AND LIPOSOMAL IRINOTECAN

CROSS-REFERENCE

This patent application claims priority to each of the following pending U.S. provisional patent applications, each incorporated herein by reference in their entirety:

62/315,129 (filed March 30, 2016), 62/324,389 (filed April 19, 2016), 62/324,986 (filed April 20, 2016), 62/338,080 (filed May 18, 2016), 62/345,506 (filed June 3, 2016) and 62/370,886 (filed August 4, 2016).

SEQUENCE LISTING

Incorporated by reference in its entirety is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One 24.0 KB ASCII (Text) file named "1119sequencelisting_ST25.txt."

TECHNICAL FIELD

The specification relates to the treatment of cancer with a combination of an EGFR inhibitor, liposomal irinotecan, leucovorin and 5-fluorouracil, including the treatment of colorectal cancer.

BACKGROUND

Despite improvements in cancer treatments, there remains a critical need to further improve therapies so as to prolong patients' lives while maintaining quality of life, particularly in the case of advanced cancers resistant to current therapeutic modalities.

Colorectal Cancer (CRC) remains a leading cause of cancer death worldwide and is the third most common cancer in men and women. The global incidence of CRC was 1.4 million in 2012 with an expected increase to 2.4 million by 2035. In 2014, an estimated 136,830 new CRC cases arose and 50,310 CRC-related deaths occurred in the US alone. Approximately 20% of patients present with advanced, metastatic disease and their 5 year survival remains poor (<10%) with current therapies, highlighting the need for improved treatments.

Treatment for mCRC is rapidly evolving. Approved agents include 5-FU, irinotecan, oxaliplatin, capecitabine, bevacizumab, aflibercept, cetuximab, panitumumab, regorafenib, and trifluridine / tipiracil hydrochloride. Optimum sequencing and/or combinations of these therapies has not been clearly defined, but aggressive therapy improves overall survival. FOLFOX based chemotherapy is a standard first line option in these patients within the United States.

Combination therapies including folinic acid (leucovorin or levoleucovorin), 5- fluorouracil, and irinotecan (FOLFIRI), folinic acid, 5-fluorouracil, innotecan and oxaliplatin (FOLFIRINOX), or a combination of folinic acid, 5-fluorouracil, and oxaliplatin (FOLFOX) are also used to treat some cancers. Bevacizumab, cetuximab and panitumumab are frequently combined with these regimens depending on patient presentation, biomarker status and physician preference. Irinotecan is 7-ethyl-10-[4-(l-piperidino)-l-piperidino]

carbonyloxycampothecin, RJPAC name (S)-4,l l-diethyl-3,4,12, 14-tetrahydro-4-hydroxy- 3, 14-dioxolH-pyrano[3',4' :6,7]-indolizino[l,2-b]quinolin-9-yl-[l,4'bipiperidine]-r- carboxylate. Irinotecan is a member of the topoisomerase I inhibitor class of drugs and is a semi-synthetic and water soluble analog of the naturally-occurring alkaloid, camptothecin. Also known as CPT-11, irinotecan is currently marketed formulated as an aqueous solution as Camptosar ^® (irinotecan hydrochloride injection). Topoisomerase I inhibitors such as irinotecan work to anest uncontrolled cell growth by inhibiting the unwinding of DNA and thereby preventing DNA replication.

The pharmacology of irinotecan is complex, with extensive metabolic conversions involved in the activation, inactivation, and elimination of the drug. Irinotecan is a prodrug that is converted by nonspecific carboxylesterases into a 100-1000 fold more active metabolite, SN-38. SN-38 is not recognized by P-glycoprotein, a drug transporter that plays an important role in acquired drug resistance by pumping certain drugs out of cells, so irinotecan is likely to be active in tumors resistant to other standard chemotherapies. In the body, SN-38 is cleared via glucuronidation, for which major pharmacogenetic variability has been described, and biliary excretion. These drug properties contribute to the marked heterogeneities in efficacy and toxicity observed clinically with irinotecan. Irinotecan hydrochloride injection is approved in the United States for treatment of metastatic colon or renal cancer and is also used to treat colorectal, gastric, lung, uterine cervical and ovarian cancers.

EGFR signaling is important for the proliferation and survival of many epithelial malignancies affecting humans. Although EGFR-targeting antibodies have been approved for use in human malignancies that appear to be driven by EGFR, there still remains a high unmet need in these tumors, as limited efficacy is attributable to de novo or acquired resistance mechanisms to these targeted therapies. One reason for this state of affairs might stem from observations that EGFR signaling is very robust due to amplification of cell signaling downstream of the receptor, thus making the need for complete inhibition of the pathway imperative for potential success in the clinic. EGFR signaling is further complicated by the presence in tumors of multiple ligands that bind to EGFR with either high or low affinity and result in activation of the pathway downstream of EGFR. Preclinical evidence obtained at Merrimack suggests that EGFR signaling driven by high affinity ligands is not always inhibited by currently approved EGFR antagonists. Additional mutations and resistance mechanisms both upstream (extracellular) and downstream of the receptor have been described by Merrimack and others.

Certain anti-EGFR monoclonal antibodies have demonstrated a lack of efficacy in patients with mCRC containing KRAS mutations, based on retrospective analyses. In these trials, patients received standard of care (i.e., BSC or chemotherapy) and were randomized to receive either an anti-EGFR antibody (cetuximab or panitumumab) or no additional therapy. In all studies, investigational tests were used to detect KRAS mutations in codon 12 or 13. The percentage of study populations for which KRAS status was assessed ranged from 23% to 92%. In CRC tumor cells with activating KRAS somatic mutations, the mutant KRAS protein is continuously active and appears independent of EGFR regulation. Retrospective subset analyses of metastatic or advanced colorectal cancer trials have not shown a treatment benefit for cetuximab in patients whose tumors had KRAS mutations in codon 12 or 13. Use of cetuximab is not recommended for the treatment of colorectal cancer with these mutations. Similarly, panitumumab is not indicated for the treatment of patients with colorectal cancer that harbor somatic mutations in exon 2 (codons 12 and 13), exon 3 (codons 59 and 61), and exon 4 (codons 117 and 146) of either KRAS or NRAS. Retrospective subset analyses across several randomized clinical trials were conducted to investigate the role of RAS mutations on the clinical effects of anti-EGFR-directed monoclonal antibodies (panitumumab or cetuximab). Anti-EGFR antibodies in patients with tumors containing RAS mutations resulted in exposing those patients to anti-EGFR related adverse reactions without clinical benefit from these agents. Additionally, in Study 3, 272 patients with RAS-mutant mCRC tumors received panitumumab in combination with FOLFOX and 276 patients received FOLFOX alone. In an exploratory subgroup analysis, OS was shorter (HR = 1.21, 95% CI 1.01-1.45) in patients with RAS- Panitumumab is not indicated for the treatment of patients with colorectal cancer that harbor somatic mutations in exon 2 (codons 12 and 13), exon 3 (codons 59 and 61), and exon 4 (codons 117 and 146) of either KRAS or NRAS.

SUMMARY

Provided are methods for treating cancer in a patient (i.e., a human patient) comprising administering to the patient liposomal irinotecan (e.g., irinotecan sucrose octasulfate salt liposome injection, also referred to as MM-398, nal-IRI, or ONIVYDE) in combination with 5-fluorouracil (5-FU) and leucovorin (together, 5-FU/LV) and an EGFR inhibitor, according to a particular clinical dosage regimen. Compositions adapted for use in such methods are also provided. In particular, a combination of the EGFR inhibitor MM- 151, MM-398 liposomal irinotecan, 5-fluorouracil and leucovorin can be used to treat metastatic colorectal cancer (mCRC).

As an active drug in mCRC, irinotecan can be combined with EGFR inhibitors to treat RAS wild type tumors. Nal-IRI (also disclosed as MM-398 or ONIVYDE ^® ) is a

nanoliposomal formulation of irinotecan. The nanoliposomal encapsulation improves the pharmacokinetics of irinotecan and results in a lower C _ma x, longer half-life, and higher levels of irinotecan and SN-38 in tumor tissue compared with standard irinotecan. Nal-IRI is approved by the FDA under the brand name ONIVYDE, in combination with 5-FU and leucovorin in gemcitabine refractory pancreatic adenocarcinoma. In a non-comparative study evaluating the use of nal-IRI + 5-FU + LV as a 2nd line therapy in mCRC (PEPCOL study), the nal-IRI containing arm met the threshold of early responses and had lower incidence of diarrhea and neutropenia than the free irinotecan arm.

