WO2017070356A1 - DOSAGE AND ADMINISTRATION OF ANTI-c-MET, ANTI-EpCAM BISPECIFIC ANTIBODIES, USES THEREOF AND MENTHODS OF TREATMENT THEREWITH - Google Patents

Abstract

Disclosed herein are methods of treating a human subject having a tumor, the method comprising co-administering to the subject: 1) an effective amount of a c-Met inhibitor and 2) an effective amount of each of at least one additional antineoplastic agent.

Description

DOSAGE AND ADMINISTRATION OF ANTI-c-MET, ANTI-EpCAM BISPECIFIC

ANTIBODIES, USES THEREOF AND METHODS OF TREATMENT THEREWITH

RELATED APPLICATIONS

This application is related to United States Provisional Application Serial Nos. 62/245,743 and 62/323,479, filed October 23, 2015 and April 15, 2016, respectively. The contents of the aforementioned applications are incorporated herein by reference in their entireties.

FIELD

Provided are methods of treating patient with cancer with targeted therapies alone or in combination with chemotherapies.

BACKGROUND

Tumor cells express receptors for growth factors and cytokines that stimulate proliferation of the cells. Antibodies to such receptors can be effective in blocking the stimulation of cell proliferation mediated by growth factors and cytokines and can thereby inhibit tumor cell proliferation and tumor growth. Commercially available therapeutic antibodies that target receptors on cancer cells include, for example, trastuzumab which targets the HER2 receptor (also known as ErbB2) for the treatment of breast cancer, and cetuximab which targets the epidermal growth factor receptor (EGFR, also known as HER1 or ErbBl) for the treatment of colorectal cancer and head and neck cancer.

Monoclonal antibodies have significantly advanced our ability to treat cancers, yet clinical studies have shown that many patients do not adequately respond to monospecific therapy. This is in part due to the multigenic nature of cancers, where cancer cells comprise multiple and often redundant pathways that trigger proliferation, and/or the cancer cells grow in an environment, such as in the presence of human growth factor (HGF), which "rescues" the cancer cells from the treatment effects of a therapeutic agent. Bi- or multi- specific antibodies capable of blocking multiple growth and survival pathways at once may better meet the challenge of blocking cancer growth, and indeed many of them are advancing in clinical development. In addition, in the treatment of cancers, the coadministration of pluralities of antineoplastic drugs (combination therapy) often provides better treatment outcomes than monotherapy.

SUMMARY

Provided herein are compositions comprising, and methods for using, c-Met inhibitors. It has now been discovered that co-administration of a c-Met inhibitor (e.g., a bispecific antibody such as MM-131, a c-Met and EpCAM co-inhibitor biomolecule described below) with one or more additional antineoplastic agents, such as irinotecan, 5-fluorouracil, paclitaxel, oxaliplatin, and carboplatin, can yield therapeutic synergy.

Accordingly, provided are methods for the treatment of a cancer in a human patient (a "subject") wherein the methods comprise co-administering to the subject a therapeutically effective amount of a c-Met inhibitor biomolecule and one or more additional antineoplastic agents.

In one embodiment, provided herein are methods of treating a human subject having a tumor, the method comprising co-administering to the subject, 1) an effective amount of a c-Met inhibitor and 2) an effective amount of each of at least one additional antineoplastic agent. The at least one additional antineoplastic agent can be an antibody-based therapeutic, such as bevacizumab, cetuximab, panitumumab, MM-141 or MM-151. The at least one additional antineoplastic agent can be a chemother apeutic, such as a taxane, a topoisomerase I inhibitor, an antimetabolite, a

camptothecin, an alkylating agent, or an anthracycline. Optionally, the chemotherapeutic is encapsulated in a liposome. The chemotherapeutic can be, e.g., paclitaxel, nab-paclitaxel, docetaxel, irinotecan, topotecan, 5-fluorouracil, oxaliplatin, doxorubicin, gemcitabine, cisplatin or carboplatin. The c-Met inhibitor can be a bispecific antibody, such as a bispecific anti-c-Met/anti-EpCAM antibody, including a bispecific anti-c-Met/anti-EpCAM antibody comprising the CDRs set forth in SEQ ID NO: 1 -6. The tumor can be c-Met amplified and/or HGF+. In some aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is irinotecan and 5-fluorouracil. In some aspects, the c-Met inhibitor is a bispecific anti-c- Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is oxaliplatin and 5- fluorouracil. In yet other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is paclitaxel and carboplatin. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and 5-fluorouracil. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and a receptor-targeted liposomal taxane. The tumor can be a gastrointestinal, head and neck, esophageal, ovarian, colorectal, non-small cell lung, squamous cell lung, pancreatic, prostate, renal, thyroid, hepatocellular carcinoma, glioma/glioblastoma, or a breast cancer tumor. The embodiment can include administering to the patient a therapeutically effective amount of the c-Met inhibitor to the patient, and wherein the therapeutically effective amount is from 1 mg/kg patient body weight to 25 mg/kg patient body weight. In some aspects, the c-Met inhibitor is a bispecific anti-c- Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is irinotecan and 5- fluorouracil. In yet other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is oxaliplatin and 5-fluorouracil. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is paclitaxel and carboplatin. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and 5-fluorouracil. In yet other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti- EpCAM antibody and the at least one additional antineoplastic agent is irinotecan and 5-fluorouracil. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is oxaliplatin and 5-fluorouracil. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is paclitaxel and carboplatin. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti- EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and 5- fluorouracil. In yet other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and a receptor-targeted liposomal taxane. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan. Finally, in other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is a receptor-targeted liposomal taxane.

In an embodiment, disclosed herein are methods of reducing the number of metastatic lesions, the method comprising co-administering to the subject an effective amount of a bispecific anti-c- Met/anti-EpCAM antibody, and optionally an effective amount of each of at least one additional antineoplastic agent.

