993, H460, H520, H1915, H2170, A549, H441, H358, HCC827, H322M, H226 and H596. Figure 7B shows a graph of the same data in each of the cell lines, visualized as curves instead. Figure 7C is a graph showing that the combination of MM-131 and MM-151 is superior to either agent alone in an HGF-overexpressing NSCLC xenograft model (H358-HGF).

Figure 8A is a graph showing the antiproliferative activity of MM- 141 (0.5 μΜ) in combination with gemcitabine in cells cultured in regular glucose medium or low glucose medium (<5 mM). Figure 8B is a graph showing that MM- 141 and metformin have an additive effect in most HRG- and/or IGF-1 positive cell lines tested.

DETAILED DESCRIPTION

Although there are many well-defined signaling pathways in tumor cells that provide druggable targets, the emergence of growth factor-mediated resistance and parallel pathway compensation frequently occurs, limiting the effectiveness of these treatment strategies. Provided herein are combinations of growth factors (ligands) and targeted investigational therapies that allow for the identification of which signaling pathways are mechanistically related. The results presented here demonstrate the use of network biology-based phenotypic screening and modeling to reveal unexpected behaviors, identify positive and negative biomarkers, and guide novel treatment strategies. Definitions

The terms "combination therapy," "co-administration," "co-administered" or

"concurrent administration" (or minor variations of these terms) include simultaneous administration of at least two therapeutic agents to a patient or their sequential administration within a time period during which the first administered therapeutic agent is still present in the patient (e.g., in the patient's plasma or serum) when the second administered therapeutic agent is administered.

The term "monotherapy" refers to administering a single drug to treat a disease or disorder in the absence of co-administration of any other therapeutic agent that is being administered to treat the same disease or disorder.

"Dosage" refers to parameters for administering a drug in defined quantities per unit time (e.g., per hour, per day, per week, per month, etc.) to a patient. Such parameters include, e.g., the size of each dose. Such parameters also include the configuration of each dose, which may be administered as one or more units, e.g., as one or more administrations, e.g., either or both of orally (e.g., as one, two, three or more pills, capsules, etc.) or injected (e.g., as a bolus or infusion). Dosage sizes may also relate to doses that are administered continuously (e.g., as an intravenous infusion over a period of minutes or hours). Such parameters further include frequency of administration of separate doses, which frequency may change over time.

"Dose" refers to an amount of a drug given in a single administration.

"Effective amount" refers to an amount (administered in one or more doses) of an antibody, protein or additional therapeutic agent, which amount is sufficient to provide effective treatment.

"c-Met," also called "MET" and hepatocyte growth factor receptor (HGFR), is a protein that in humans is encoded by the MET gene, as described, e.g., in U.S. Patent No. 7,605,127.

"EpCAM" refers to epithelial cell adhesion molecule, a protein that in humans is encoded by the EPCAM gene. EpCAM has also been designated as TACSTD1 (tumor- associated calcium signal transducer 1), CD326 (cluster of differentiation 326), and the 17- 1A antigen. EpCAM is a pan-epithelial differentiation antigen that is expressed by most carcinomas. Exemplary human EpCAM nucleic acid and protein sequences are set forth in RefSeqGene Gene ID: 4072 and GenBank Accession Number: NP_002345.2, respectively.

"EGFR" refers to Epidermal Growth Factor Receptor, which is also known as ErbB l, HER-1, mENA, and PIG61. EGFR is known to bind ligands including epidermal growth factor (EGF), transforming growth factor a (TGFa), amphiregulin, heparin-binding EGF (hb- EGF), betacellulin, epiregulin and has Gene ID 1956 (Herbst, R. S., and Shin, D. M., Cancer 94 (2002) 1593-1611; Mendelsohn, J., and Baselga, J., Oncogene 19 (2000) 6550-6565). EGFR is transmembrane glycoprotein that is a member of the protein kinase superfamily that regulates numerous cellular processes via tyrosine -kinase mediated signal transduction pathways, including, but not limited to, activation of signal transduction pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitogenesis, and metastasis (Atalay, G., et al., Ann Oncology 14 (2003) 1346-1363; Tsao, A. S., and Herbst, R. S., Signal 4 (2003) 4-9; Herbst, R. S., and Shin, D. M., Cancer 94 (2002) 1593- 1611; Modjtahedi, H., et al., Br. J. Cancer 73 (1996) 228-235). Binding of the ligand to EGFR induces receptor dimerization and tyrosine autophosphorylation, which leads to cell proliferation. Multiple alternatively spliced transcript variants that encode different protein isoforms have been found for this gene. The aa sequences for human EGFR isoforms a-d precursors are provided at Genbank Accession Nos. NP_005219.2, NP_958439.1,

NP_958440.1 and NP_958441.1.

"ErbB3" refers to ErbB3 protein, as described in U.S. Pat. No. 5,480,968. The human ErbB3 protein sequence is shown in SEQ ID NO:4 of U.S. Pat. No. 5,480,968, wherein the first 19 amino acids (aas) correspond to the leader sequence that is cleaved from the mature protein. ErbB3 is a member of the ErbB family of receptors, other members of which include ErbB l (EGFR), ErbB2 (HER2/Neu) and ErbB4. ErbB3 itself lacks tyrosine kinase activity, but is itself phosphorylated upon dimerization of ErbB3 with another ErbB family receptor, e.g., ErbB l (EGFR), ErbB2 and ErbB4, which are receptor tyrosine kinases. Ligands for the ErbB family receptors include heregulin (HRG), betacellulin (BTC), epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF), transforming growth factor alpha (TGF-a ), amphiregulin (AR), epigen (EPG) and epiregulin (EPR). The aa sequence of human ErbB3 is provided at Genbank Accession No. NP_001973.2 (receptor tyrosine -protein kinase erbB-3 isoform 1 precursor) and is assigned Gene ID: 2065.

"IGF-1R" or "IGF1R" refers to the receptor for insulin-like growth factor 1 (IGF-1, formerly known as somatomedin C). IGF-1R also binds to, and is activated by, insulin-like growth factor 2 (IGF-2). IGF1-R is a receptor tyrosine kinase, which, upon activation by IGF-1 or IGF-2, is auto-phosphorylated. The aa sequence of human IGF-1R precursor is provided at Genbank Accession No. NP_000866 and is assigned Gene ID: 3480.

"Tandem Fc bispecific antibodies" or "TFcBAs" are molecules that comprise a Tandem Fc, which is a polypeptide moiety that comprises a first Fc region and a second Fc region, each of said first Fc region and second Fc region having a C-terminus and an N- terminus; the first Fc region and the second Fc region are linked as a single polypeptide chain through a TFc linker having a C-terminus and an N-terminus (i.e., the C-terminus of the first Fc region is linked by a peptide bond to the N-terminus of the TFc linker, the C-terminus of which TFc linker is in turn linked by a peptide bond to the N-terminus of the second Fc region). A TFcBA may comprise at least two binding sites (at least a first binding site and a second binding site). Each such binding site binds specifically to a specific part of a cell surface receptor. Exemplary cell surface receptors are those that are expressed or

overexpressed by cancer cells. Exemplary binding sites include antibody-derived binding sites that bind specifically to an extracellular domain of a cell surface receptor. Exemplary TFcBAs are disclosed in WO 2014/138449, the contents of which are herein incorporated by reference.