As both cetuximab and irinotecan represent established treatments, in a combination regimen or in combination with 5-FU/leucovorin (comprising the FOLFIRI regimen), the present disclosure provides methods of treating colorectal cancer by administering both MM- 151 (a next-generation EGFR antibody) and ONIVYDE (also called MM-398 or nal-IRI) in combination with 5-FU and leucovorin.

1. In some embodiments, a therapeutically effective combination of the anti-EGFR therapy MM-151 can be administered in combination with liposomal irinotecan, 5- fluorouracil and leucovorin for the treatment of patients diagnosed as having colorectal cancer (CRC), including patients diagnosed as having metastatic CRC (mCRC), containing KRAS mutations. The CRC can be mCRC with tumor cells containing RAS mutations. For example, the patient can be diagnosed with mCRC that harbors somatic mutations in exon 2 (codons 12 or 13), exon 3 (codons 59 and 61) and/or exon 4 (codons 117 and 146) of either KRAS or NRAS. In another embodiment, the patient can be diagnosed with mCRC that harbors a BRAF mutation. In one example, the BRAF mutation could be the BRAF V600E mutation. In another example, the BRAF mutation is a somatic mutation in one or more of codons 464, 466, 469, 595, 596, and 601. In another embodiment, the patient can be diagnosed with mCRC that harbors an EGFR mutation. For example, the EGFR mutation can be a somatic mutation in one or more codons of exon 12. In another embodiment, the patient can be diagnosed with mCRC that harbors a somatic mutation in the region coding for the catalytic subunit of PI3K, PIK3CA. For example, the PIK3CA mutation is mutation in one or more of codons 542, 545, and 1047. a m

MM-151 is a combination of three fully human IgGl monoclonal antibodies. MM- 151 antagonizes high-affinity EGFR ligands more effectively than approved inhibitors, including cetuximab and panitumumab, and has been observed to elicit a greater decrease in signal amplification. MM-151 has been evaluated in a phase I study of solid tumors and demonstrated a safety profile that was comparable to other EGFR inhibitors. The

recommended phase II dose (RP2D) identified in the MM-151 monotherapy phase I study was 10.5 mg/kg QW and this is the starting dose for MM-151 in this study.

MM-151 was combined with nal-IRI in a xenograft experiment with the LoVo colorectal cancer cell line. The data showed that this combination enhances anti-tumor activity as compared to either therapy alone. MM-151 was combined with nal-IRI and 5-FU in a xenograft experiment with the LFM1215 colorectal cancer cell line. The data showed that this combination enhances anti-tumor activity as compared to the combination of cetuximab, irinotecan (at equivalent SN-38 exposure), and 5-FU. These results, combined with the preliminary efficacy data seen in the PEPCOL study, suggest that the combination of MM- 151 + nal-IRI + 5-FU + LV may be a viable therapeutic option in mCRC.

In one aspect, a method for treatment (e.g. effective treatment) of colorectal cancer in a patient is provided, the method comprising intravenously administering to a human patient in need thereof a single administration of 6.0, 7.5, 9.0 or 10.5 mg/kg of MM-151 once per week, following an initial priming dose of 225 mg MM-151 in week 1 and a second priming dose of 450 mg of MM-151 in week 2; and a single administration once every two weeks of: a single administration of 70 mg/m ² irinotecan (free base) encapsulated in a MM-398 irinotecan liposome over 90 minutes, a single administration of 200 mg/m ² of the (1) form of leucovorin, and a single administration of 2400 mg/m ² of 5-fluorouracil over 46 hours, to treat the colorectal cancer in the patient.

In another aspect, a method for treatment of colorectal cancer in a patient is provided, the method comprising co-administering to the patient an effective amount each of liposomal irinotecan, 5-fluorouracil (5-FU), and leucovorin, wherein the method comprises at least one cycle of administration, wherein the cycle is a period of 2 weeks, and wherein for each cycle (in any order of administration, unless otherwise indicated):

(a) once-weekly administration of 10.5 mg/kg MM-151 in combination with (b) a single administration of liposomal irinotecan once every two weeks administered to patients not homozygous for the UGT1A1 *28 allele on day 1 of each cycle at a dose of 70 mg/m ² irinotecan (free base) in an MM-398 irinotecan liposome, and to patients homozygous for the UGT1A1 *28 allele on day 1 of cycle 1 at a dose of 50 mg/m ² irinotecan (free base) in an MM-398 irinotecan liposome, and on day 1 of each subsequent cycle at a dose of ranging from 50 mg/m ² to 70 mg/m ² (e.g., 60 mg/m ² or 70 mg/m ² or 80 mg/m ² ) irinotecan (free base) in an MM-398 irinotecan liposome;

(c) a single administration of 5-FU administered at a dose of 2400 mg/m ² once every two weeks in combination with the MM-398 irinotecan liposome and

(d) leucovorin administered once every two weeks at a dose of 200 mg/m ² (/ form, or levo-leucovorin) or 400 mg/m ² (/ + d racemic form) in combination with the MM-398 irinotecan liposome and the 5-FU.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the anti-tumor activity of MM-398 in an orthotopic pancreatic tumor model expressing luciferase (L3.6pl).

Figure 2 is a graph showing accumulation of SN-38 in tumors following treatment with free irinotecan or liposomal irinotecan (MM-398).

Figure 3 is a graph showing the effect of MM-398 on Carbonic Anhydrase IX Staining in a HT29 Xenograft Model.

Figure 4 shows the effect of MM-398 on perfusion of small molecule Hoechst stain.

Figure 5 summarizes the pharmacokinetics of MM-398 in q3w (irinotecan, liposome + free drug).

Figure 6 summarizes the pharmacokinetics of MM-398 in q3w (SN-38).

Figure 7 summarizes the pharmacokinetic parameters of individual MM-151 monoclonal antibodies in patients treated with MM-151 monotherapy.

Figure 8A shows the simulated serum concentrations of MM-151 in patients administered with two priming doses of MM-151 followed by 10.5 mg/kg qlw dosing schedule.

Figure 8B is a graph showing the effect of weekly MM-151 dose intensity on the probability to experience rash event in patients treated with MM-151. Top: any grade rash; Bottom: grade 3 or higher. Error bars indicate standard errors of the mean.

Figure 9 shows the mean plasma concentrations of total irinotecan and SN-38 following the administration of either nal-IRI (100 mg/mg ² ) based on the amount of irinotecan trihydrate hydrochloride) or irinotecan HC1 (300 mg/m ² ) in study PEP0206. Figure 10 is a graph showing model predictions of tumor and plasma drug metabolites (CPT- 11 and SN-38) in patients treated with nal-IRI.

Figures 11A and 11B show levels of CPT-11 and SN-38, respectively, measured in patient tumor biopsies and plasma samples.

Figure 12 is a schematic of a clinical treatment program.

Figure 13 is a schematic of a clinical treatment program.

Figure 14 is a sequence listing for the P1X antibody component of MM-151.

Figure 15 is a sequence listing for the P2X antibody component of MM-151.

Figure 16 is a sequence listing for the P3X antibody component of MM-151.

Figure 17 is a cartoon schematic that shows the MM-151 oligoclonal design with the three component IgGl antibodies— P1X, P2X, and P3X— bound to the EGFR extracellular domains (marked with Roman numerals I, II, III, and IV). P1X and P3X have binding epitopes on EGFR extracellular domain III while P2X has a binding epitope on EGFR extracellular domain I.

Figure 18 is a graph showing the results of a ligand antagonism cell binding assay, demonstrating the EGF ligand blocking ability of P1X (dark gray triangle), P2X (black square) or P3X (light gray circle) alone at low doses as compared to EGF ligand alone (black diamond). The concentrations for the antibodies (P1X = 0.97 nM; P2X = 2.00 nM; P3X = 4.68 nM) represent a sub -saturating concentration (approx. EC90 concentration) of cell binding. The EGFR-expressing A431 cell line was incubated with one dose of single antibody for 1 hr. followed by a dilution series of biotin-labeled EGF ligand and the amount of cell bound biotin-EGF ligand measured by quantitative flow cytometry (MFI = mean fluorescence intensity).

Figure 19 is a graph showing the results of a phospho-EGFR (pEGFR) inhibition experiment, demonstrating pEGFR inhibition by single-agent treatment with P1X (dark gray triangle), P2X (black square) or P3X (light gray circle) antibody at the indicated doses. A431 cells were incubated with antibody for 1 hr. followed by 10 min treatment with 8 nM EGF ligand and the amount of pEGFR measured by ELISA (MFI = mean fluorescence intensity).

Figure 20 demonstrates improved activity of nal-IRI + MM-151 TC combination compared to the two agents alone in LoVo subcutaneous CRC xenograft model.