In an embodiment, disclosed herein are methods of treating a human subject having a tumor, the method comprising co-administering to the subject an effective amount of a c-Met inhibitor and an effective amount of an EGFR inhibitor. In certain aspects, the c-Met inhibitor is a bispecific antibody. In other aspects, the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody. In other aspects, the bispecific anti-c-Met/anti-EpCAM antibody comprises CDRs set forth in SEQ ID NOs: l-6. In certain aspects, the EGFR inhibitor is a bipecific antibody. In other aspects, the EGFR inhibitor is an oligoclonal antibody. In other aspects, the oligoclonal anti-EGFR antibody comprises the amino acid sequences set forth in SEQ ID NOs: 11-16. In certain aspects, the methods disclosed herein further comprise administering to the subject an effective amount of each of at least one additional antineoplastic agent. In certain aspects, the tumor is HGF+. In certain aspects, the tumor is EGFR+. In other aspects, the tumor is HGF+ and EGFR+.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB show heat maps displaying the results of quantification of dose-response curves from an in vitro viability screen of therapeutic agents in combination with HGF and/or MM- 131. Figures 1C and ID show how the addition of HGF and/or MM-131 changes the efficacy of each therapeutic agent. Figure 2A shows a bar graph of the % inhibition of viability from each therapeutic agent, either in the presence of HGF (red bars) or combined with HGF and MM-131 (blue bars), in the c-Met overexpressing lung cancer cell line NCI-H441. Figure 2B shows three examples (docetaxel, SN-38 and doxorubicin) of relative viability dose-response curves (normalized to media control) comparing each therapeutic agent alone (green), in the presence of InM HGF (red) or with HGF and MM-131 (blue). The dotted line indicates the media control. Figure 2C shows a bar graph showing the % inhibition of viability from each therapeutic agent, either alone (green bars) or combined with MM- 131 (light blue bars), in the c-Met amplified gastric cancer cell line MKN-45. Figure 2D shows three examples (oxaliplatin, SN-38 and 5-FU) of relative viability dose-response curves (normalized to media control) comparing each therapeutic agent alone (green) and in combination with MM-131 (light blue). The dotted line indicates the media control.

Figures 3A and 3B show the plotted data of tumor volume mean and standard error of the mean for each measurement of NCI-H441 xenografts in nu/nu mice treated weekly with either MM- 131 alone or in combination with docetaxel at 5 mg/kg (Figure 3A) or 10 mg/kg (Figure 3B).

Figures 4A-4C show the plotted data of tumor volume mean and standard error of the mean for each measurement of MKN-45 xenografts in NOD/SCID mice treated weekly with MM-131 alone or in combination with irinotecan and 5-fluorouracil (Figure 4A), oxaliplatin and 5-fluorouracil

(Figure 4B), and paclitaxel and carboplatin (Figure 4C).

Figure 5A shows the conditional probability of 29 colorectal cancer cells to respond (increase in viability) to one ligand and also respond to a second one. Figure 5B shows the effect of treatment with MM-151, MM-131 or the combination of both in 29 colorectal cancer cells in the presence of EGF and HGF.

Figure 6A 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 6B shows a graph of the same data in each of the cell lines, visualized as curves instead. Figure 6C 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). DETAILED DESCRIPTION

Methods and Compositions

Methods of combination therapy and combination compositions for treating cancer or reducing the number of metastatic lesions in a patient are provided. In these methods, the cancer patient is treated with both a bispecific anti-c-Met and anti-EpCAM antibody and one or more additional antineoplastic agents, e.g., an antibody-based therapeutic, such as bevacizumab, cetuximab, panitumumab, MM-141 or MM-151 ; or a chemotherapeutic, such as a taxane, a topoisomerase I inhibitor, an antimetabolite, a camptothecin, an alkylating agent, or an anthracycline.

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.

"Additional antineoplastic agents" is used herein to indicate any drug that is useful for the treatment of a tumor or cancer tumor.

"c-Met" or "c-MET" refers to Mesenchymal-Epithelial Transition (MET) factor, which is also known as Hepatocyte Growth Factor Receptor (HGFR), Scatter Factor (SF) receptor, AUTS9, and RCCP2. c-Met corresponds to Gene ID 4233, and has tyrosine -kinase activity. The primary single chain precursor protein is post-translationally cleaved to produce the alpha and beta subunits, which are disulfide linked to form the mature receptor. Two transcript variants encoding different isoforms have been found for this gene. HGF is the only known ligand for c-Met. The aa sequence of the human c-Met isoform a precursor is provided at Genbank Accession No. NP_001120972.1 and isoform b precursor is provided at Genbank Accession No. NP_000236.2.

"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.

"EpCAM" refers to epithelial cell adhesion molecule. Exemplary human EpCAM nucleic acid and protein sequences are set forth in RefSeqGene Gene ID: 4072 and GenBank Accession Number: NP_002345.2, respectively.