As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or preventative measures described herein. The methods of "treatment" employ administration to a subject, the combination disclosed herein in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. As used herein, the term "agent," refers to an active molecule, e.g., a therapeutic protein, e.g., a drug.

As used herein, "antineoplastic agent" refers to agents that have the functional property of inhibiting the development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia.

Inhibition of metastasis is frequently a property of antineoplastic agents.

As used herein, "effective treatment" refers to treatment producing a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method. Effective treatment may refer to alleviation of at least one symptom of cancer.

As used herein, the term "effective amount" refers to an amount of an agent that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount can be administered in one or more administrations.

As used herein, the term "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a

formulation of the molecules disclosed herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, is being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

As used herein, adjunctive or combined administration (coadministration) includes simultaneous administration of the agents in the same or different dosage form, or separate administration of the agents (e.g., sequential administration). For example, the agents can be formulated for separate administration and administered concurrently or sequentially. Such concurrent or sequential administration preferably results in the agents being simultaneously present in treated patients.

As used herein, the terms "fixed dose", "flat dose" and "flat-fixed dose" are used interchangeably and refer to a dose that is administered to a patient without regard for the weight or body surface area (BSA) of the patient. The fixed or flat dose is therefore not provided as a mg/kg dose, but rather as an absolute amount of the agent.

As used herein, "Q2W" refers to administration once every two weeks.

As used herein, "PO" refers to oral administration (per os).

As used herein, the term "subject" or "patient" is a human patient (e.g., a patient having cancer).

As used herein, "RT-PCR" indicates reverse transcription followed by PCR of the resulting reverse transcripts.

As used herein, "sample", "tumor cell sample," "cancer cell sample" "patient sample", or "sample from a patient", as used herein, is meant a sample comprising tumor cells from the patient. Such a sample may be, e.g., from a biopsy of a tumor, a tissue sample, or circulating tumor cells from the blood.

MM-1X1 antibodies

MM- 121

"MM-121 " or "seribantumab" is a human monoclonal anti-ErbB3 IgG2 (see, e.g., U.S. Patent Nos. 7,846,440; 8,691,771 and 8,961,966; 8,895,001, U.S. Patent Publication Nos., 20110027291, 20140127238, 20140134170, and 20140248280), as well as international publication Nos. WO/2013/023043, WO/2013/138371, WO/2012/103341, and US

Provisional Patent Application serial No. 62/090,780, the teachings of which are expressly incorporated herein by reference). The amino acid sequences of the heavy and light chains are set forth in SEQ ID NOs: 34 and 35, respectively; those of the heavy and light chain variable regions of MM- 121 are set forth in SEQ ID NOs: 36 and 37, respectively; and those of the VH CDRl-3 and VL CDRl-3 are set forth in SEQ ID NOs: 28-30 and 31-33, respectively.

Accordingly, in certain embodiments, the anti-ErbB3 antibody for use in the methods disclosed herein comprise the heavy and light chain variable region CDRl, CDR2, and CDR3 domains having the amino acid sequence set forth in SEQ ID NOs: 28-30 and 31-33, respectively. In another embodiment, the anti-ErbB3 antibody comprises a VH and VL region comprising the amino acid sequences set forth in SEQ ID NOs: 36 and 37,

respectively. In another embodiment, the anti-ErbB3 antibody comprises VH and/or VL regions comprising the amino acid sequences set forth in SEQ ID NO: 36 and/or SEQ ID NO: 37, respectively. In another embodiment, the anti-ErbB3 antibody comprises heavy and/or light chains comprising the amino acid sequences set forth in SEQ ID NO: 34 and/or SEQ ID NO: 35, respectively. In another embodiment, an antibody is used that competes for binding with and/or binds to the same epitope on human ErbB3 as the above-mentioned antibodies. In a particular embodiment, the epitope comprises residues 92- 104 of human ErbB3 (SEQ ID NO: 38). In another embodiment, the antibody competes with seribantumab for binding to human ErbB3 and has at least 90% variable region amino acid sequence identity with the above-mentioned anti-ErbB3 antibodies (see, e.g., US Patent No. 7,846,440 and US Patent Publication No. 20100266584).

MM-131

"MM- 131" refers to a TFcBA with a Fab moiety binding to c-Met and a scFv moiety binding to EpCAM. MM- 131 is described in WO2014/138449, which is incorporated herein by reference in its entirety. MM-131 is a TFcBA that comprises two polypeptide chains, a large chain and a Fab light chain, each chain having a C-terminus and an N-terminus, the TFcBA comprising a first binding site comprised by a Fab moiety comprising the Fab light chain and a Fab heavy chain, which Fab heavy chain is at the N-terminus of the large chain, which Fab moiety specifically binds to cMET, and which TFcBA further comprises a second binding site comprised by a single chain Fv (scFV) moiety at the C-terminus of the large chain, which scFv moiety specifically binds to EpCAM, wherein: (a) the Fab heavy chain and the scFv moiety are linked through a Tandem Fc ('TFc");

(b) the TFc is comprised by the large chain and has a first Fc region and a second Fc region which are linked through a TFc linker to form a contiguous polypeptide; and

(c) the first and the second Fc regions associate to form an Fc dimer.

The sequence of the Fab light chain comprises three LCDRs, set forth as SEQ ID NOs:

503, 504, and 505 in WO2014/138449 (and set forth herein as SEQ ID NOs: 44-46, respectively). The sequence of the Fab heavy chain comprises three HCDRs, set forth as SEQ ID NOs: 506, 507, and 508 in WO2014/138449 (and set forth herein as SEQ ID NOs: 41-43, respectively). The sequence of the TFcBA large chain is set forth in SEQ ID NO: 39. The sequence of the Fab light chain is set forth in SEQ ID NO: 40.

MM-141

"MM- 141" or "istiratumab" refers to a recombinant fully human bispecific anti-IGF- 1R and anti-ErbB3 tetravalent antibody (also known as PBA P4-G1-M1.3). The complete tetrameric structure of the IgGl -based molecule is composed of two heavy chains (720 amino acids each) and two kappa light chains (214 amino acids each) held together by intrachain and inter-chain disulfide bonds. The variable regions of the heavy and light chains encode anti-IGF-lR modules. The C-terminus of the heavy chain encodes anti-ErbB3 scFv modules. MM-141-P5G5 is the designation for Master Cell Bank which produces MM-141.

Istiratumab has two pairs of polypeptide chains, each pair of said two pairs comprising a heavy chain joined to a light chain by at least one heavy-light chain bond, wherein each light chain comprises the amino acid sequence set forth in SEQ ID NO: 1 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 2. SEQ ID NOs: 1 and 2 correspond to SEQ ID NOs: 204 and 226, respectively, as set forth in U.S. Patent No.