Figure 21 shows the anti-tumor activity of nal-IRI (5 mg/kg) compared to irinotecan (25 mg/kg) at similar SN-38 tumor exposure in a LIM1215 subcutaneous CRC xenograft model.

Figure 22 shows the effects of irinotecan+5-FU+cetuximab and nal-IRI+5-FU+MM-151 TC combinations in LIM1215 subcutaneous CRC xenografts. Doses of 5-FU, cetuximab, and MM-151 TC were kept constant, (Figure 22 A) low doses of nal-IRI (1.25 mg/kg) and irinotecan (6.25 mg/kg) and (Figure 22B) high doses of nal-IRI (5 mg/kg) and irinotecan (25 mg/kg) were utilized for comparison at which tumor SN-38 were comparable at these dose levels.

Figure 23 provides the amino acid sequences of the Complementarity Determining Regions (CDRs) of an antibody used in Example 8.

Figure 24 is a diagram showing detectable pretreatment somatic mutations within the CRC patient subset.

Figure 25 is a summary of the 24 colorectal cancer patients discussed in Example 17 and discloses the treatment regimens each patient received.

Figure 26 is a table detailing the patient treatment parameters, the duration of treatment, the best overall response, duration of treatment and mutation status (wild type, "WT" or mutant, "MT") for 24 of 29 patients within the CRC efficacy cohort who were evaluated for RECIST response in the clinical trial described in Example 16

DETAILED DESCRIPTION

I. Definitions

As used herein, the term "subject" or "patient" is a human cancer patient.

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. A beneficial effect can also take the form of arresting, slowing, retarding, or stabilizing of a deleterious progression of a marker of a cancer. Effective treatment may refer to alleviation of at least one symptom of a cancer. Such effective treatment may, e.g., reduce patient pain, reduce the size and/or number of lesions, may reduce or prevent metastasis of a cancer tumor, and/or may slow growth of a cancer tumor.

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. In reference to cancers, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor development. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and may stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

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 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., taken at a single administration, e.g., orally (e.g., as one, two, three or more pills, capsules, etc.) or injected (e.g., as a bolus). 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.

As used herein, "cancer" refers to a condition characterized by abnormal, unregulated, malignant cell growth. In one embodiment, the cancer is colorectal cancer. The terms "resistant" and "refractory" refer to tumor cells that survive treatment with a therapeutic agent. Such cells may have responded to a therapeutic agent initially, but subsequently exhibited a reduction of responsiveness during treatment, or did not exhibit an adequate response to the therapeutic agent in that the cells continued to proliferate in the course of treatment with the agent.

II. Irinotecan sucrose sulfate liposome injection (ONIVYDE. MM-398. PEP02. nal-IRI)

Nal-IRI can be administered by IV infusion over 90 minutes (±10 minutes) every two weeks at a dose of 70 mg/m ² (free base), on Days 1 and 15 of each 28-day cycle. The first cycle Day 1 is a fixed day; subsequent doses should be administered on the first day of each cycle +/- 2 days. Prior to administration, the appropriate dose of nal-IRI must be diluted in 5% Dextrose Injection solution (D5W) or normal saline to a final volume of 500 mL. Care should be taken not to use in-line filters or any diluents other than D5W or normal saline. Nal-IRI can be administered at a rate of up to 1 mL/sec (30 mg/sec).

The actual dose of nal-IRI to be administered will be determined by calculating the patient's body surface area at the beginning of each cycle. A +/- 5% variance in the calculated total dose will be allowed for ease of dose administration. Since nal-IRI vials are single use vials, site staff must not store any unused portion of a vial for future use and they must discard unused portions of the product.

All patients can be pre-medicated prior to nal-IRI infusion and 5-FU/LV infusion with standard doses of dexamethasone and a 5-HT3 antagonist, or equivalent other anti-emetics according to standard institutional practices for irinotecan and 5-FU administration. Atropine may be prescribed prophylactically, according to standard institutional practices, for patients who experienced acute cholinergic symptoms in the previous cycles.

Patients will be tested for UGT1A1 *28 status during screening, however the result of the test is not required prior to the initial dose of nal-IRI. All patients will begin dosing at 70 mg/m ² (free base), however future doses may be reduced for patients who are positive (i.e. homozygous) for UGT1A1 *28 7/7 genotype. Depending on the overall safety profile seen after the first dose, the dose may be reduced to 50 mg/m ² (free base) after discussion between the PI, Sponsor and Medical Monitor.

As provided herein, irinotecan is administered in a stable liposomal formulation as irinotecan sucrose sulfate liposome injection (otherwise termed "irinotecan sucrose octasulfate salt liposome injection" or "irinotecan sucrosofate liposome injection"), the formulation referred to herein as "MM-398" (also known as PEP02, see US 8,147,867). MM-398 may be provided as a sterile, injectable parenteral liquid for intravenous injection. 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. An MM-398 liposome is a unilamellar lipid bilayer vesicle of approximately 80-140 nm in diameter that encapsulates an aqueous space which contains irinotecan complexed in a gelated or precipitated state as a salt with sucrose octasulfate. The lipid membrane of the liposome is composed of phosphatidylcholine, cholesterol, and a polyethyleneglycol- derivatized phosphatidyl-ethanolamine in the amount of approximately one

polyethyleneglycol (PEG) molecule for 200 phospholipid molecules.

This stable liposomal formulation of irinotecan has several attributes that may provide an improved therapeutic index. The controlled and sustained release improves activity of this schedule-dependent drug by increasing duration of exposure of tumor tissue to drug, an attribute that allows it to be present in a higher proportion of cells during the S-phase of the cell cycle, when DNA unwinding is required as a preliminary step in the DNA replication process. The long circulating pharmacokinetics and high intravascular drug retention in the liposomes can promote an enhanced permeability and retention (EPR) effect. EPR allows for deposition of the liposomes at sites, such as malignant tumors, where the normal integrity of the vasculature (capillaries in particular) is compromised resulting in leakage out of the capillary lumen of particulates such as liposomes. EPR may thus promote site-specific drug delivery of liposomes to solid tumors. EPR of MM-398 may result in a subsequent depot effect, where liposomes accumulate in tumor associated macrophages (TAMs), which metabolize irinotecan, converting it locally to the substantially more cytotoxic SN-38. This local bioactivation is believed to result in reduced drug exposure at potential sites of toxicity and increased exposure at cancer cells within the tumor.

Nal-IRI comprises irinotecan encapsulated in a nanoliposome drug delivery system (nanoliposomal irinotecan; nal-IRI). The active ingredient of the nal-IRI injection, irinotecan, is a member of the topoisomerase I inhibitor class of drugs and is a semi-synthetic and water soluble analog of the naturally-occurring alkaloid, camptothecin. Topoisomerase I inhibitors work to arrest uncontrolled cell growth by preventing the unwinding of DNA and therefore preventing replication. The pharmacology of irinotecan is complex, with extensive metabolic conversions involved in the activation, inactivation, and elimination of the drug. Irinotecan is a pro-drug that is converted by nonspecific carboxylesterases into a 100-1000 fold more active metabolite, SN-38. SN-38 is cleared via glucuronidation, (for which major

pharmacogenetic differences have been shown), and biliary excretion. These drug properties contribute to the marked differences in efficacy and toxicity observed in clinical studies with irinotecan.

Drug carrier technologies represent a rational strategy to improve the pharmacokinetics and biodistribution of irinotecan while protecting it from premature metabolism. Nal-IRI employs a novel intraliposomal drug stabilization technology for encapsulation of irinotecan into long-circulating liposome-based nanoparticles with high drug load and high in vivo stability. The stable nanoliposome formulation of irinotecan has several attributes that may provide an improved therapeutic index. The controlled and sustained release should improve activity of this schedule-dependent drug by increasing duration of exposure of tumor tissue to drug, an attribute that allows it to be present in a higher proportion of cells during the more sensitive S-phase of the cell cycle. The improved pharmacokinetics, high intravascular drug retention in the liposomes, and enhanced permeability and retention (EPR) effect may result in site-specific drug delivery to solid tumors. Stromal targeting results from the subsequent depot effect, where liposomes accumulating in tumor associated macrophages (TAMs) release the active drug and convert it locally to the substantially more cytotoxic SN-38. The preferentially local bioactivation should result in reduced exposure to potential sites of toxicity and increased exposure to neighboring cancer cells within the tumor.