"PBA" refers to a polyvalent bispecific antibody, an artificial hybrid protein comprising at least two different binding moieties or domains and thus at least two different binding sites (e.g., two different antibody binding sites), wherein one or more of the pluralities of the binding sites are covalently linked, e.g., via peptide bonds, to each other. A preferred PBA described herein is an anti-IGF-lR+anti-ErbB3 PBA (e.g., as disclosed in U.S. Patent No. 8,476,409), which is a polyvalent bispecific antibody that comprises one or more first binding sites binding specifically to human IGF- 1R protein, and one or more second binding sites binding specifically to human ErbB3 protein. An anti-IGF-lR+anti-ErbB3 PBA is so named regardless of the relative orientations of the anti-IGF-lR and anti-ErbB3 binding sites in the molecule, whereas when the PBA name comprises two antigens separated by a slash (/) the antigen to the left of the slash is amino terminal to the antigen to the right of the slash. A PBA may be a bivalent binding protein, a trivalent binding protein, a tetravalent binding protein or a binding protein with more than 4 binding sites. An exemplary PBA is a tetravalent bispecific antibody, i.e., an antibody that has 4 binding sites, but binds to only two different antigens or epitopes. Exemplary bispecific antibodies are tetravalent "anti-IGF-lR/anti- ErbB3" PBAs and "anti-ErbB3 /anti- IGF-1R" PBAs. Typically the N-terminal binding sites of a tetravalent PBA are Fabs and the C-terminal binding sites are scFvs. Exemplary IGF-lR+ErbB3 PBAs comprising IgGl constant regions each comprise two joined essentially identical subunits, each subunit comprising a heavy and a light chain that are disulfide bonded to each other, (SEQ ID NOs hereinafter refer to sequences set forth in U.S. Patent No. 8,476,409, which is herein incorporated by reference in its entirety) e.g., M7-G1-M78 (SEQ ID NO: 284 and SEQ ID NO: 262 are the heavy and light chain, respectively), P4-G1-M1.3 (SEQ ID NO: 226 and SEQ ID NO: 204 are the heavy and light chain, respectively), and P4-G1-C8 (SEQ ID NO: 222 and SEQ ID NO: 204 are the heavy and light chain, respectively), are exemplary embodiments of such IgGl-(scFv)2 proteins. When the immunoglobulin constant regions are those of IgG2, the protein is referred to as an IgG2- (scFv)2. Other exemplary IGF-lR+ErbB3 PBAs comprising IgGl constant regions include (as described in U.S. Patent No. 8,476,409) SF-G1-P1,SF-G1-M1.3, SF-G1-M27, SF-G1-P6, SF-G1- B69, P4-G1-C8, P4-G1-P1, P4-G1-M1.3, P4-G1-M27, P4-G1-P6, P4-G1-B69, M78-G1-C8, M78- Gl-Pl, M78-G1-M1.3, M78-G1-M27, M78-G1-P6, M78-G1-B69, M57-G1-C8, M57-G1-P1, M57- G1-M1.3, M57-G1-M27, M57-G1-P6, M57-G1-B69, P1-G1-P4, P1-G1-M57, P1-G1-M78, M27-G1- P4, M27-G1-M57, M27-G1-M78, M7-G1-P4, M7-G1-M57, M7-G1-M78, B72-G1-P4, B72-G1- M57, B72-G1-M78, B60-G1-P4, B60-G1-M57, B60-G1-M78, P4M-G1-M1.3, P4M-G1-C8, P33M- G1-M1.3, P33M-G1-C8, P4M-G1-P6L, P33M-G1-P6L, P1-G1-M76.

"TFcA" refers to a tandem Fc antibody. A TFcA may be a monovalent or monospecific TFcA, e.g., comprising a single binding site. A TFcA may also be a bispecific TFcA, which is referred to herein as a TFcBA. A TFcA may be monoclonal.

"TFcBA" refers to a tandem Fc bispecific antibody, an artificial hybrid protein comprising at least two different binding moieties or domains and thus at least two different binding sites (e.g., two different antibody binding sites), wherein one or more of the pluralities of the binding sites are covalently linked, e.g., via peptide bonds, to each other. An exemplary TFcBA described herein is an anti-c-Met+anti-EpCAM TFcBA, which is a polyvalent bispecific antibody that comprises a first binding site binding specifically to a c-Met protein, e.g., a human c-Met protein, and one or more second binding sites binding specifically to an EpCAM protein, e.g., a human EpCAM protein. When a TFcBA name comprises two antigens separated by a plus sign (+) this indicates that the binding sites for the two antigens may be in either relative amino to carboxy orientation in the molecule, whereas when the TFcBA name comprises two antigen binding site names separated by a slash (/) the antigen binding site to the left of the slash is amino terminal to the antigen binding site to the right of the slash. A TFcBA may be a bivalent binding protein, a trivalent binding protein, a tetravalent binding protein or a binding protein with more than 4 binding sites. An exemplary TFcBA is a bivalent bispecific antibody, i.e., an antibody that has 2 binding sites, each binding to a different antigen or epitope. In certain embodiments, the N-terminal binding site of a TFcBA is a Fab and the C-terminal binding site is a scFv.

The term "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 below as SEQ ID NOs: 1-3, 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 below as SEQ ID NOs:4-6, respectively). The sequence of the Fab light chain is set forth below as SEQ ID NO: 7. The sequence of the TFcBA large chain is set forth below as SEQ ID NO: 8.

NO:

1 RASQGISSWL

2 AASSLQS

3 LQANSFPPT

4 GGSISSSVYY

5 VIYPSGNTYY SPSLKS

6 TIYDLFDI

DIQMTQSPSS VSASVGDRVT ITCRASQGIS SWLAWYQQKP GKAPKLLIYA ASSLQSGVPS

RFSGSGSGTD FTLTISSLQP EDFATYYCLQ ANSFPPTFGG GTKVEIKRTV AAPSVFIFPP

7

SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT

LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

QLQLQESGPG LVKPSETLSL TCTVSGGSIS SSVYYWSWIR QPPGKGLEWI GVIYPSGNTY

YSPSLKSRVT ISVDTSKNQF SLKLSSVTAA DTAVYYCART IYDLFDIWGQ GTMVTVSSAS

TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL

YSLSSWTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPSC PAPEFLGGPS

VFLFPPKPKD TLMI SRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSK

YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSREEMT

KNQVSLSCAV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ

GNVFSCSVMH EALHNHYTQK SLSLSKSCDK TGGGGSGGGG SGGGGSGGGG SGGGGSGGGG

SGGGGSGGGG SCPSCPAPEF LGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSQEDPEVQ

o o

FNWYVD GVE V HNAKTKPREE QFNSDYRWS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK

TISKAKGQPR EPQVYTLPPS REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT

PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS GECGGGGSGG

GGSDIVMTQS PLSLPVTPGE PASISCRSTK SLLHSDGITY LYWYLQKPGQ SPQLLIYQLS

NLASGVPDRF SSSGSGTDFT LKI SRVEAED EGVYYCAQNL EIPRTFGCGT KLEIKRTGGG

GSGGGGSGGG GSGGGGSQVQ LVQSGAEVKK PGESVKISCK ASGYTFTNYG MNWVRQAPGQ

CLKWMGWINT YTGESTYADD FKGRFAFSLD TSASTAYLQL SSLRSEDTAV YFCARFAIKG

DYWGQGTLVT VSS

The term "MM- 141" refers to anti-IGF-lR+anti-ErbB3 PBA P4-G1-M1.3 having 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:204 and each heavy chain comprises the amino acid sequence set forth in SEQ ID NO:226, wherein SEQ ID NOs: 204 and 226 are those as set forth in U.S. Patent No.8,476,409 (which is herein incorporated by reference in its entirety). For the sake of convenience, the sequence of the light chain of MM-141 is set forth below as SEQ ID NO: 9 and the sequence of each heavy chain is set forth below as SEQ ID NO: 10 Table 2: Sequences of MM-141