8,476,409 (which is herein incorporated by reference in its entirety). In one embodiment, istiratumab comprises a linker having the amino acid sequence set forth in SEQ ID NO: 3, which corresponds to SEQ ID NO: 53 as set forth in PCT/US2010/052712 (which is herein incorporated by reference in its entirety). MM-/ 5/

"MM-151" is disclosed in US Patent No. 8,691,231, herein incorporated by reference in its entirety. MM-151 is an oligoclonal anti-epidermal growth factor receptor (EGFR) antibody, comprising a plurality of species of monoclonal anti-EGFR antibodies, one against each of at least two extracellular epitopes of EGFR, one of the species of monoclonal anti- EGFR antibody inhibiting the binding of ligand to EGFR. MM-151 comprises a triple combination of P1X+P2X+P3X, wherein the heavy and light chain variable region sequences of PIX are set forth below as SEQ ID NOs: 22 and 23, respectively; the heavy and light chain variable region sequences of P2X are set forth below as SEQ ID NOs: 24 and 25,

respectively; and the heavy and light chain variable region sequences of P3X are set forth below as SEQ ID NOs: 26 and 27, respectively. Accordingly, in certain embodiments, the oligoclonal anti-EGFR antibody for use in the methods disclosed herein comprises a triple combination of anti-EGFR antibodies, wherein the antibodies comprise the VH CDRl-3 and VL CDRl-3 sequences of PIX (SEQ ID NOs: 4-9), P2X (SEQ ID NOs: 10-15), and P3X (SEQ ID NOs: 16-21). In another embodiment, the oligoclonal antibody comprises a triple combination of anti-EGFR antibodies, wherein the antibodies comprise the VH and VL sequences set forth in (a) SEQ ID NOs: 22 and 23, (b) 24 and 25, and (c) 26 and 27.

Methods of use

Methods of combination therapy for treating cancer in a patient are provided herein.

In one aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is HRG- positive, the method comprising administering to the patient a therapeutically effective amount of an anti-ErbB3 antibody (e.g., MM-121), a c-Met inhibitor (MM-131), a bispecific anti-IGF-lR and anti-ErbB3 antibody (MM-141), or an EGFR inhibitor (MM-151). In one embodiment, the patient is administered a therapeutically effective amount of an anti-ErbB3 antibody (e.g., MM-121). In another embodiment, the patient is administered a

therapeutically effective amount of a bispecific anti-IGF-lR and anti-ErbB3 antibody (e.g., MM-141).

In another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is HGF- positive, the method comprising administering to the patient a therapeutically effective amount of a c-Met inhibitor (e.g., MM-131), a bispecific anti-IGF-lR and anti-ErbB3 antibody (e.g., MM-141), or an EGFR inhibitor (e.g., MM-151).

In yet another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is EGF-positive, the method comprising administering to the patient a therapeutically effective amount of an EGFR inhibitor (e.g., MM-151) or a c-Met inhibitor (e.g., MM-131).

In another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is IGF-2- positive, the method comprising administering to the patient a therapeutically effective amount of a c-Met inhibitor (e.g., MM-131), a bispecific anti-IGF-lR and anti-ErbB3 antibody (e.g., MM-141), or an EGFR inhibitor (e.g., MM-151).

In another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is both HRG- and IGF- 1 -positive, the method comprising administering to the patient a therapeutically effective amount of an anti-ErbB3 antibody (e.g., MM-121), a c-Met inhibitor (e.g., MM- 131), a bispecific anti-IGF-lR and anti-ErbB3 antibody (e.g., MM-141), or an EGFR inhibitor (e.g., MM-151).

In other aspects, a patient with cancer is treated with a combination of an anti-ErbB3 antibody (e.g., MM-121), a c-Met inhibitor (e.g., MM-131), a bispecific anti-IGF-lR and anti-ErbB3 antibody (e.g., MM-141), and/or an EGFR inhibitor (e.g., MM-151). For example, in one aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is HRG-positive, the method comprising administering to the patient a therapeutically effective amount of a c-Met inhibitor (e.g., MM- 131) and an anti-ErbB3 antibody (e.g., MM- 121).

In another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is HRG- positive, the method comprising administering to the patient a therapeutically effective amount of a c-Met inhibitor (e.g., MM- 131) and a bispecific anti-IGF-lR and anti-ErbB3 antibody (e.g., MM-141).

In another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is EGF- positive, the method comprising administering to the patient a therapeutically effective amount of a c-Met inhibitor (e.g., MM- 131) and an anti-ErbB3 antibody (e.g., MM- 121).

In another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is EGF- positive, the method comprising administering to the patient a therapeutically effective amount of a c-Met inhibitor (e.g., MM- 131) and a bispecific anti-IGF-lR and anti-ErbB3 antibody (e.g., MM-141).

In another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is EGF- positive, the method comprising administering to the patient a therapeutically effective amount of a c-Met inhibitor (e.g., MM-131) and an EGFR inhibitor (e.g., MM-151).

In another aspect, provided herein is a method of treating a cancer in a human patient, wherein the patient is determined or has been determined to have a tumor that is HGF- positive, the method comprising administering to the patient a therapeutically effective amount of a c-Met inhibitor (e.g., MM-131) and an EGFR inhibitor (e.g., MM-151).

In some embodiments, the anti-ErbB3 antibody comprises VH and VL CDR sequences set forth in SEQ ID NOs: 28-30 and 31-33, respectively. In some embodiments, the anti-ErbB3 antibody comprises VH and VL sequences set forth in SEQ ID NOs: 36 and 37, respectively. In one embodiment, the anti-ErbB3 antibody is MM- 121.

In some embodiments, the c-Met inhibitor is a bispecific antibody. In some embodiments, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody. In further embodiments, the bispecific anti-c-Met/anti-EpCAM antibody comprises VH and VL CDR sequences set forth in SEQ ID NOs: 41-43 and 44-46. In one embodiments, the bispecific anti-c-Met/anti-EpCAM antibody is MM-131.

In some embodiments, the bispecific anti-IGF-lR and anti-ErbB3 antibody is istiratumab (i.e., MM-141).

In some embodiments, the EGFR inhibitor is a bispecific antibody. In some embodiments, the EGFR inhibitor is an oligoclonal antibody. In further embodiments, the oligoclonal anti-EGFR antibody comprises a triple combination of antibodies comprising the VH CDRl-3 and VL CDRl-3 sequences of P1X (SEQ ID NOs: 4-9), P2X (SEQ ID NOs: 10- 15), and P3X (SEQ ID NOs: 16-21). In another embodiment, the oligoclonal antibody comprises a triple combination of anti-EGFR antibodies, wherein the antibodies comprise the VH and VL sequences set forth in (a) SEQ ID NOs: 22 and 23, (b) 24 and 25, and (c) 26 and 27. In one embodiment, the oligoclonal antibody is MM- 151.

In some embodiments, positivity for the above-identified ligand is determined using an FDA-approved test. For example, in one embodiment, the patient has an HRG-positive cancer, wherein HRG positivity is determined by a HRG RNA in situ hybridization assay or by RT-PCR assay (e.g., a quantitative RT-PCR assay). In one embodiment, the patient has a positive in situ hybridization test for HRG with a score of >1+.

In another aspect, the treatment methods described herein comprise administering an anti-ErbB3 antibody (e.g., MM-121), a c-Met inhibitor (e.g., MM-131), MM- 141, or an

EGFR inhibitor (e.g., MM-151), or a combination thereof, in combination with one or more other antineoplastic agents (e.g., other small molecule drugs, for example, those shown in Table 1).