Pharmacogenetics of Irinotecan Glucuronidation

The enzyme produced by the UGT1 Al gene, UDP-glucuronosyltransferase 1, is responsible for bilirubin metabolism and also mediates SN-38 glucuronidation, which is the initial step in the predominant metabolic clearance pathway of this active metabolite of irinotecan. Besides its anti -tumor activity, SN-38 is also responsible for the severe toxicity sometimes associated with irinotecan therapy. Therefore, the glucuronidation of SN-38 to the inactive form, SN-38 glucuronide, is an important step in the modulation of irinotecan toxicity.

Mutational polymorphisms in the promoter of the UGT1 Al gene have been described in which there is a variable number of thymine adenine (ta) repeats. Promoters containing seven thymine adenine (ta) repeats (found in the UGT1A1 *28 allele) have been found to be less active than the wild-type six repeats, resulting in reduced expression of UDP- glucuronosyltransferase 1. Patients who carry two deficient alleles of UGT1A1 exhibit reduced glucuronidation of SN-38. Some case reports have suggested that individuals who are homozygous for UGT1A1 *28 alleles (referred to as having the UGT1A1 7/7 genotype, because both alleles are UGT1A1 *28 alleles that contain 7 ta repeats, as opposed to the wild- type UGT1 Al 6/6 genotype in which both alleles contain 6 ta repeats) and who have fluctuating elevation in serum bilirubin, (e.g., Gilbert's Syndrome patients), may be at greater risk of toxicity upon receiving standard doses of irinotecan. This suggests that there is a link between homozygosity of the UGT1A1 *28 allele, bilirubin levels and irinotecan toxicity.

The metabolic transformation of MM-398 to SN-38 (e.g., in plasma) includes two critical steps: (1) the release of irinotecan from the liposome and (2) the conversion of free irinotecan to SN-38. While not intending to be limited by theory, it is believed that once irinotecan leaves the liposomes, it is catabolized by the same metabolic pathways as conventional (free) irinotecan. Therefore the genetic polymorphisms in humans predictive for the toxicity and efficacy of irinotecan and those of MM-398 can be considered similar. Nonetheless, due to the smaller tissue distribution, lower clearance, higher systemic exposure and longer elimination half-life of SN-38 of the MM-398 formulation compared to free irinotecan, the deficient genetic polymorphisms may show more association with severe adverse events and/or efficacy.

Patients with Reduced UGT1A1 Activity

Individuals who are homozygous for the UGT1A1 *28 allele (UGT1A1 7/7 genotype) have been shown to be at increased risk for neutropenia following initiation of irinotecan treatment. According to the prescribing information for irinotecan (Camptosar®), in a study of 66 patients who received single-agent irinotecan (350 mg/m ² once every-3 -weeks), the incidence of grade 4 neutropenia in patients homozygous for the UGT1A1 *28 allele was as high as 50%, and in patients heterozygous for this allele (UGT1 Al 6/7 genotype) the incidence was 12.5%. Importantly, no grade 4 neutropenia was observed in patients homozygous for the wild-type allele (UGT1A1 6/6 genotype). In other studies, a lower prevalence of life threatening neutropenia is described. For this reason, patients who are enrolled in the phase III study described in the Examples herein and are homozygous for the UGT1A1 *28 allele (UGT1A1 7/7 genotype) will have MM-398 treatment initiated at a lower dose than patients with one (e.g., UGT1 Al 6/7) or two (UGT1 Al 6/6) wild-type alleles.

Additional genotypic modifiers of irinotecan metabolism

Although the UGT1A1 *28 allele is relatively common in Caucasians (estimates 10%)), the prevalence is varied in other ethnic groups. Furthermore, additional UGT1A1 genotypes are found with higher prevalence for example in Asian populations and these could be important for the metabolism of irinotecan in these populations. For example, the UGT1 Al *6 allele is more prevalent in Asians. This allele is not associated with a ta repeat, but with a Gly71 Arg mutation that reduces enzyme activity. In previous and ongoing studies of MM-398, pharmacogenetic information has been collected on patients being enrolled. In a study referred to as the PEP0203 study, the relationship of genetic polymorphism of UGT1A family and of DP YD (dihydropyrimidine dehydrogenase, an enzyme associated with catabolism of 5-FU) with pharmacokinetic parameters of MM-398 and toxicity did not provide a clear correlation with the small sample size of subjects evaluated. However, it was observed that patients with UGTlAl *6/*28 combined polymorphism had higher dose- normalized AUCs of SN-38 and experienced DLT.

III. 5-Fluorouracil (5-FU) and Leucovorin

5-FU can be administered at a dose of 2400 mg/m ² as an IV infusion over 46-48 hours on Days 1 and 15 of each 28-day cycle. 5-FU should be reconstituted per the instructions on the package insert, SmPC or standard institutional guidelines for reconstitution of leucovorin.

5-FU can be administered after leucovorin and last in the treatment regimen. Actual dose of 5-FU and leucovorin to be administered will be determined by calculating the patient's body surface area prior to each cycle. A +/- 5% variance in the calculated total dose will be allowed for ease of dose administration.

Stomatitis and esophagopharyngitis (which may lead to sloughing and ulceration), diarrhea, anorexia, nausea, emesis and leukopenia are commonly seen with treatment;

alopecia and dermatitis, in the form of pruritic rash usually appearing on the extremities, may also be seen (see US package insert or SmPC).

5-Fluorouracil is a pyrimidine antagonist that interferes with nucleic acid

biosynthesis. The deoxyribonucleotide of the drug inhibits thymidylate synthetase, thus inhibiting the formation of thymidylic acid from deoxyuridylic acid, thus interfering in the synthesis of DNA. It also interferes with RNA synthesis.

Leucovorin (also called folinic acid) acts as a biochemical cofactor for 1 -carbon transfer reactions in the synthesis of purines and pyrimidines. Leucovorin does not require the enzyme dihydrofolate reductase (DHFR) for conversion to tetrahydrofolic acid. The effects of methotrexate and other DHFR-antagonists are inhibited by leucovorin. Leucovorin can potentiate the cytotoxic effects of fluorinated pyrimidines (i.e., fluorouracil and floxuridine). After 5-FU is activated within the cell, it is accompanied by a folate cofactor, and inhibits the enzyme thymidylate synthetase, thus inhibiting pyrimidine synthesis. Leucovorin increases the folate pool, thereby increasing the binding of folate cofactor and active 5-FU with thymidylate synthetase.

Leucovorin can be administered at a dose of 400 mg/m ² of the 1 + d racemic form, as an IV infusion over 30 minutes (±5 minutes), on Days 1 and 15 of each 28-day cycle.

Leucovorin should be reconstituted per the instructions on the package insert, SmPC or standard institutional guidelines for reconstitution of leucovorin.

Leucovorin should be administered after MM-151 and prior to the 5-FU infusion. Actual dose of 5-FU and leucovorin to be administered will be determined by calculating the patient's body surface area prior to each cycle. A +/- 5% variance in the calculated total dose will be allowed for ease of dose administration.

Leucovorin has dextro- and levo-isomers, only the latter one being pharmacologically useful. As such, the bioactive levo-isomer ("levoleucovorin") has also been approved by the FDA for treatment of cancer. The dosage of levoleucovorin is typically half that of the racemic mixture containing both dextro (d) and levo (/) isomers.

IV. MM-151 (preferred EGFR inhibitor)

EGFR signaling is important for the proliferation and survival of many epithelial malignancies. Although EGFR targeting antibodies have been approved for use in human malignancies driven by the EGFR pathway, there remains a high unmet need in these tumors, as limited efficacy is attributable to de novo or acquired resistance mechanisms to these targeted therapies. One reason for this state of affairs might stem from observations that EGFR signaling is very robust due to amplification of cell signaling downstream of the receptor, thus making the need for complete inhibition of the pathway imperative for potential success in the clinic. EGFR signaling is further complicated by the presence in tumors of multiple ligands that bind to EGFR with either high or low affinity and result in activation of the pathway downstream of EGFR. Preclinical evidence obtained at Merrimack suggests that EGFR signaling driven by high affinity ligands is not always inhibited by currently approved EGFR antagonists.

MM-151 is a novel mixture of three fully human monoclonal antibody EGFR antagonists designed to optimally inhibit EGFR dependent signaling. MM-151 antibodies have non-overlapping epitopes and can simultaneously engage the same EGFR receptor molecule. Preclinical studies suggest that MM- 151 leads to downregulation of EGFR expression via internalization and degradation of EGFR. Preclinical studies demonstrate that MM-151 is more effective in inhibiting EGFR signaling and proliferation driven by both high and low affinity ligands, compared to currently approved EGFR targeting monoclonal antibodies which inhibit only low-affinity ligand pathway activation. Preclinical studies also demonstrate that MM-151 leads to antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), with the latter being a unique property of the antibody mixture that is not observed with currently approved EGFR-targeting antibodies. In vivo studies also showed that treatment with MM-151 results in inhibition of tumor growth of xenograft models from multiple indications including colorectal, non-small cell lung, head and neck, and triple-negative breast cancer.