SEQ Sequence

ID

NO:

D IQMTQSP S S LSASLGDRVT I TCRASQGI S SYLAWYQQKP GKAPKLL IYA KSTLQSGVP S

RFSGSGSGTD FTLT I S SLQP EDSATYYCQQ YWTFPLTFGG GTKVE IKRTV AAP SVF IFPP

Q

SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQD SKD STYSLS STLT

LSKADYEKHK VYACEVTHQG LS SPVTKSFN RGEC

EVQLLQSGGG LVQPGGSLRL SCAASGFMFS RYPMHWVRQA PGKGLEWVGS I SGSGGATPY

ADSVKGRFT I SRDNSKNTLY LQMNSLRAED TAVYYCAKDF YQI LTGNAFD YWGQGTTVTV

S SASTKGP SV FPLAP S SKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ

S SGLYSLS SV VTVP S S SLGT QTYI CNVNHK P SNTKVDKKV EPKSCDKTHT CPPCPAPELL

GGP SVFLFPP KPKDTLMI SR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ

YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAP IEKT I SKAKGQPRE PQVYTLPP SR

1 Π

EEMTKNQVSL TCLVKGFYP S D IAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS

RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GGGGGSGGGG SGGGGSQVQL VQSGGGLVQP

GGSLRLSCAA SGFTFDDYAM HWVRQAPGKG LEWVAGI SWD SGSTGYADSV KGRFT I SRDN

AKNSLYLQMN SLRAEDTALY YCARDLGAYQ WVEGFDYWGQ GTLVTVS SAS TGGGGSGGGG

SGGGGSGGGG S SYELTQDPA VSVALGQTVR I TCQGDSLRS YYASWYQQKP GQAPVLVIYG

KNNRP SGIPD RFSGSTSGNS ASLT I TGAQA EDEADYYCNS RDSPGNQWVF GGGTKVTVLG

The term "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 PIX is a human IgGl having a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2; P2X is a human IgGl having a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 4; and P3X is a human IgGl having a heavy chain variable region comprising SEQ ID NO: 5 and a light chain variable region comprising SEQ ID NO: 6, which sequences are set forth in US Patent No. 8,691 ,231. The P1X:P2X:P3X molar ratio is 2:2: 1. For the sake of convenience, the heavy and light chain variable region sequences of PIX are set forth below as SEQ ID NOs: 11 and 12, respectively; the heavy and light chain variable region sequences of P2X are set forth below as SEQ ID NOs: 13 and 14, respectively; and the heavy and light chain variable region sequences of P3X are set forth below as SEQ ID NOs: 15 and 16, respectively.

Table 3: Sequences of MM-151

SEQ Sequence ID

NO:

MGFGLSWLFL VAI LKGVQCQ VQLVQSGAEV KKPGS SVKVS CKASGGTFS S YAI SWVRQAP

11 GQGLEWMGS I IP IFGTVNYA QKFQGRVT I T ADESTSTAYM ELS SLRSEDT AVYYCARDP S VNLYWYFDLW GRGTLVTVS S

MGTPAQLLFL LLLWLPDTTG D IQMTQSP ST LSASVGDRVT I TCRASQS I S SWWAWYQQKP

12 GKAPKLL IYD AS SLESGVP S RFSGSGSGTE FTLT I S SLQP DDFATYYCQQ YHAHPTTFGG GTKVE IK

13 MGFGLSWLFL VAI LKGVQCQ VQLVQSGAEV KKPGS SVKVS CKASGGTFGS YAI SWVRQAP GQGLEWMGS I IP IFGAANPA QKSQGRVT I T ADESTSTAYM ELS SLRSEDT AVYYCAKMGR GKVAFD IWGQ GTMVTVS S

14 MGTPAQLLFL LLLWLPDTTG D IVMTQSPDS LAVSLGERAT INCKS SQSVL YSPNNKNYLA WYQQKPGQPP KLL IYWASTR ESGVPDRFSG SGSGTDFTLT I S SLQAEDVA VYYCQQYYGS P ITFGGGTKV E IK

15 MGFGLSWLFL VAI LKGVQCQ VQLVQSGAEV KKPGASVKVS CKASGYAFTS YGINWVRQAP GQGLEWMGWI SAYNGNTYYA QKLRGRVTMT TDTSTSTAYM ELRSLRSDDT AVYYCARDLG GYGSGSVPFD PWGQGTLVTV S S

16 MGTPAQLLFL LLLWLPDTTG E IVMTQSPAT LSVSPGERAT LSCRASQSVS SNLAWYQQKP GQAPRLL IYG ASTRATGIPA RFSGSGSGTE FTLT I S SLQS EDFAVYYCQD YRTWPRRVFG GGTKVE IK

Combination therapies with additional anti-cancer agents

As herein provided, bispecific antibodies {e.g., MM-131) are co-administered with one or more additional antineoplastic agents (e.g., an antibody-based therapeutic or a chemotherapeutic, such as a taxane, a topoisomerase I inhibitor, an antimetabolite, a camptothecin, an alkylating agent, or an anthracycline), to provide effective treatment to human patients having a tumor (e.g., a

gastrointestinal, head and neck, esophageal, ovarian, colorectal, non-small cell lung, squamous cell lung, pancreatic, prostate, renal, thyroid, hepatocellular carcinoma, glioma/glioblastoma, or a breast cancer tumor).

In certain combination therapy methods, one or more of the following therapeutic agents is co-administered to the patient with an anti-c-Met/anti-EpCAM antibody.

5-Fluorouracil (5-FU Adrucil®, Carac®, Efudix®, Efudex® and Fluoroplex®) is a pyrimidine analog that works through irreversible inhibition of thymidylate synthase. 5-Fluorouracil has been given systemically for anal, breast, colorectal, oesophageal, stomach, pancreatic and skin cancers (especially head and neck cancers).