In one embodiment, no more than three other antineoplastic agents are administered within the treatment cycle. In another embodiment, no more than two other antineoplastic agents are administered within the treatment cycle. In another embodiment, no more than one other antineoplastic agent is administered within the treatment cycle. In another embodiment, no other antineoplastic agent is administered within the treatment cycle.

As used herein, adjunctive or combined administration (coadministration) includes simultaneous administration of one or more of the antibodies described herein (e.g., MM-121, MM-131, MM- 141, or MM-151, or a combination thereof), and one or more antineoplastic agents (e.g., an agent shown in Table 1) in the same or different dosage form, or separate administration of one or more of the antibodies described herein (e.g., MM-121, MM-131, MM-141, or MM-151, or a combination thereof), and one or more antineoplastic agents (e.g., sequential administration). Such concurrent or sequential administration preferably results in both one or more of the antibodies described herein (e.g., MM-121, MM-131, MM-141, or MM-151, or a combination thereof), and the one or more agents being simultaneously present in treated patients.

In another embodiment, provided herein is a method of treating a patient with cancer comprising administering MM-141 and an agent that reduces mitochondrial respiration in cells by activating AMPK, and also reduces glucose production by the liver. In a particular embodiment, the agent that reduces glucose production is metformin. Accordingly, provided herein is a method of treating a patient with cancer comprising administering to the patient a therapeutically effective amount of MM-141 and metformin. In one embodiment, MM-141 is administered at a dose of 2.8 g/ml q2w by IV infusion, and metformin hydrochloride is dosed (self-dosed by the patient) at two doses of 1000 mg each, twice daily P.O. In certain embodiments, the patients selected for treatment by the methods disclosed herein may have or are at risk of developing type 2 diabetes.

Metformin hydrochloride (CAS No. 1115-70-4) is an oral antihyperglycemic drug approved for use in the management of type 2 diabetes, marketed under the name, e.g.,

GLUCOPHAGE®.. Metformin hydrochloride is a white to off-white crystalline compound with a molecular formula of C4H11N5 · HC1 and a molecular weight of 165.63. Metformin hydrochloride is freely soluble in water and is practically insoluble in acetone, ether, and chloroform. The pKa of metformin is 12.4. The pH of a 1% aqueous solution of metformin hydrochloride is 6.68. GLUCOPHAGE tablets contain 500 mg, 850 mg, or 1000 mg of metformin hydrochloride.

The usual starting dose of GLUCOPHAGE® (metformin hydrochloride tablets) is 500 mg twice a day or 850 mg once a day, given with meals. Dosage increases should be made in increments of 500 mg weekly or 850 mg every 2 weeks, up to a total of 2000 mg per day, given in divided doses. Patients can also be titrated from 500 mg twice a day to 850 mg twice a day after 2 weeks. For those patients requiring additional glycemic control,

GLUCOPHAGE® may be given to a maximum daily dose of 2550 mg per day. Doses above 2000 mg may be better tolerated given three times a day with meals. When treating a patient for cancer who also has been diagnosed with diabetes, adjustments to the dose may be made that the discretion of the health care practitioner.

Metformin activates the enzyme AMPK (AMP-activated protein kinase), which plays an important role in insulin signaling, systemic energy balance, and the metabolism of glucose and fats. Activated AMPK may slow cancer growth by reducing the amount of sugar available for cancer cells to consume (by lowering sugar output from the liver, increasing sugar uptake from the blood, and maintaining insulin sensitivity). Metformin also inhibits mTOR (mammalian target of rapamycin), which is responsible for cell growth, including tumor cell growth. Metformin can also inhibit mTOR directly (independent of AMPK activation), slowing tumor growth. Some studies suggest that metformin may also kill cancer stem cells, which are often the cell type in tumors that is most resistant to both chemotherapy and radiation therapy. Metformin may also prevent precancerous cells from evolving into cancer cells, thus suggesting a prophylactic use for the drug. In addition, metformin reduces the amount of circulating estrogen and testosterone, both of which can stimulate the growth of hormone-dependent tumors (i.e. breast cancer, prostate cancer).

In another embodiment, provided herein is a method of treating a patient with cancer comprising administering to the patient a therapeutically effective amount of MM-141, metformin, and gemcitabine.

Gemcitabine (Gemzar®) is indicated as a first line therapy for pancreatic

adenocarcinoma and is also used in various combinations to treat ovarian, breast and non- small-cell lung cancers. Gemcitabine HC1 is 2'-deoxy-2',2'-difluorocytidine

monohydrochloride (-isomer) (MW=299.66) and is administered parenterally, typically by i.v. infusion.

In another aspect, provided herein is a method of treating cancer in a patient with a tumor that is HRG-positive who previously received drug treatment for the tumor (e.g., treatment with one or more drugs listed in Table 1) and developed resistance to the treatment, the method comprising administering to the patient a therapeutically effective amount of (a) MM-121, (b) MM-131, (c) MM-141, (d) MM-151, (e) MM-131 and MM-121, or (f) MM-131 and MM-141.

In another aspect, provided herein is a method of treating cancer in a patient with a tumor that is HGF-positive (as assessed, e.g., with RNA in-situ hybridization) who previously received drug treatment for the tumor (e.g., treatment with one or more drugs listed in Table 1) and developed resistance to the treatment, the method comprising administering to the patient a therapeutically effective amount of (a) MM- 131, (b) MM- 141, (c) MM- 151, or (d) MM-131and MM- 151.

In another aspect, provided herein is a method of treating cancer in a patient with a tumor that is EGF-positive who previously received drug treatment for the tumor (e.g., treatment with one or more drugs listed in Table 1) and developed resistance to the treatment, the method comprising administering to the patient a therapeutically effective amount of (a) MM-151, (b) MM-131, (c) MM-131 and MM-121, (d) MM-131 and MM-141, or (e) MM- 131 and MM-151.

In another aspect, provided herein is a method of treating cancer in a patient with a tumor that is IGF-2-positive (as assessed, e.g., with RNA in-situ hybridization) who previously received drug treatment for the tumor (e.g., treatment with one or more drugs listed in Table 1) and developed resistance to the treatment, the method comprising administering to the patient a therapeutically effective amount of MM-151.

In another aspect, provided herein is a method of treating cancer in a patient with a tumor that is both HRG- and IGF- 1 -positive (as assessed, e.g., with RNA in-situ

hybridization) who previously received drug treatment for the tumor (e.g., treatment with one or more drugs listed in Table 1) and developed resistance to the treatment, the method comprising administering to the patient a therapeutically effective amount of MM-131 or MM-141.

Also provided are methods of treating cancer in a human patient who previously received antineoplastic therapy and developed resistance to the antineoplastic therapy. For example, in one embodiment, the method comprises treating cancer in a human patient in need thereof who previously received antineoplastic therapy and developed resistance to the antineoplastic therapy by administering MM-121, MM-131, MM-141, or MM-151, or combinations thereof, and optionally another antineoplastic agent. In another embodiment, the human patient is treated following disease progression or recurrence after prior treatment with antineoplastic therapy. In another embodiment, the human patient is treated after failure of an antineoplastic therapy. In another embodiment, the cancer is identified as a cancer that has acquired resistance to antineoplastic therapy.