MM-151 contains three distinct antibodies: P1X, P2X and P3X. All are colorless, liquid solutions that are formulated in 20mM Histidine, 10% sucrose and 0.02% Polysorbate 80, pH 6.0. The molecular weights of the drug P1X, P2X and P3X are 145, 148 and 146 respectively. P1X, P2X and P3X drug substances are combined in a ratio of 1 : 1 :0.5 to form the final MM-151 drug product. Similar to the three drug substances, MM-151 drug product is formulated in 20mM Histidine, 10% sucrose and 0.02% Polysorbate 80, pH 6.0. The final concentration of drug product is 25 mg/ml per 10ml vial.

P IX is a full EGF ligand antagonist and binds an epitope on domain III of EGFR with subnanomolar affinity (1 lpM) resulting in significant inhibition of pEGFR. MM-151 P1X is a recombinant human IgGl monoclonal antibody. The complete tetrameric structure of the IgGl molecule is composed of two heavy chains (451 amino acids each) and two kappa light chains (214 amino acids each) held together by intra-chain and inter-chain disulfide bonds. The MM-151 P1X amino acid sequence predicts a molecular weight of 145 ko for the intact IgGl, which is within 0.2% of the actual molecular weight as experimentally determined by mass spectroscopy. MM-151 P1X is produced using a recombinant CHO cell system. The heavy and light chain sequences of the P1X antibody are provided in FIG. 14.

P2X is also a full EGF ligand antagonist and binds a distinct epitope different from that of P1X located on domain I of EGFR with subnanomolar affinity (70pM) resulting in significant inhibition of pEGFR. MM-151 P2X is a recombinant human IgGl monoclonal antibody. The complete tetrameric structure of the IgGl molecule is composed of two heavy chains (449 amino acids each) and two kappa light chains (220 amino acids each) held together by intra-chain and inter-chain disulfide bonds. The predicted molecular weight of intact glycosylated MM-151 P2X is 148 kD, which is within 0.2% of the actual molecular weight as experimentally determined by mass spectroscopy. MM-151 P2X is produced using a recombinant CHO cell system. The heavy and light chain sequences of the P2X antibody are provided in FIG. 15.

P3X is a partial EGF antagonist and binds a third epitope on domain III distinct from those of P1X and P2X. P3X binds EGFR at a lower affinity than P1X and P2X (360pM) resulting in moderate inhibition (20%) of pEGFR. MM-151 P3X is a recombinant human IgGl monoclonal antibody. The complete tetrameric structure of the IgGl molecule is composed of two heavy chains (453 amino acids each) and two kappa light chains (215 amino acids each) held together by intra-chain and inter-chain disulfide bonds. The MM-151 P3X amino acid sequence predicts a molecular weight of 146 kD for the intact IgGl, which is within 0.2% of the actual molecular weight as experimentally determined by mass spectroscopy. MM-151 P3X is produced using a recombinant CHO cell system. The heavy and light chain sequences of the P3X antibody are provided in FIG. 16.

The CDR regions of the MM151 antibodies are provided in the table below, and in publication WO2013/006547.

Table 1 CDR regions for MM151 Anti-EGFR Oligoclonal Antibody Preparation

Together, the mixture of these three EGFR antagonists is expected to increase the relative potency of MM-151 compared to currently approved antibody based EGFR antagonists by completely blocking EGFR pathway activation in tumor cells through acting on three distinct epitopes of the target receptor. In in vitro studies, the mixture of the three antibodies inhibited EGF -mediated signaling of both the target receptor, EGFR, and its downstream effector, ERK. As EGFR is one of the key growth factor receptors used by tumors cells to proliferate and migrate to distal sites, MM-151 has the potential to treat a number of patients with unmet medical needs in oncology.

The individual components of MM-151, PIX, P2X, and P3X, are fully human anti- EGFR IgGl monoclonal antibodies that bind to three distinct epitopes on the extracellular domain of EGFR. Surface plasma resonance experiments showed that PIX, P2X and P3X can associate simultaneously; that they associate with EGFR domains III, I and II respectively (Figure 16) and that the order of association does not matter. Additionally, PIX and P2X have been shown to be potent ligand antagonists, while P3X only partially blocks EGF ligand binding. However, together the mixture acts as potent EGF ligand blockers (Figure 17). PIX and P2X are potent inhibitors of pEGFR signaling, whereas P3X only partially blocks EGF -induced receptor signaling, (Figure 19).

MM-151 is administered by IV infusion every week. The first two doses of Cycle 1 are priming doses and the dose levels are 225 mg for priming dose 1 and 450 mg for priming dose 2. Subsequent doses of MM-151 are administered according to the dose levels outlined above. MM-151 should not be administered as a bolus or a push.

MM-151 is also described in Patent Cooperation Treaty (PCT) patent application PCT/US2012/045235, filed July 2, 2012, and published as WO2013/006547, incorporated herein by reference in its entirety.

V. Administration

Liposomal irinotecan is administered intravenously, either alone or in combination with 5-fluorouracil (5-FU) and/or leucovorin. In one embodiment, liposomal irinotecan is administered prior to 5-FU and leucovorin. In another embodiment, leucovorin is administered prior to 5-FU. In another embodiment, liposomal irinotecan is administered intravenously over 90 minutes. In another embodiment, 5-FU is administered intravenously over 46 hours. In another embodiment, leucovorin is administered intravenously over 30 minutes. In various embodiments the liposomal irinotecan is MM-398.

The concentration of MM-398 will be 43 mg/10 mL irinotecan free base as a white to slightly yellow, opaque, liposomal dispersion in a single-dose vial irinotecan in the form of the sucrosofate salt, encapsulated in liposomes for intravenous infusion. Nal-IRI must be stored refrigerated at 2 to 8°C, with protection from light.

Nal-IRI is administered by IV infusion over 90 minutes every two weeks at a dose of 70 mg/m ² (free base).

5-FU is a commercially available product that can be supplied at multiple

concentration levels and vial sizes depending on the source country. It must be stored at room temperature with protection from light. 5-FU is administered by IV infusion over 46 hours every two weeks at a dose of 2400 mg/m ² .

LV is a commercially available product that can be supplied at multiple concentration levels and vial sizes depending on the source country. It must be stored at room temperature with protection from light. Leucovorin (1 + d racemic form) will be administered by IV infusion over 30 minutes every two weeks at a dose of 400 mg/m ² .

V. Patient Populations

In one embodiment, a patient treated using the methods and compositions disclosed herein exhibits evidence of recurrent or persistent colorectal cancer following primary chemotherapy.

In another embodiment, the patient has had and failed at least one prior platinum based chemotherapy regimen for management of primary or recurrent disease, e.g., a chemotherapy regimen comprising carboplatin, cisplatin, or another organoplatinum compound.

In an additional embodiment, the patient has failed prior treatment with gemcitabine or become resistant to gemcitabine.

In one embodiment a resistant or refractory tumor is one where the treatment-free interval following completion of a course of therapy for a patient having the tumor is less than 6 months (e.g., owing to recurrence of the cancer) or where there is tumor progression during the course of therapy.

VII. Treatment Protocols

Suitable treatment protocols include, for example, those wherein the patient is administered an effective amount of liposomal irinotecan, wherein the treatment comprises at least one cycle, wherein the cycle is a period of 3 weeks, and wherein for each cycle the liposomal irinotecan is administered on day 1 of the cycle at a dose of 120 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride), except if the patient is homozygous for the UGT1A1 *28 allele, wherein liposomal irinotecan is administered on day 1 of cycle 1 at a dose of 80 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride). In one embodiment, the dose of liposomal irinotecan administered to the patient homozygous for the UGT1A1 *28 allele is increased after one cycle in increments of 20 mg/m ² , up to a maximum of 120 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride).

In another embodiment, the treatment protocol includes administering to the patient an effective amount each of liposomal irinotecan, 5-fluorouracil (5-FU), and leucovorin, wherein the treatment comprises at least one cycle, wherein the cycle is a period of 2 weeks, and wherein for each cycle: (a) liposomal irinotecan is administered on day 1 of the cycle at a dose of 80 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride), except if the patient is homozygous for the UGT1A1 *28 allele, wherein liposomal irinotecan is administered on day 1 of cycle 1 at a dose of 60 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride); (b) 5-FU is administered at a dose of 2400 mg/m ² ; and (c) leucovorin is administered at a dose of 200 mg/m ² (/ form) or 400 mg/m ² (/ + d racemic form). In a particular embodiment, the dose of liposomal irinotecan administered to the patient homozygous for the UGT1A1 *28 allele is increased after one cycle to 80 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride).