Carboplatin (cis-diammine(l,l-cyclobutanedicarboxylato)platinum(II) (trade names Paraplatin® and Paraplatin-AQ®) is a chemotherapy drug used against mainly ovarian carcinoma, lung, head and neck cancers as well as endometrial, esophageal, bladder, breast and cervical, central nervous system, or germ cell tumors; and osteogenic sarcomas (CAS No. 41575-94-4).

Cisplatin (also known as cisplatinum, platamin, neoplatin, cismaplat or cis- diamminedichloroplatinum(II)) is a chemotherapy drug for treating solid malignancies. Sarcomas, some carcinomas (e.g. small cell lung cancer, and ovarian cancer), lymphomas, bladder cancer, cervical cancer, and germ cell tumors can be treated with cisplatin (CAS No. 15663-27-1)

Docetaxel (Taxotere®) is an anti-mitotic chemotherapy used for the treatment of breast, advanced non-small cell lung, metastatic androgen-independent prostate, advanced gastric and locally advanced head and neck cancers.

Doxorubicin (trade name Adriamycin®, Rubex®; PEGylated liposomal form trade name

Doxil®; non-PEGylated liposomal form trade name Myocet®, Caelyx®), also known as hydroxydaunorubicin and hydroxydaunomycin, is a drug used in cancer chemotherapy. It is used to treat cancers such as hematological malignancies (blood cancers, such as leukemia and lymphoma), carcinomas and soft tissue sarcomas (CAS No. 23214-92-8).

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 HCl is 2'-deoxy-2',2'-difluorocytidine monohydrochloride (-isomer) (MW=299.66) and is administered parenterally, typically by i.v. infusion.

Irinotecan HCl (trade name Camptosar®, Campto®) is a topoisomerase 1 -inhibitor, mainly used in the treatment of colon cancer. It is often used in the FOLFIRI regimen, consisting of infusion of 5-fluorouracil, leucovorin, and irinotecan (CAS No. 100286-90-6).

Nab-paclitaxel (Abraxane®) is a nanoparticulate albumin-bound formulation of paclitaxel (Paclitaxel CAS No. 33069-62-4).

Nanoliposomal irinotecan (irinotecan sucrosofate liposome injection: MM-398) is a stable nanoliposomal formulation of irinotecan. MM-398 is described, e.g., in U.S. Patent No. 8,147,867. MM-398 may be administered, for example, on day 1 of the cycle at a dose of 120 mg/m², except if the patient is homozygous for allele UGTlAl*, wherein nanoliposomal irinotecan is administered on day 1 of cycle 1 at a dose of 80 mg/m². The required amount of MM-398 may be diluted, e.g., in 500mL of 5% dextrose injection USP and infused over a 90 minute period.

Oxaliplatin (trade name Eloxatin®; [(lR,2R)-cyclohexane-l,2-diamine](ethanedioato-

0,0')platinum(II)), is a platinum-based antineoplastic agent used in cancer chemotherapy, such as the treatment of colorectal cancers (CAS No. 63121-00-6).

Paclitaxel (Taxol®) is an anti-mitotic chemotherapy used for the treatment of lung, ovarian, breast and head and neck cancers. Topotecan (trade name Hycamtin®) is a chemotherapeutic agent that is a topoisomerase inhibitor. It can be used to treat ovarian cancer, cervical cancer, lung cancer, neuroblastomas, brainstem gliomas, and Ewing's sarcoma (CAS No. 123948-87-8).

Bevacizumab (trade name Avastin®) is an angiogenesis inhibitor. Bevacizumab is a recombinant humanized monoclonal antibody that blocks angiogenesis by inhibiting vascular endothelial growth factor A (VEGF-A) (CAS No. 216974-75-3).

Cetuximab (trade name Erbitux®) is an epidermal growth factor receptor (EGFR) inhibitor used for the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer. Cetuximab is a chimeric (mouse/human) monoclonal antibody (CAS No. 205923- 56-4).

Panitumumab (trade name Vectibix®) is a fully human monoclonal antibody specific to epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1 and HERl in humans). It is commonly used to treat colorectal cancers (CAS No. 339177-26-3).

Standard-of-care regimens in MM-131 relevant indications is shown in Table 4. For highly cytotoxic drugs, two-drug cytotoxic regimens may present advantages over three -drug cytotoxic regimens because of lower toxicity. Note that in the table due to a variety of chemotherapeutic-based regimens that can be used in gastric cancer, there is no internationally accepted regimen.

Table 4: Standard-of-care regimens in MM-131 relevant indications

Outcomes As shown in the Examples herein, co-administration of an anti-c-Met/anti-EpCAM antibody with one or more additional antineoplastic agents (e.g., irinotecan, 5-fluorouracil, oxaliplatin, paclitaxel, and carboplatin) provides improved efficacy compared to treatment with the antibody alone or with the one or more additional antineoplastic agents in the absence of antibody therapy. Preferably, a combination of an anti-c-Met/anti-EpCAM antibody with one or more additional antineoplastic agents exhibits therapeutic synergy. Also shown in the Examples herein is that human growth factor, HGF, frequently reduces sensitivity to standard-of-care agents (such as irinotecan, 5- fluorouracil, oxaliplatin, paclitaxel, and carboplatin), but that MM-131 reverses HGF' s effect. It is also found that MM-131 is effective in combination therapy to treat c-Met amplified cancers.

"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, independently active constituent of the combination used at its optimum dose (T. H. Corbett et al., 1982, Cancer Treatment Reports, 66, 1187). When assessing therapeutic synergy of combinations comprising leucovorin and 5-FU, the combination of leucovorin and 5-FU is treated as a single agent, since leucovorin is not independently active (i.e., is not active when administered as monotherapy) and only works by potentiating the activity of the 5-FU. 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 with the an antibody, e.g., a bispecific antibody, or treatment with other antineoplastic agent(s) in the absence of antibody therapy. 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.