In one embodiment, the patient to be treated with the methods disclosed herein has lung cancer, such as non-small cell lung cancer (NSCLC). In another embodiment, the patient has colorectal cancer. In another embodiment, the patient has gastric cancer. In another embodiment, the patient has endometrial cancer. In another embodiment, the patient has esophageal cancer.

Also provided herein is a method of selecting a cancer patient for treatment with one or more of the antibodies described herein (e.g., MM-121, MM-131, MM-141, or MM-151, or combinations thereof), by determining the level of HRG, HGF, EGF, IGF-1, and/or IGF-2 in the patient, for example, using an FDA-approved test. In one embodiment, the patient has an HRG-positive cancer, wherein HRG positivity is determined by a HRG RNA in situ hybridization assay or by RT-PCR assay (e.g., a quantitative RT-PCR assay). In one embodiment, the patient has a positive in situ hybridization test for HRG with a score of >1+. In another embodiment, the patient has an HGF-, EGF-, IGF-1, and/or IGF-2-positive cancer, wherein ligand positivity is determined by immunological and immunochemical methods such as flow cytometry (e.g., FACS analysis), enzyme-linked immunosorbent assays (ELISA), including chemiluminescence assays, radioimmunoassay, immunoblot (e.g., Western blot), and immunohistology/immunohistochemical methods, or other suitable methods such as mass spectroscopy. For example, antibodies to the respective ligand can be used to determine the presence and/or expression level of the ligand in a sample directly or indirectly using, for instance, immunohistology and/or immunohistochemistry. For instance, paraffin sections can be taken from a biopsy, fixed to a slide and combined with one or more labeled and/or unlabeled antibodies by suitable methods. Outcomes

"Therapeutic synergy" refers to a phenomenon where treatment of patients with a combination of therapeutic agents manifests a therapeutically superior outcome to the outcome achieved by each individual constituent of the combination used at its optimum dose (T. H. Corbett et al., 1982, Cancer Treatment Reports, 66, 1187). In this context a

therapeutically superior outcome is one in which the patients either a) exhibit fewer incidences of adverse events while receiving a therapeutic benefit that is equal to or greater than that where individual constituents of the combination are each administered as monotherapy at the same dose as in the combination, or b) do not exhibit dose-limiting toxicities while receiving a therapeutic benefit that is greater than that of treatment with each individual constituent of the combination when each constituent is administered in at the same doses in the combination(s) as is administered as individual components. In xenograft models, a combination, used at its maximum tolerated dose, in which each of the constituents will be present at a dose generally not exceeding its individual maximum tolerated dose, manifests therapeutic synergy when decrease in tumor growth achieved by administration of the combination is greater than the value of the decrease in tumor growth of the best constituent when the constituent is administered alone.

Thus, in combination, the components of such combinations have an additive or superadditive effect on suppressing tumor growth, as compared to monotherapy, e.g., with istiratumab or with gemcitabine or with nab-paclitaxel, or with one or two of these three compared to the triple combination. By "additive" is meant a result that is greater in extent (e.g., in the degree of reduction of tumor mitotic index or of tumor growth or in the degree of tumor shrinkage or the frequency and/or duration of symptom-free or symptom-reduced periods) than the best separate result achieved by monotherapy with each individual component, while "superadditive" is used to indicate a result that exceeds in extent the sum of such separate results. In one embodiment, the additive effect is measured as slowing or stopping of tumor growth. The additive effect can also be measured as, e.g., reduction in size of a tumor, reduction of tumor mitotic index, reduction in number of metastatic lesions over time, increase in overall response rate, or increase in median or overall survival. Compositions

In another aspect, provided herein are compositions, e.g. , pharmaceutical

compositions, for use in the methods of treating cancer described herein. The compositions provided herein contain one or more of the antibodies disclosed herein, formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the compositions are administered by intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g. , by injection or infusion), and the carrier is suited for such administration. Depending on the route of administration, the antibody may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate proteins.

Pharmaceutical compositions may be administered alone or in combination therapy, i.e. , combined with other agents. For example, in some embodiments, the combination therapy can include an antibody disclosed herein with at least one additional therapeutic agent, such as an anti-cancer (antineoplastic) agent. In other embodiments, the composition does not contain any other therapeutic agent (e.g., no other anti-neoplastic agent).

Pharmaceutical compositions can also be administered in conjunction with another anti- cancer treatment modality, such as radiation therapy and/or surgery.

A composition of the present disclosure can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

To administer a composition provided herein by certain routes of administration, it may be desirable to coat the antibody with, or co-administer the antibody with, a material to prevent its inactivation. For example, the antibody may be administered to a patient in an appropriate carrier, for example, in liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in- water CGF emulsions as well as conventional liposomes.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any excipient, diluent or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions provided herein is contemplated. Supplementary active compounds (e.g. , additional anti-cancer agents) can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions. The composition, if desired, can also contain minor amounts of wetting or solubility enhancing agents, stabilizers, preservatives, or pH buffering agents. In many cases, it will be useful to include isotonic agents, for example, sodium chloride, sugars, polyalcohols such as mannitol, sorbitol, glycerol, propylene glycol, and liquid polyethylene glycol in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Kits and Unit Dosage Forms

Further provided are kits that include a pharmaceutical composition containing the antibodies disclosed herein, or combinations thereof, including a pharmaceutically-acceptable carrier, in a therapeutically effective amount adapted for use in the preceding methods. The kits include instructions to allow a practitioner (e.g., a physician, nurse, or physician's assistant) to administer the composition contained therein to treat cancer, such as an ErbB2 expressing cancer.

Optionally, instruments or devices necessary for administering the pharmaceutical composition(s) may be included in the kits. For instance, in one embodiment, a kit may provide one or more pre-filled syringes containing an amount of istiratumab, or an amount of istiratumab and metformin, that allows for convenient delivery of a single dose of 2.8 g istiratumab or a single dose of 2.8 g istiratumab and a single dose of 1000 mg metformin to a patient.

Furthermore, the kits may also include additional components such as instructions or administration schedules for a patient suffering from a cancer to use the pharmaceutical composition(s) containing istiratumab or istiratumab and metformin.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, methods, and kits of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

EXAMPLES

The following Examples should not be construed as limiting the scope of this disclosure.

Example 1: Screen for effects of combinations of targeted therapies and ligands on cell viability

This Example describes a high throughput viability screen in 3D cancer cell cultures to assess the effect of ligands and RTK-directed antibodies in combination with targeted investigational therapies in cancer cells. The screen was conducted in a 3D cell culture system (Scivax) with cell viability (CellTiter Glo) as a readout. A library of 90 small molecules targeting various components of signaling and metabolic pathways was used in combination with growth factors and therapeutic antibodies, e.g., istiratumab. The ability of RTK ligands to modify the activity of targeted agents, such as MM- 121, MM- 131, MM- 141, and MM- 151, in a panel of lung (NCI-H441, H358, HCC827), gastric (OE33, OE19, MKN- 45, NCI-87, SNU-5), colon (LIM1215, NCI-H747, LOVO, LS 174T), and ovarian

(OVCAR8) cancer cell lines was assessed. A schematic of the study design is provided in Figure 1. For the analysis and visualization of the 3D data cube, 2D slices corresponding to cell lines and drugs in a particular ligand background (left and middle heat maps) were analyzed. Raw viability data were normalized to DMSO and media control for each cell line across the entire data cube. To analyze the effect of adding ligands, the difference between the ligand conditions and media control (right heat map) was calculated. The color scale indicates the differential effect of adding ligand to treatment. Black dots indicate statistical significance (P < 0.05) and relevance based on rank sum tests and changes of at least 20%.