VII. Outcomes

Provided herein are methods for treating cancer in a patient comprising administering an anti-EGFR antibody such as MM-151 (e.g., once weekly) to the patient in combination with administration once every two weeks of liposomal irinotecan (MM-398) in combination with 5-fluorouracil (5-FU) and leucovorin, according to a particular clinical dosage regimen. Responses to therapy may include:

Pathologic complete response (pCR): absence of invasive cancer in the breast and lymph nodes following primary systemic treatment.

Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) which has reduction in short axis to <10 mm;

Partial Response (PR): At least a 30% decrease in the sum of dimensions of target lesions, taking as reference the baseline sum diameters;

Stable Disease (SD): Neither sufficient shrinkage to qualify for partial response, nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum diameters while on study; or

Meanwhile, non-CR/Non-PD denotes a persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits. Progressive Disease (PD) denotes at least a 20% increase in the sum of dimensions of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of 5 mm. The appearance of one or more new lesions is also considered progression.

In one embodiment the patient so treated exhibits pCR, CR, PR, or SD.

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

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

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

In some embodiments, administration of effective amounts of liposomal irinotecan, 5- FU and leucovorin according to any of the methods provided herein produce at least one therapeutic effect selected from the group consisting of reduction in size of a breast tumor, reduction in number of metastatic lesions appearing over time, complete remission, partial remission, stable disease, increase in overall response rate, or a pathologic complete response. In some embodiments, the provided methods of treatment produce a comparable clinical benefit rate (CBR = CR+ PR+ SD > 6 months) better than that achieved by the same combinations of anti-cancer agents administered without concomitant MM-398 administration. In other embodiments, the improvement of clinical benefit rate is about 20% 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to the same combinations of anticancer agents administered without concomitant MM-398 administration.

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

EXAMPLES

Example 1: Activity of MM-398 in an Orthotopic Pancreas Tumor Model Expressing Luciferase (L3.6pl)

The anti-tumor activity of MM-398 was assessed in an orthotopic pancreatic cancer model (L3.6pl), a highly hypoxic preclinical tumor model. Approximately 2.5 x 10 ^"5 L3.6pl pancreatic tumor cells were implanted by direct injection into the pancreas. The

bioluminescence images (BLI) were followed over time for tumor burden

detection/quantitation. MM-398 and free irinotecan were dosed at a dose of 20 mg/kg/dose weekly for three weeks. As shown in Figure 1, MM-398 (liposomal CPT11) had significant anti-tumor activity, as compared to a control (HBS) and free CPT11.

Example 2: Accumulation of SN-38 in Tumors Following Treatment with Free

Irinotecan or Liposomal Irinotecan (MM-398)

It was hypothesized that the anti-tumor activity observed in the orthotopic pancreatic cancer model is due to the effect of macrophages in converting irinotecan to the more active SN-38 locally. To test this hypothesis, human colon cancer cells (HT-29) were injected subcutaneously into SCID mice, 40 mg/kg of free irinotecan or MM-398 was injected intravenously when the tumors reached 1000 mm ³ in size. Tumor-bearing mice were sacrificed at different time points, tumors from both groups were extracted and the concentrations of SN-38 were measured.

As shown in Figure 2, there was a 20-fold increase in the tumor AUCSN-38 for MM- 398 as compared to free irinotecan. The long duration of exposure allows for prolonged exposure of the slow proliferating cancer cells to the active metabolite as they progress through the cell cycle. In addition, this activity was also hypothesized to result from a reduction in intra-tumoral hypoxia, and the subsequent downstream effects on angiogenesis, metastasis, and the immunosuppressive environment in tumors.

Example 3: Effect of MM-398 on Carbonic Anhydrase IX Staining in a HT29

Xenograft Model

To test whether MM-398 reduces markers of hypoxia, experiments were conducted in a human colon cancer cell (HT-29) model. Specifically, HT-29 cells were injected subcutaneously into nude mice, on day 13 either PBS control or 1.25, 2.5, 5, 10 or 20 mg/kg MM-398 was injected intravenously. MM-398 was dosed once a week for 4 weeks at the indicated doses. Tumors from both groups (n = 5) were extracted 24 hours after the last dose. Frozen tumor sections were used for immunohistochemical staining of Carbonic Anhydrase IX (CAIX). Quantification of CAIX staining was performed using Definiens ^® (Definiens AG, Munich) software.

As shown in Figure 3, MM-398 reduced markers of hypoxia. Specifically, the graphs in Figure 3 show the percentage of cells that stained with medium (middle third) or high (top third) intensity for CAIX. Representative samples from each group are shown as well as the group average (mean +/- stdev). MM-398 treatment modifies the tumor microenvironment by decreasing the percentage of both medium and high CAIX positive cells in a dose- dependent manner. As hypoxia is a hallmark of resistant and aggressive disease, a reduction in hypoxia is expected to make tumor cells more sensitive to chemotherapies.

Example 4: MM-398 Increases Perfusion of Hoechst Stain

In addition to changing the chemosensitivity of tumor cells through modification of the tumor microenvironment, lowering hypoxia can indicate improved tumor vascularization, which can facilitate delivery of small molecule therapies. MM-398 treatment led to increased microvessel density 6 days after treatment as measured by CD31 (platelet endothelial cell adhesion molecule) staining in an HT29 xenograft study. To further assess the effect of MM- 398 on small molecule tumor vascularization, a Hoechst 33342 perfusion experiment was conducted. Specifically, a primary pancreatic tumor was grown in NOD-SCID mice and given one dose of MM-398 (20mg/kg). After 24 hours, Hoechst 33342 stain was

administered 20 minutes prior to sacrificing the animal. As shown in Figure 4, the increase in stain intensity in treated mice was statistically significant, p < 0.001. These data indicate that MM-398 modifies the tumor microenvironment in a manner that should make tumors more susceptible to agents such as 5-FU/LV, through decreasing tumor hypoxia and increasing small molecule perfusion.

Example 5: MM-398 Pharmacokinetics in Humans (Phase I)

The pharmacokinetic profile of MM-398 single agent was investigated in a phase I clinical study (PEP0201) in patients at 60, 120 or 180mg/m ² dose levels and in a phase II clinical trial in gastric cancer patients (PEP0206) at 120mg/m ² (based on the amount of irinotecan trihydrate hydrochloride). Plasma levels of total irinotecan, SN-38 and encapsulated irinotecan were measured in these studies.

The peak serum concentrations of total irinotecan (C _ma x) ranged from 48-79 μg/ml for 120mg/m ² of MM-398 (based on the amount of irinotecan trihydrate hydrochloride), which was approximately 50-fold higher than 125mg/m ² free irinotecan. The total irinotecan half- life (ti/ ₂ ) for MM-398 ranged from 21 to 48 hours, which was approximately 2-3 folds higher than 125mg/m ² of free irinotecan. Overall, total irinotecan exposure at one week (AUC 0-T) ranged from 1200- 3000 (μg*h/ml) at a dose of 120 mg/m ² of MM-398 (based on the amount of irinotecan trihydrate hydrochloride), approximately 50-100 fold higher than 300mg/m ² of free irinotecan. In contrast, SN38 Cmax levels at 120mg/m ² of MM-398 (based on the amount of irinotecan trihydrate hydrochloride) ranged from 9 to 17 ng/ml, which was approximately 50% less than free irinotecan at 125mg/m ² . Overall, exposure of SN38 at one week (AUC 0- T) ranged from 474 to 997 ng*/ml and was only 1-2 fold higher than achieved by free irinotecan at 300mg/m ² . For both SN38 and total irinotecan, AUC increased less than proportionally with dose of MM-398. The PK parameters of encapsulated irinotecan almost matched that of total irinotecan indicates that most of irinotecan remained encapsulated in the liposomes during circulation. The MM-398 PK parameters were not significantly changed when combined with 5-FU/LV. Figures 5 and 6 summarize the PK findings in previous studies of MM 398.