One non-limiting example of a measure by which effectiveness of a therapeutic treatment can be quantified is by calculating the log 10 cell kill, which is determined according to the following equation:

loglO cell kill = T C (days)/3.32 x Td

in which T C represents the delay in growth of the cells, which is the average time, in days, for the tumors of the treated group (T) and the tumors of the control group (C) to have reached a predetermined value (1 g, or 10 mL, for example), and Td represents the time, in days necessary for the volume of the tumor to double in the control animals. When applying this measure, a product is considered to be active if loglO cell kill is greater than or equal to 0.7 and a product is considered to be very active if loglO cell kill is greater than 2.8. Using this measure, a combination, used at its own maximum tolerated dose, in which each of the constituents is present at a dose generally less than or equal to its maximum tolerated dose, exhibits therapeutic synergy when the loglO cell kill is greater than the value of the loglO cell kill of the best constituent when it is administered alone. In an exemplary case, the loglO cell kill of the combination exceeds the value of the loglO cell kill of the best constituent of the combination by at least 0.1 log cell kill, at least 0.5 log cell kill, or at least 1.0 log cell kill.

Kits and Unit Dosage Forms

Further provided are kits that include a pharmaceutical composition containing a bispecific anti-c-Met and anti-EpCAM antibody, 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 an ErbB2 expressing cancer.

Preferably, the kits include multiple packages of the single -dose pharmaceutical

composition(s) containing an effective amount of a bispecific anti-c-Met and anti-EpCAM antibody for a single administration in accordance with the methods provided above. Optionally, instruments or devices necessary for administering the pharmaceutical composition(s) may be included in the kits. For instance, a kit may provide one or more pre -filled syringes containing an amount of a bispecific anti-c-Met and anti-EpCAM antibody that is about 100 times the dose in mg/kg indicated for administration in the above methods.

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 a bispecific anti-c-Met and anti-EpCAM antibody.

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.

The following Examples utilize several different methodologies. The materials and methods used in these Examples are detailed below.

In Vitro Experiments

Cell culture and reagents

The following cell lines, obtained from the American Type Culture Collection (Manassas, VA), were used in the screen: NCI-H747, CAR-1, CCK81, LIM1215, NCI-H441, MKN-45, SW948, SNU-5, HCC827, CX-1, SW620, HT-29, SW1417, NCI-N87, HCT116, NCI-H1993, A549, and RKO. Cells were cultured in RPMI medium (Life Technologies; Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 1% penicillin-streptomycin (pen/strep, Life Technologies) at 37°C and 5% C0₂. Recombinant human hepatocyte growth factor (HGF) was obtained from Peprotech (Rocky Hill, NJ; catalog #100-39). Docetaxel (catalog #S7787), paclitaxel (catalog #S1150), SN-38 (irinotecan, catalog #S4908), doxorubicin (catalog #S1208), oxaliplatin (catalog #S1224), and 5-fluorouracil (5-FU, catalog #S1209) were purchased from Selleck (Houston, TX) and stock solutions were prepared in DMSO. Cetuximab was obtained from Myoderm

(Norristown, PA). MM-131 was manufactured in-house by the Merrimack Pharmaceuticals

(Cambridge, MA) Protein Engineering Department and stored at 4°C. CellTiter-Glo® was obtained from Promega (Madison, WI) and reconstituted fresh for each experiment. Cells were grown in exponential phase using standard cell culture media containing 10% FBS, and were passaged at least twice before the start of each experiment. On the day of the experiment, cells were visually assessed under a microscope to confirm that they were between 60% and 80% confluent. Cells were detached from the culture plate through the addition of 0.05% trypsin-EDTA (Life Technologies - Gibco®), and once a majority of cells were detached (as assessed visually by microscope) the trypsin was inactivated using cell culture media containing 4% FBS.

Spheroid growth assay

To measure cell viability in a three-dimensional spheroid culture, cells were seeded into 384- well low-binding multi-spheroid culture plates (Scivax USA Inc. ; Woburn, MA) in the relevant growth medium supplemented with 4% FBS and 1% pen/strep. To allow for spheroid formation, plates were incubated for 48 hours, after which cells were treated with ligand (InM HGF) and inhibitors in 4% FBS containing medium. Following 48 hours of incubation, media, ligands, and drugs were replenished, and the plates were incubated for an additional 24 hours. Cell viability was determined through measuring well luminescence on an Envision plate reader (Perkin Elmer;

Waltham, MA) after incubation with CellTiter-Glo® reagent for 10 minutes.

Data analysis

Data were pre-processed in Microsoft Excel and visualized in Prism (GraphPad; La Jolla, CA). The combination data was further analyzed in MATLAB (MathWorks; Natick, MA), using a customized code.

In Vivo Experiments

Cell Lines

MKN-45 (ATCC) and NCI-H441cells were cultured in T-75 flasks under a humidified atmosphere of 5% C0₂ at 37°C in RPMI 1640 medium (Sigma; St. Louis, MO) supplemented with 10% fetal bovine serum (Life Technologies, catalog #16140-071), 1% penicillin streptomycin (Life Technologies, catalog #151140-112), and 1% L-Glutamine (Life Technologies, catalog #25030-081) . Cells were harvested by exposure to 0.25% trypsin EDTA (Life Technologies, catalog #25299-056), washed twice with phosphate -buffered saline (PBS; Life Technologies, catalog #14190-136), and resuspended in growth factor reduced Matrigel (Corning; Corning, NY; #356231) at a 1 : 1 ratio with PBS. Viability (>90 ) was verified by Trypan Blue (Life Technologies, catalog #15250-061).

Therapeutic agents

MM-131 (12 mg/kg), oxaliplatin (Curascript; Lake Mary, FL; NDC 67457-469-10), 5- Fluorouracil (5-FU, Curascript, NDC 16729-276-03), Carboplatin (Curascript, NDC 25021-202-45), and Docetaxel (Curascript, NDC 16729-267-63) were diluted in sterile PBS (Life Technologies - Gibco 14190-136) before intraperitoneal (i.p.) or intravenous (i.v.) administration. Paclitaxel (LC LABS; Woburn, MA; P-9600, 40mg/ml) stock solution was diluted in ethanol (Sigma E7023) at 37 °C for 15 min. Cremofor EL® (Sigma C5135) was added at a 1 : 1 ratio in ethanol and diluted in sterile PBS before intraperitoneal injection. Irinotecan HC1 lOmg solution was diluted in 5% Dextrose

(Sigma 49163). The resulting solution was heated to 60-70 °C for 3-5 minutes and filtered through sterile 0.2μπι syringe filters.