The experimental data from this screen was used in a network model to estimate the information flow through receptors and downstream pathways in individual cell types. The model assigns weights corresponding to the strength of the information flow to each edge in the network. The results are visualized by changing line width and color of the network edges according to the estimated edge weights.

A summary of the small molecule library and targets are shown in Table 1 below. A schematic of antibodies corresponding to MM- 121, MM- 131, MM- 141, and MM- 151 is provided in Figure 2.

Table 1:

Everolimus (RAD001) 159351-69-6 mTOR

SNS-032 (BMS-387032) 345627-80-7 CDK

Barasertib (AZD1152-HQPA) 722544-51-6 Aurora Kinase

PLX-4720 918505-84-7 Raf

Roscovitine (Seliciclib,CYC202) 186692-46-6 CDK

Regorafenib (BAY 73-4506) 755037-03-7 c-RET,VEGFR

Vemurafenib (PLX4032, RG7204) 918504-65-1 Raf

GSK1059615 958852-01-2 PI3K,mTOR

Rigosertib (ON-01910) 1225497-78-8 PLK

Ruxolitinib (INCB018424) 941678-49-5 JAK

VX-745 209410-46-8 p38 MAPK

LY2228820 862507-23-1 p38 MAPK

Fasudil (HA-1077) HC1 105628-07-7 ROCK,Autophagy

Tie2 kinase inhibitor 948557-43-5 Tie-2

H 89 2HC1 130964-39-5 PKA

PF-573228 869288-64-2 FAK

AZD1480 935666-88-9 JAK

PF-4708671 1255517-76-0 S6 Kinase

SGI-1776 free base 1025065-69-3 Pirn

Mubritinib (TAK 165) 366017-09-6 HER2

Apatinib 811803-05-1 VEGFR

Degrasyn (WP1130) 856243-80-6 DUB,Bcr-Abl

Chrysophanic Acid 481-74-3 mTOR,EGFR

Imatinib (STI571) 152459-95-5 PDGFR

NU7441 (KU-57788) 503468-95-9 DNA-PK,PI3K

Trametinib (GSK1120212) 871700-17-3 MEK

Ibrutinib (PCI-32765) 936563-96-1 BTK

A-769662 844499-71-4 AMPK

Milciclib (PHA-848125) 802539-81-7 CDK

Dinaciclib (SCH727965) 779353-01-4 CDK

TAE226 (NVP-TAE226) 761437-28-9 FAK

PF-562271 717907-75-0 FAK

Go 6983 133053-19-7 PKC

SC-514 354812-17-2 IKB/IKK

AP26113 1197958-12-5 ALK

CX-6258 HC1 1353859-00-3 Pirn

GSK2334470 1227911-45-6 PDK-1

PF-3758309 898044-15-0 PAK

VE-822 1232416-25-9 ATM/ATR

AZD1208 1204144-28-4 Pirn

AZD3463 1356962-20-3 ALK 10058-F4 403811-55-2 c-Myc

SSR128129E 848318-25-2 FGFR

SKI II 312636-16-1 S IP Receptor

RKI-1447 1342278-01-6 ROCK

Losmapimod (GW856553X) 585543-15-3 p38 MAPK

AZD2858 486424-20-8 GSK-3

NMS-P937 (NMS1286937) 1034616-18-6 PLK

WZ4003 1214265-58-3 AMPK

GNE-9605 1536200-31-3 LRRK2

EHT 1864 754240-09-0 Rho

Filgotinib (GLPG0634) 1206161-97-8 JAK

PQ 401 196868-63-0 IGF-1R

VE-821 1232410-49-9 ATM/ATR

BYL719 1217486-61-7 PI3K

Bosutinib (SKI-606) 380843-75-4 Src

Cabozantinib (XL184, BMS- FLT3,Tie-2,c-Kit,c-

849217-68-1

907351) Met,VEGFR,Axl

Pazopanib HC1 (GW786034 HC1) 635702-64-6 VEGFR,PDGFR,c-Kit

Axitinib 319460-85-0 c-Kit,VEGFR,PDGFR

AZD8055 1009298-09-2 mTOR

GDC-0941 957054-30-7 PI3K

The heap maps shown in Figure 3 demonstrate that HRG, HGF, and EGF broadly desensitize cells to targeted agents in ligand-responsive cell lines. Some ligands, such as HRG and HGF had a stronger effect in promoting resistance to targeted therapies in these cell lines.

Moreover, as shown in Figures 4A-4F, MM-121, MM- 131, MM- 141, and MM- 151 restored sensitivity in the respective biomarker contexts, and ligand presence could be considered positive or negative biomarkers in certain contexts. Each graph illustrates the differential effect of all antibody molecules in a different ligand context (control, HRG, HGF, EGF, IGF-2, and HRG/IGF-1). The data were normalized to DMSO in each cell line and ligand context. Dots represent different cell lines. The asterisks indicate significance of the effect compared to control based on a t-test (P < 0.05). The grey boxes show 25% and 75% quartiles. For instance, while MM-121 significantly restored sensitivity in HRG treated cells, it did not in HGF, EGF, or IGF-2 treated cells. Similarly, MM- 131 restored sensitivity in HRG, HGF, and HRG/IGF-1 treated cells; MM- 141 in HRG, HGF, and HRG/IGF-1 treated cells, and MM-151 in HGF, EGF, and IGF-2 treated cells. Notably, MM-151 had an effect on cells which were not treated with ligand (Figure 4A).

Example 2: Generation of pathway connectivity models

Using the data obtained in Example 1, pathway connectivity models were developed in order to identify pathway crosstalk and novel and effective combination strategies (Figures 5A-5H). The experimental data was used in addition with a network model to estimate the information flow through receptors and pathways for individual cell types. A simple model that assigns weights, corresponding to the strength of information flow, to each edge in the network was used. The ligand and drug perturbations provide the required information for this task. The results of the network analysis is visualized by changing the line width and color of the network edges according to the estimated edge weights. The average information flow network revealed a stronger signaling via PI3K as opposed to MAPK or JAK/STAT. Downstream PI3K, strong signaling via mTOR and subsequent survival signaling was observed. Cell cycle and survival signaling contributed similarly to the observed cell viability read out. From the heterogeneity of information flow, network flow via PI3K and subsequent mTOR were more conserved between cell lines that flow though other network edges. The strongest variability was observed between mTOR and cell cycle, as well as between survival and cell viability, indicating that cells might modulate the signaling pattern differently.