Example 6: MM-398 Phase 1 Dose Escalation Study

A regimen combining fluorouracil, leucovorin, and MM-398 was studied in a phase I trial of solid tumors in 16 subjects, of whom 5 were patients with pancreatic cancer. The objective tumor response rate, duration of response, and disease control rate were efficacy endpoints of the study. Among the 15 efficacy-evaluable patients, 2 (13.3%) had confirmed PR, 9 (60.0%) had SD, and 4 (26.7%) had PD. The overall disease control rate was 73.3%. Partial response was observed in one gastric cancer patient (at 80mg/m ² dose level, based on the amount of irinotecan trihydrate hydrochloride) and one breast cancer patient (at 100 mg/m ² dose level, based on the amount of irinotecan trihydrate hydrochloride), with the duration of response of 142 and 76 days, respectively. Among the 6 patients who received the MTD dose of 80 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride), there were 1 PR, 4 SD and 1 PD. The tumor response rate and disease control rate were 16.7%) and 83.3%, respectively. The main DLTs were grade 3 diarrhea, leucopenia, neutropenia and febrile neutropenia. The MTD for MM-398 was 80mg/m ² (based on the amount of irinotecan trihydrate hydrochloride). In the phase I dose-escalation study of MM-398 in combination with 5-FU/LV in advanced solid tumors (PEP0203), a total of 401 episodes of AE were reported from the 16 treated subjects (safety population), of which 74 (18.4%) were of CTC grade 3 or above. Among all AEs, 231 (57.6%) were considered by the investigators to be treatment-related. The most common treatment-related AEs, included nausea (81.3%), diarrhea (75.0%), vomiting (68.8%), fatigue (43.8%), mucositis (43.8%), leucopenia (37.5%), neutropenia (37.5%)), weight loss (37.5%), anemia (31.3%), and alopecia (31.3%). Acute cholinergic diarrhea was rarely observed. Table 2 provides the incidence of treatment-emergent adverse events by maximum CTC grade and by causality (incidence > 20%), as seen in the PEP0203 study. Table 3 provides the incidence of grade 3 or higher treatment-emergent adverse events seen in the 5 pancreatic cancer patients treated in the PEP0203 study.

Table 2: Incidence of treatment-emergent adverse events by maximum CTC grade and by causality (incidence > 20%) in the PEP0203 Study

Table 3: Incidence of Grade 3 or higher treatment-emergent adverse events in pancreatic cancer patients in the PEP0203 Study (irinotecan dose based on the amount of irinotecan trihydrate hydrochloride)

Example 7: MM-151 Phase 1 study

MM-151 has been assessed in one clinical study to date, MM-151-01-01-01. This study was a phase I study that evaluated MM-151 as a monotherapy and in combination with innotecan, at different dosing levels and at dosing frequencies of QW, Q2W and Q3W. The study has completed, and preliminary signs of meaningful clinical benefit and an acceptable safety profile were observed, warranting further evaluation in metastatic CRC. Rationale for MM-151 starting dose

Table 4: Summary of Studies Conducted with nal-IRI + 5-FU + LV

(Irinotecan dose based on the amount of irinotecan trihydrate hydrochloride)

The starting dose of 10.5 mg/kg is the QW RP2D that was identified in the phase I study.

Nal-IRI + 5-FU + LV clinical experience

The combination of nal-IRI + 5-FU + LV has been investigated in three clinical studies to date. One of these studies (NAPOLI-l)15 provided the basis for approval of this combination in gemcitabine-refractory pancreatic patients.

Nal-IRI + 5-FU + LV PK in humans

The pharmacokinetic profile of single agent nal-IRI has been investigated in several studies, in which plasma levels of total irinotecan, SN-38 and encapsulated irinotecan were measured. In a single phase II clinical study (Study PEP0206), direct comparison of the pharmacokinetics of irinotecan and SN-38 in patients administered with nal-IRI or

conventional (i.e. non-liposomal) irinotecan HCl (Camptosar ^® ) was evaluated. Compared to the administration of irinotecan HCl 300 mg/m ² q3w, administration of nal-IRI 100 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride) q3w resulted in higher exposure of total irinotecan (Cmax: 13.4 fold, AUCo-∞: 46.2 fold, ti/ ₂ : 2.0 fold), and higher SN-38 ti/ ₂ (3 fold) and marginally higher AUCo-∞ (1.4 fold), however, SN-38 Cmax was reduced by 5.3 fold (Figure 4). In other PK studies of single agent nal-IRI, similar findings were observed when compared to standard doses of irinotecan HCl. Based on population pharmacokinetic analysis, no significant association was observed between the PK parameters of total irinotecan and SN-38 following nal-IRI monotherapy and when co-administered with 5- FU/LV. This result is consistent with the lack of drug interaction noted between irinotecan HCl and 5-FU (Camptosar US label). The pharmacokinetic parameters of total irinotecan and total SN-38 following the administration of nal-IRI 70 mg/m ² (free base) as a single agent or part of combination chemotherapy are presented in Table 5.

Figure 9 is a graph showing the mean plasma concentrations of total irinotecan and SN-38 following the administration of either nal-IRI (100mg/m ² ) (based on the amount of irinotecan trihydrate hydrochloride) or irinotecan HCl (300mg/m ² ) in Study PEP0206.

Gastric cancer patients received either nal-IRI at a dose of 100 mg/m ² (blue line) (based on the amount of irinotecan trihydrate hydrochloride) or nal-IRI at a dose of 300 mg/m ² (red line) (based on the amount of irinotecan trihydrate hydrochloride) every 3 weeks. Total irinotecan (top) and its active metabolite, SN-38 (bottom) were measured during Cycle 1. Error bars indicate 95% confidence interval. Dotted lines indicate lower limit of quantification (LLOQ); total irinotecan measurements consists of two LLOQ values because of two different irinotecan assay was used to measure low and high range of concentrations. The

concentrations less than LLOQ values were set to the corresponding LLOQ.

irinotecan increases with dose. Additionally, the Cmax of total SN-38 increases

proportionally with dose; however, the AUC of total SN-38 increases less than proportionally with dose.

The above PK results obtained from patients treated with either nal-IRI or irinotecan HC1 confirmed the pre-clinical observation that nal-IRI extended plasma PK of both CPT-11 and SN-38 compared to treatment with irinotecan HC1. Further, a phase I clinical study of nal-IRI monotherapy (protocol MM-398-01-01-02; NCT# 01770353) investigated tumor levels of both CPT-11 and SN-38 following treatment with nal-IRI using post-treatment biopsies. Based on model predictions, SN-38 levels in tumor were expected to be higher than in plasma, suggesting local conversion of CPT-11 to SN-38 in the tumor microenvironment with nal-IRI (Figure 10). Predictions were confirmed by measuring levels of CPT-11 and SN-38 in tumor biopsy samples collected from patients 72 hours post-dose, demonstrating 5- fold higher levels of SN-38 in the tumor than the plasma (Figure 11 A-l IB). Collectively the evidence suggests that the prolonged systemic exposure to CPT-11 and SN-38 leads to prolonged levels of SN-38 in tumor tissue, which in turn leads to prolonged DNA damage to tumor cells, suggesting an advantage of nal-IRI compared to irinotecan HC1.

Figure 10, Figures 11 A and 1 IB are graphs showing the clinical evidence for local activation and accumulation of SN-38 in tumor tissue. Figure 10 illustrates the mechanistic tumor PK model of nal-IRI predicted higher SN-38 levels in tumor compared to plasma when 70mg/m ² (based on the amount of irinotecan free base) nal-IRI is administered. The range of actual data, collected from a phase I study of patients (n=12) with advanced solid tumors following the administration of 70mg/m ² (based on the amount of irinotecan free base) nal-IR, is indicated by in Figure 11 A (CPT-11) and Figure 1 IB (SN-38), as measured from patient tumor and plasma samples collected 72h post-nal-IRI infusion, which coincide with the levels as predicted by the mechanistic model. Nal-IRI + 5-FU + LV safety in humans

It has been shown in animal and human PK studies that once irinotecan is released from the nal-IRI liposomes, the conversion of irinotecan to SN-38 is similar to that of the unencapsulated irinotecan. The safety of nal-IRI, therefore, may be indirectly compared with the safety of irinotecan, primarily based on a qualitative comparison of adverse reactions, as reported in the Camptosar US label for irinotecan. The comparison is qualitative, as both irinotecan and nal-IRI have been used in different doses and schedules as monotherapy and combination therapy with other chemotherapeutic agents; therefore, quantitative comparisons are difficult. The most common adverse reactions of irinotecan and nal-IRI are similar and consist mainly of gastrointestinal events and myelosuppression.