In vivo tumor response studies

MKN-45 cells were implanted subcutaneously into the flank of NOD/SCID female mice and NCI-H441 cells were implanted subcutaneously into the flank of female athymic nude mice. When average tumor volume reached 100 to 200 mm³, animals were randomized to treatment groups according to tumor volume and body weight. Relevant treatments were administered to the animals once every week via i.v. or i.p. administration. Tumor dimensions were measured twice weekly with calipers, and tumor volumes were calculated using the formula: (JI/6)*L*W². All mice were from Charles River Laboratories, Wilmington, MA. All in vivo mouse experimental protocols were approved and maintained in accordance with the Institutional Animal Care and Use Committee (IACUC) procedures and guidelines.

Example 1: Screening for ligand and drug responses in vitro.

An in vitro viability screen of therapeutic agents in combination with HGF and/or MM-131 was performed. Heat maps shown in Figures 1A and IB demonstrate the results of quantification of dose-response curves. These data were generated by calculating the log2 of the mean fold change between the area-under-the -curve (AUCs) for either each therapeutic agent alone compared to each therapeutic agent in the presence of InM HGF (Figure 1A) or each therapeutic agent with HGF compared to each therapeutic agent in combination with ΙμΜ MM-131 (Figure IB). Scatter plots

(Figures 1C and ID) demonstrate how the addition of HGF and/or MM-131 changes the efficacy of each therapeutic agent (color coded). Scatter plot data were generated by plotting the percent inhibition for either each therapeutic agent alone compared to the therapeutic agent in the presence of InM HGF (Figure 1C) or each agent with HGF compared to each agent in combination with MM-131 (Figure ID). Example 2: MM-131 sensitizes c-Met driven tumor cells to therapeutic agents

The bar graph in Figure 2A demonstrates the percent inhibition of viability for the c-Met overexpressing lung cancer cell line NCI-H441 either in the presence of HGF (red bars) or combined with HGF and MM-131 (blue bars) for each therapeutic agent. Three examples (docetaxel, SN-38 and doxorubicin) of relative viability dose-response curves (normalized to media control) comparing each therapeutic agent alone (green), in the presence of InM HGF (red) or with HGF and ΙμΜ MM-131 (blue) are shown in Figure 2B. The dotted line indicates the media control.

The bar graph in Figure 2C demonstrates the percent inhibition of viability for the c-Met amplified gastric cancer cell line MKN-45 either alone (green bars) or in combination with MM-131 (light blue bars) for each therapeutic agent. Three examples (oxaliplatin, SN-38 and 5-FU) of relative viability dose-response curves (normalized to media control) comparing each therapeutic agent alone (green) and in combination with Ι μΜ MM-131 (light blue) are shown in Figure 2D. The dotted line indicates the media control. Example 3: Tumor growth inhibition of NCI-H441 NSCLC xenografts with MM-131 in combination with docetaxel

NCI-H441 xenografts were established subcutaneously in nu/nu mice (n=8) and treated weekly with either MM-131(12 mg/kg, i.p.) alone or in combination with docetaxel at 5 mg/kg, i.v. (Figure 3A) or 10 mg/kg, i.v. (Figure 3B). Tumor dimensions were measured every 3-4 days with calipers and volumes were calculated using the formula: (n/6)*L*W2. Plotted data of tumor volumes represents the mean and standard error of the mean for each measurement. The tumor volumes in the combination arm were significantly (*p<0.05) lower than either drug alone (p = 0.00459, Figure 3A and p= 0.0139, Figure 3B). Example 4: Tumor growth inhibition of MKN45 xenograft with MM-131 in combination with multiple standard of care regimens in gastric cancer

MKN-45, a c-Met amplified gastric xenograft model, was established subcutaneously in NOD/SCID mice (n=8) and treated weekly with MM-131(12 mg/kg, i.p.) alone or in combination with a FOLFIRI based regimen of irinotecan HC1 (25 mg/kg, i.v,) and 5-FU (50 mg/kg, i.p.) (Figure 4A); a FOLFOX based regimen of oxaliplatin (5 mg/kg, i.v.) and 5-FU (50 mg/kg, i.p.) (Figure 4B); a regimen of Paclitaxel (5 mg/kg, i.p.) and carboplatin (50 mg/kg, i.p) (Figure 4C). Tumor dimensions were measured every 3-4 days with calipers and volumes were calculated using the formula:

(JI/6)*L*W2. Plotted data of tumor volumes represents the mean and standard error of the mean for each measurement. The tumor volumes in the combination arm were significantly (*p<0.05) lower than either drug alone (p= 0.0010, Figure 4A; p=0.0017, Figure 4B; p=0.0031, Figure 4C). Example 5: Screening for ligand combinations which promote tumor cell growth and inhibiting the growth of cancer cells with MM- 131 in combination with MM-151

To identify ligand combinations which promote tumor cell growth, 29 colorectal cell lines (OUMS23, LS411N, DLD1, SW948, HCT8, LSI 80, LS174T1, HCT15, SW48, RKO, COLO201, HCC2998, MDST8, HCT116, HT29, COCM1, COLO205, KM12, LOVO, SW620, WIDR, SW403, CXI, SW1417, C2BBE1, CCK81, NCIH747, CAR1, and LIM215) were cultured in the presence of EGF, HRG, IGF-1, or HGF. The cell lines were seeded at 5,000 cells per well in low-binding Nanoculture 96-well plates (Scivax Corporation) and grown in the appropriated medium

supplemented with 4% fetal bovine serum and Pen-Strep at 37 °C. Forty-eight hours later, after which time spheroids had formed, the cells were incubated with ligands: EGF (5 nM), HRG (5 nM), IGF-1 (50 nM), or HGF (1 nM) for 96 h at 37 °C. Relative live cell densities were then determined by luminescence using the CellTiter Glo reagent (Promega).