Overall, ligand-mediated resistance varied depending on the cancer type. For example, HGF and EGF play particularly strong roles in gastric and colorectal cancer cells, respectively. Interestingly, there were a few cases in which a ligand reduces cell viability compared to control, but had no effect on drug response. In most cases where a ligand rendered cells insensitive to a certain drug treatment, a combination with the appropriately matched therapeutic antibody re-sensitized cells to the drug, suggesting a combination strategy could potentially be used to overcome ligand-mediated resistance. Among the findings that are observed in multiple cell lines, istiratumab combined well with metformin in colorectal and lung cancer cell lines (as discussed below in Example 4). Example 3: MM-1X1 combinations overcome ligand-mediated resistance

As predicted by the inferred network model for MKN45, Met inhibition is insufficient to block cell viability if ligands for alternative pathways are present (i.e., compensation by alternative pathways) (Figure 6A). As shown in Figure 6B, the combination of MM- 131 with MM- 151, MM- 121, or MM- 141 generally has a greater effect on killing cells relative to treatment with MM- 131 alone, suggesting that co-targeting of Met and EGFR or Met and ErbB3 more effectively blocks downstream survival pathways. The effect is also context dependent. For instance, while MM- 131 and the combination of MM- 131 + MM- 151 did not overcome resistance conferred by HRG treatment, MM- 131 +MM-121 and MM- 131 + MM- 141 substantially reduced cell viability. Similarly, while MM-131 and the combination of MM-131 + MM-121 and MM-131 + MM-141 reduced cell viability in EGF treated cells, MM-131 + MM- 151 had an even stronger effect.

As shown in Figures 7A-7B, HGF is a resistance ligand for MM- 151. Here, the combination of MM- 151 + MM-131 restored sensitivity of the indicated NSCLC cell types to the anti-cancer agents. Figures 7A-7B show cell viability data for a panel of 13 NSCLC cell lines. The dots in Figure 7 A indicate changes of more than 20% as well as significance based on a rank sum test based on four technical replicates. The data is normalized to untreated control for each cell line. MM- 151 treatment reduced cell viability for some cell lines (for instance for HCC827). Treatment with HGF increased cell viability both in the presence and absence of MM- 151 and can therefore be considered as resistance ligand to MM- 151. Co- treatment with the MM-131 and MM-151 combination restored sensitivity (e.g., for HCC827) and led to overall increased reduction of cell viability in some cases (e.g., for H23 and H596). As shown in Figure 7C, the combination of MM-131 + MM-151 was superior to either agent alone in an HGF-overexpressing NSCLC xenograft model (H358-HGF). H358-HGF overexpressing tumor xenografts were established by subcutaneous injection of 200μί of a cell suspension consisting of 5 x 10⁶ of H358-HGF cancer cells, diluted 1: 1 in Matrigel® (BD Biosciences), into single sites on flank of recipient 4-5 week old female nu/nu mice. Tumor formation was monitored twice weekly and once the average measured tumor volume reached 150 - 200mm , mice were randomized into groups of 10 and treatment was initiated. Overall, the average tumor volume per group was equivalent across all groups at the beginning of the treatments. Treatment was initiated on day 17 after tumor implantation. MM-131 was administered intraperitoneally once a week (q7d, 12mg/ml); and MM-151 tool compound (labelled MM-151 in graphs; consisting of 25E+P2X+P3X) was administered as follows: 25E (6.25 mg/kg) and P3X (3.125 mg/kg), intraperitoneally q7d. In addition, P2X (8.75 mg/kg) was co-administered intraperitoneally with the q7d dosing of 25E and P3X. Plotted data of tumor volume represents mean and standard error of the mean for each measurement. Tumor volumes in the combination arm were significantly (*p<0.05) lower than either drug alone.

Example 4: MM-141 activity is enhanced by glucose deprivation

This Example shows that MM-141 activity is enhanced by glucose deprivation.

HCC38 cells were cultured in regular medium or low glucose medium, and treated with or without gemcitabine and with or without MM-141. As shown in Figure 8A, MM-141 had no effect on cell proliferation when treated with in regular medium or low glucose (<5 mM) medium in the absence of gemcitabine. However, the antiproliferative activity of MM-141 in combination with gemcitabine was enhanced when cells were deprived of glucose (<5 mM). Given that MM-141 activity is enhanced by glucose deprivation, whether a combination of MM-141 and metformin (a DPP4 inhibitor) could target metabolic pathways in cancer cells was determined. As shown in Figure 8B, MM-141 and metformin had an additive effect in most of the HRG- and/or IGF-1 positive cell lines tested.

Example 5: Treatment of a patient with cancer with a combination of Istiratumab and Metformin

This Example discloses a method of treatment of a patient having a cancer, e.g., colorectal cancer or lung cancer, with istiratumab and the DPP4 inhibitor metformin.

Patients are dosed with istiratumab at 2.8 g/ml q2w by IV infusion, and metformin

hydrochloride is dosed (self-dosed by the patient) at two doses of 1000 mg each, twice daily P.O. SUMMARY OF SEQUENCE LISTING

Protein (MM- 151)

P2X V_H CDR3 MGRGKV

Protein (MM- 151)

P2X V_L CDRl QSVLYSPNNKNYLA

Protein (MM- 151)

P2X V_L CDR2 WASTR

Protein (MM- 151)

P2X V_L CDR3 QQYYGSP

Protein (MM- 151)

P3X V_H CDRl SYGIN

Protein (MM- 151)

P3X V_H CDR2 ISAYNGNTYY

Protein (MM- 151)

P3X V_H CDR3 DLGGYGSGS

Protein (MM- 151)

P3X V_L CDRl QSVSSNLA

Protein (MM- 151)

P3X V_L CDR2 GASTR

Protein (MM- 151)

P3X V_L CDR3 QDYRTWPR

Protein (MM- 151)

P1X V_H Protein (MM- 151) MGFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGSSVKVSCKAS

GGTFSSYAISWVRQAPGQGLEWMGSI IPIFGTVNYAQKFQGRVT ITADESTSTAYMELSSLRSEDTAVYYCARDPSVNLYWYFDLWGR GTLVTVSS

P1X V_LProtein (MM-151) MGTPAQLLFLLLLWLPDTTGDIQMTQSPSTLSASVGDRVTITCR

ASQSISSWWAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSG TEFTLTI SSLQPDDFATYYCQQYHAHPTTFGGGTKVEIK

P2X V_H Protein (MM-151) MGFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGSSVKVSCKAS

GGTFGSYAISWVRQAPGQGLEWMGSI IPIFGAANPAQKSQGRVT ITADESTSTAYMELSSLRSEDTAVYYCAKMGRGKVAFDIWGQGT MVTVSS

P2X V_L Protein (MM-151) MGTPAQLLFLLLLWLPDTTGDIVMTQSPDSLAVSLGERATINCK

SSQSVLYSPNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCQQYYGSPITFGGGTKVEI K

P3X V_H Protein (MM-151) MGFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKVSCKAS

GYAFTSYGINWVRQAPGQGLEWMGWI SAYNGNTYYAQKLRGRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARDLGGYGSGSVPFDPW GQGTLVTVSS

P3X V_L Protein (MM-151) MGTPAQLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCR

ASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSG TEFTLTI SSLQSEDFAVYYCQDYRTWPRRVFGGGTKVEIK

Heavy Chain CDRl HYVMA

(CDRHl) of MM-121

Heavy Chain CDR2 SISSSGGWTLYADSVKG (CDRH2) of MM-121

Heavy Chain CDR3 GLKMATIFDY

(CDRH3) of MM-121

Light Chain CDR1 TGTSSDVGSYNWS

(CDRL1) of MM-121

Light Chain CDR2 EVSQRPS

(CDRL2) of MM-121

Light Chain CDR3 CSYAGSSIFVI

(CDRL3)ofMM-121

Heavy Chain of Antibody EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGKG MM-121 LEWVSS

ISSSGGWTLYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVY YCTRGLKMATIFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VQFNWYVD GVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNG KEYKCKVSNKGLPAP IEKTI SKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light Chain of MM-121 QSALTQPASVSGSPGQSITISCTGTSSDVGSYNWSWYQQHPGK

APKLI I YEVSQRPSGVSNRFSGSKSGNTASLTI SGLQTEDEADY YCCSYAGSS IFVIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQA NKATLVC L VS DF YP GAVT VAWKAD G S P VKVGVE TTKPSKQSNNK YAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

Heavy Chain Variable EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGKG Region (VH) of MM-121 LEWVSS I SSSGGWTLYADSVKGRFTI SRDNSKNTLYLQMNSLRA

EDTAVYYCTRGLKMATIFDYWGQGTLVTVSS

Light Chain Variable QSALTQPASVSGSPGQSITISCTGTSSDVGSYNWSWYQQHPGK Region (VL) of MM-121 APKLI I YEVSQRPSGVSNRFSGSKSGNTASLTI SGLQTEDEADY

YCCSYAGSS IFVIFGGGTKVTVL

ErbB3 protein SEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEWMGNL

EIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRWRG

TQVYDGKFAIFVMLNYNTNSSHALRQLRLTQL

TEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIWKDNGRSC

PPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCC

HDECAGGCSGPQDTDCFACRHFNDSGACVPRC

PQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFWDQTSCVR

ACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSN

IDGFVNCTKILGNLDFLITQGDPWHKIPALDP

EKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYN

RGFSLLIMKNLNVTSLGFRSLKEI SAGRI YI SANRQLCYHHSLN

WTKVLRGPTEERLDIKHNRPRRDCVAEGKVCD

PLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAH

EAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSC

PHGVLGAKGP I YKYPDVQNECRPCHENCTQGC

KGPELQDCLGQTLVLIGKTHLTMALTVIAGLWIFMMLGGTFLY

WRGRRIQNKRAMRRYLERGES IEPLDPSEKANKVLARIFKETEL

RSLKVLGSGVFGTVHKGVWIPEGESIKIPVCI

KVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQL

VTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHG MVHRNLAARNVLLKSPSQVQVADFGVADLLPP

DDKQLLYSEAKTP IKWMALES IHFGKYTHQSDVWSYGVTVWELM

TFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWM

IDENIRPTFKELANEFTRMARDPPRYLVIKRE

SGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATT

TLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQES

AVSGSSERCPRPVSLHPMPRGCLASESSEGHV

TGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTP

LSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTE

EEDEDEEYEYMNRRRRHSPPHPPRPSSLEELG

YEYMDVGSDLSASLGSTQSCPLHPVP IMPTAGTTPDEDYEYMNR

QRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYAR

LKTLRSLEATDSAFDNPDYWHSRLFPKANAQR

T

Large chain (MM- 131) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSVYYWSWIRQPPG (CDRs are underlined) KGLEWIGVIYPSGNTYYSPSLKSRVTISVDTSKNQFSLKLSSVT

AADTAVYYCARTIYDLFDIWGQGTMVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPSCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCW VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSKYRWSVLT VLHQDWLNGKEYKCKVSNKGLPSS IEKTI SKAKGQPREPQVYTL PPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSKSCDKTGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSCPSCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCV WDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSDYRWSVL TVLHQDWLNGKEYKCKVSNKGLPSS IEKTI SKAKGQPREPQVYT LPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSGECGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRS TKSLLHSDGITYLYWYLQKPGQSPQLLIYQLSNLASGVPDRFSS SGSGTDFTLKI SRVEAEDEGVYYCAQNLEIPRTFGCGTKLEIKR TGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGESVKISCK ASGYTFTNYGMNWVRQAPGQCLKWMGWINTYTGESTYADDFKGR FAFSLDTSASTAYLQLSSLRSEDTAVYFCARFAIKGDYWGQGTL VTVSS

Light chain (MM- 131) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAP (CDRs are underlined) KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

LQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-cMET VH CDR1 GGSISSSVYY

(MM- 131)

anti-cMET VH CDR2 VIYPSGNTYYSPSLKS

(MM-131)

anti-cMET VH CDR3 TIYDLFDI

(MM-131)

anti-cMET VL CDR1 RASQGISSWL

(MM-131)

anti-cMET VL CDR2 AASSLQS (MM- 131)

46 anti-cMET VL CDR3 LQANSFPPT

(MM-131)

Equivalents and Incorporation by reference

Those skilled in the art will recognize, or be able to ascertain and implement using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combinations of the embodiments disclosed in the dependent claims are contemplated to be within the scope of the disclosure. The disclosure of each and every U.S. and foreign patent and pending patent application and publication referred to herein is specifically incorporated by reference herein in its entirety for all purposes.

Claims

We claim:

1. A method of treating cancer in a patient, wherein the patient has a tumor that is HRG- positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-121, (b) MM-131, (c) MM-141, (d) MM-151, (e) MM-131 and MM-121, or (f) MM-131 and MM-141.

2. A method of treating a cancer in a patient, wherein the patient has a tumor that is HGF-positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-131, (b) MM-141, (c) MM-151, or (d) MM- 13 land MM-151.

3. A method of treating a cancer in a patient, wherein the patient has a tumor that is EGF-positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-151, (b) MM-131, (c) MM-131 and MM-121, (d) MM-131 and MM-141, (e) MM-131 and MM-151.

4. A method of treating a cancer in a patient, wherein the patient has a tumor that is IGF- 2-positive, the method comprising administering to the patient a therapeutically effective amount of (a) MM-131, (b) MM-141, or (c) MM-151.

5. A method of treating a cancer in a patient, wherein the patient has a tumor that is both HRG- and IGF- 1 -positive, the method comprising administering to the patient a

therapeutically effective amount of (a) MM-121, (b) MM-131, (c) MM-141, or (d) MM-151.

6. The method of any one of claims 1-5, further comprising administering an effective amount of at least one additional antineoplastic agent.

7. The method of any one of claims 1-5, wherein positivity for HRG, HGF, EGF, IGF2, or IGF-1 is determined using an FDA-approved test.

8. The method of claim 7, wherein positivity for HRG, HGF, EGF, IGF2, or IGF-1 is determined by in situ hybridization or RT-PCR.

9. A method for treating a cancer in a patient, the method comprising co-administering to the subject a therapeutically effective amount of metformin and a bispecific anti-IGF-lR and anti-ErbB3 antibody.

10. The method of claim 9, wherein the metformin is metformin hydrochloride.

11. The method of claim 9, wherein the bispecific anti-IGF-lR and anti-ErbB3 antibody is istiratumab.

12. The method of claim 11, wherein the istiratumab is administered at a dose of 2.8 g/ml, q2w.

13. The method of claim 10, wherein the metformin hydrochloride is administered at a dose of 2000 mg daily.

14. The method of claim 13, wherein the 2000 mg daily dose comprises two doses of 1000 mg each, administered about 12 hours apart.

15. The method of any one of claims 9-14, further comprising administering a

therapeutically effective amount of gemcitabine.