As shown in Figure 5A, responders to EGF usually responded to HGF (high conditional probability). For example, the black square in the second row (row 2) and last column (column 4) represents the probability that cells that respond to EGF also respond to HGF. Response was defined as >=15 stimulation by the ligand compared to controls. Euclidean distance is used as the distance metric for clustering

To determine whether the combination of MM-131 and MM-151 have an additive or a synergistic inhibitory effect on cancer cell growth, the 29 colorectal cell lines were seeded at 5,000 cells per well in low-binding Nanoculture 96-well plates (Scivax Corporation) and grown in the appropriated medium supplemented with 4% fetal bovine serum and Pen-Strep at 37 °C. Forty-eight hours later, after which time spheroids had formed, cells were incubated with ligands and/or drugs for 96 h at 37 °C. Relative live cell densities were then determined by luminescence using the CellTiter Glo (CTT) reagent (Promega). Cells were treated with ligands EGF (5 nM) and HGF (1 nM), ligands plus MM-151 or MM-131 (1 μΜ each), or with ligands plus both MM-151 and MM-131.

As shown in Figure 5B, approximate 80% (23 out of 29) of the colorectal cancer cells benefitted (measured as a decrease in cell viability) from the combination of MM-151+MM-131 when both ligands (EGF and HGF) were present. The patterns correspond to the CTG response of each cell line to combination therapy (top row) or monotherapies (second and third rows) in the presence of both ligands. CTG response in the presence of inhibitor(s) + ligands was normalized to CTG response in the presence of ligands alone. The cell lines with asterisks below them correspond to cell lines that responded at least 15% better to the combination therapies compared to the best monotherapy.

Example 6: MM-131 in combination with MM-151 overcomes HGF-mediated resistance

As shown in Figures 6A-6B, 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 6A-6B show cell viability data for a panel of 13 NSCLC cell lines. The dots in Figure 6 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 6C, 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 - 200 mm , 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.

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

CLAIMS What is claimed is:

1. A method of treating a human subject having a tumor, the method comprising coadministering to the subject, 1) an effective amount of a c-Met inhibitor and 2) an effective amount of each of at least one additional antineoplastic agent.

2. The method of claim 1 , wherein the at least one additional antineoplastic agent is an antibody-based therapeutic.

3. The method of claim 2, wherein the antibody-based therapeutic is bevacizumab, cetuximab, panitumumab, MM-141 or MM-151.

4. The method of claim 1 , wherein the at least one additional antineoplastic agent is a chemotherapeutic.

5. The method of claim 4, wherein the chemotherapeutic is a taxane, a topoisomerase I inhibitor, an antimetabolite, a camptothecin, an alkylating agent, or an anthracycline, optionally wherein the chemotherapeutic is encapsulated in a liposome.

6. The method of claim 4, wherein the chemotherapeutic is paclitaxel, nab-paclitaxel, docetaxel, irinotecan, topotecan, 5-fluorouracil, oxaliplatin, doxorubicin, gemcitabine, cisplatin or carboplatin, optionally wherein the chemotherapeutic is encapsulated in a liposome.

7. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific antibody.

8. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody.

9. The method of claim 8, wherein the bispecific anti-c-Met/anti-EpCAM antibody comprises CDRs set forth in SEQ ID NOs: l-6.

10. The method of any of the preceding claims, wherein the tumor is c-Met amplified.

11. The method of any of the preceding claims, wherein the tumor is HGF+.

12. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is irinotecan and 5-fluorouracil.

13. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is oxaliplatin and 5-fluorouracil.

14. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is paclitaxel and carboplatin.

15. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and 5-fluorouracil.

16. The method of claim 1, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti- EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and a receptor-targeted liposomal taxane.

17. The method of any of the preceding claims, wherein the tumor is a gastrointestinal, head and neck, esophageal, ovarian, colorectal, non-small cell lung, squamous cell lung, pancreatic, prostate, renal, thyroid, hepatocellular carcinoma, glioma/glioblastoma, or a breast cancer tumor.

18. The method of any of the preceding claims, wherein the method comprises administering to the patient a therapeutically effective amount of the c-Met inhibitor to the patient, and wherein the therapeutically effective amount is from 1 mg/kg patient body weight to 25 mg/kg patient body weight.

19. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is irinotecan and 5-fluorouracil.

20. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is oxaliplatin and 5-fluorouracil.

21. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is paclitaxel and carboplatin.

22. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and 5-fluorouracil.

23. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is irinotecan and 5-fluorouracil.

24. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is oxaliplatin and 5-fluorouracil.

25. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is paclitaxel and carboplatin.

26. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and 5-fluorouracil.

27. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan and a receptor-targeted liposomal taxane.

28. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is liposomal irinotecan.

29. The method of any of the preceding claims, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-EpCAM antibody and the at least one additional antineoplastic agent is a receptor-targeted liposomal taxane.

30. A method of reducing the number of metastatic lesions, the method comprising coadministering to the subject an effective amount of a bispecific anti-c-Met/anti-EpCAM antibody, and optionally an effective amount of each of at least one additional antineoplastic agent.

31. A method of treating a human subject having a tumor, the method comprising co- administering to the subject, 1) an effective amount of a c-Met inhibitor and 2) an effective amount of an EGFR inhibitor.

32. The method of claim 31 , wherein the c-Met inhibitor is a bispecific antibody.

33. The method of claim 32, wherein the c-Met inhibitor is a bispecific anti-c-Met/anti-

EpCAM antibody.

34. The method of claim 33, wherein the bispecific anti-c-Met/anti-EpCAM antibody comprises CDRs set forth in SEQ ID NOs: l-6.

35. The method of claim 31 , wherein the EGFR inhibitor is a bipecific antibody.

36. The method of claim 35, wherein the EGFR inhibitor is an oligoclonal antibody.

37. The method of claim 36, wherein the oligoclonal anti-EGFR antibody comprises the amino acid sequences set forth in SEQ ID NOs: 11-16.

38. The method of any one of claims 31-37, further comprising administering to the subject, 3) an effective amount of each of at least one additional antineoplastic agent.

39. The method of any one of claims 31-37, wherein the tumor is HGF+.

40. The method of any one of claims 31-37, wherein the tumor is EGFR+.

41. The method of any one of claims 31-37, wherein the tumor is HGF+ and EGFR+.