The common adverse reactions (>30%) observed in clinical studies with irinotecan in combination with other agents are: nausea, vomiting, abdominal pain, diarrhea, constipation, anorexia, mucositis, neutropenia, leukopenia (including lymphocytopenia), anemia, thrombocytopenia, asthenia, pain, fever, infection, abnormal bilirubin, and alopecia. The common adverse reactions (>30%) observed in single agent irinotecan therapy in clinical studies are: nausea, vomiting, abdominal pain, diarrhea, constipation, anorexia, neutropenia, leukopenia (including lymphocytopenia), anemia, asthenia, fever, body weight decreasing, and alopecia (Camptosar US label).

With respect to liposomal irinotecan, nal-IRI, when used in combination with 5-FU and leucovorin, the most common adverse reactions (>20 %) observed in clinical trials considered to be related are: diarrhea, nausea, vomiting, decreased appetite, neutropenia, fatigue, anemia, stomatitis and pyrexia. The overall safety profile of nal-IRI is presented in detail in the related Investigator Brochure. Additionally, Table 6 summarizes > Grade 3 safety data from the NAPOLI-1 trial comparing nal-IRI + 5-FU/LV (at a dose of 80 mg/m ² based on the amount of irinotecan hydrochloride trihydrate, given on an every 2 week schedule), or nal-IRI monotherapy (at a dose of 120 mg/m ² based on the amount of irinotecan trihydrate hydrochloride given on an every 3 week schedule), with 5-FU/LV alone (given weekly for 4 weeks followed by 2 weeks of rest) in the same population of patients who had received prior gemcitabine therapy.

Table 6: Summary of Grade 3 or Higher Adverse Events in NAPOLI-1 Study

Rationale for nal-IRI + 5-FU + LV dose levels

Nal-IRI, 5-FU and LV are all approved drugs and the dose levels used in this study are the approved dose levels for each therapy as defined in the package insert. In this phase I study, the doses of nal-IRI, 5-FU and LV will remain constant and the dose of MM-151 will be adjusted until an MTD is identified.

Rationale for combining MM-151 + nal-IRI + 5-FU + LV in CRC

Irinotecan is a well-established therapy in the frontline treatment of mCRC, alone or in combination, and can be combined with EGFR inhibitors to treat RAS wild type tumors; Nal-IRI (Onivyde™) is a nanoliposomal formulation of irinotecan. The nanoliposomal encapsulation improves the pharmacokinetics of irinotecan and results in a lower Cmax, longer half-life, and higher levels of irinotecan and SN-38 in tumor tissue compared with standard irinotecan. Nal-IRI is approved by the FDA in combination with 5-FU and leucovorin in gemcitabine refractory pancreatic adenocarcinoma under the brand name Onivyde. In a non-comparative study evaluating the use of nal-IRI + 5-FU + LV as a 2nd line therapy in mCRC (PEPCOL study), the nal-IRI containing arm met the threshold of early responses and had lower incidence of diarrhea and neutropenia than the FOLFIRI containing arm. Combination treatment of FOLFIRI and an EGFR inhibitor as first line treatment in mCRC are in accordance with National Comprehensive Cancer Network (NCCN) guidelines. MM-151 is an EGFR inhibitor that combines three fully human IgGl monoclonal antibodies. MM-151 antagonizes high-affinity EGFR ligands more effectively than another EGFR inhibitor, cetuximab, and has been observed to elicit a ~65-fold greater decrease in signal amplification. MM-151 has been evaluated in a phase I study of solid tumors and it demonstrated a safety profile that was comparable to other EGFR inhibitors, in monotherapy and in combination with irinotecan.

MM-151 was combined with nal-IRI in a LoVo colon xenograft experiment. The data showed that this combination enhances anti-tumor activity by overcoming EGFR insensitivity. This result, combined with the preliminary efficacy data seen in the PEPCOL study, suggests that the combination of MM-151 + nal-IRI + 5-FU + LV may be a viable therapeutic option in mCRC.

Potential toxicities of MM-151 + nal-IRI + 5-FU + LV

The nal-IRI+5-FU+LV regimen was studied in the NAPOLI-1 trial and the most common adverse reactions (>20 %) observed in clinical trials considered to be related are: diarrhea, nausea, vomiting, decreased appetite, neutropenia, fatigue, anemia, stomatitis and pyrexia.

In addition, we would expect to see the known class toxicities of an EGFR inhibitor in addition to the nal-IRI+5-FU+LV regimen such as skin reaction, acneiform rash, liver toxicity, fatigue, diarrhea, stomatitis, electrolyte imbalances, paronychia, and vomiting as were seen in the FIRE-3 trial that combined FOLFIRI + cetuximab. In the phase I MM-151- 01-01-01 study the most common related treatment emergent adverse events (>20 %) in the combination of MM-151 and irinotecan were: anemia, neutropenia, diarrhea, nausea, vomiting, stomatitis, fatigue, mucosal inflammation, asthenia, infusion related reaction, weight loss, rash, dry skin, and alopecia.

Example 8

The following example employs a bin 1 antibody, "ca" which comprises the CDR regions of the Heavy and Light Chains in Figure 23. This antibody is described in the patent entitled "Antibodies Against Epidermal Growth Factor Receptor (EGFR) and Uses Thereof, U.S. Patent No. 9,044,460 as antibody "ca 34", which is herein incorporated by reference. In vivo studies of MM-151 alone and in combination with irinotecan were also performed in a colorectal cancer patient-derived xenograft (ST094). This study utilizes the MM-151 preclinical tool compound ("MM-151 TC") in which the antibody ca was substituted for antibody PIX. PIX, but not ca cross reacts with (binds to) mouse EGFR as well as binding to human EGFR, and the binding of PIX to the mouse antigen depletes the amount of this antibody in the mouse circulation, altering exposure of the human tumor to the antibody. Results obtained with ca are predictive of results that would be obtained in human patients with PIX, except that the antibody trio comprising PIX would be expected to be more active than the trio comprising ca in its stead. In this model, MM-151 and to a greater extent, the MM-151/irinotecan combination decreased tumor growth relative to the PBS control.

Example 9: Phase II Trial

Figure 12 is a scheme of a human clinical trial. The study can have two parts, a dose finding phase to determine the MTD and an expansion cohort after the MTD is identified. The dose levels to be used in this study are summarized in Table 7.

Table 7: Dose levels of MM-151, nal-IRI, 5-FU, and LV (irinotecan dose based on amount of irinotecan free base):

ΪΙΊΟΓ to initiating the first dose in each method, there is a two week Priming Phase. In this Priming Phase, MM-151 is given at fixed doses of 225 mg and 450 mg on Weeks 1 and 2 respectively. Nal-IRI, 5-FU and Leucovorin are all administered in Week 1 of the Priming Phase at their normal doses listed in the table 7 above. ² Dose levels -1, -2, and -3 will be used if there is toxicity observed in Method 1 that limits further enrollment. ³ 1 + d racemic form of LV will be used

30 - 90 minutes prior to infusion, patients will be administered their MM-151 premedication with all of the following:

• acetaminophen 650 mg PO or IV, and

• diphenhydramine 25-50 mg PO or IV, and

• methylprednisolone (Solumedrol®) 125 mg IV

The first two doses of MM-151 are administered during a two week priming phase. The first priming dose is given as a fixed dose of 225 mg in week 1 and the second priming dose is given as a fixed dose of 450 mg in week 2. Subsequent dose levels are given as weight based dose levels defined per cohort.

Table 8: MM-151 Administration Rates

151 + nal-IRI + 5-FU + LV in patients with metastatic colorectal cancer that are RAS- wildtype.

The secondary objectives of this study are:

• To characterize the adverse event profile (including DLTs)

• To determine the pharmacokinetic parameters

To determine the immunogenicity parameters

The primary objectives of the phase lib portion of the study are:

• To further characterize the safety and tolerability of the MM-151 + nal-IRI + 5-FU + LV combination in patients with metastatic colorectal cancer that are RAS- wildtype

• To characterize observed biomarkers from tissue, blood and/or urine and describe the impact of treatment on these biomarkers

The exploratory objectives of this study are:

• To assess preliminary efficacy as measured by objective response rate, disease control rate and progression free survival.

• To assess the relationship between biomarkers from tissue, blood and/or urine with toxicity and efficacy parameters

To be eligible for inclusion into the study patients must have/be: • Pathologically documented, definitively diagnosed, colorectal adenocarcinoma that is locally advanced or metastatic and surgically un-resectable

• Wild type RAS gene in tumor tissue assessed via extended RAS testing for KRAS/NRAS (documentation of previously existing mutational status from an accredited laboratory will be accepted)

• Measureable disease in accordance with RECIST vl .1

• Adequate end organ function and bone marrow reserves:

• Received no more than one line of prior treatment for metastatic disease with one oxaliplatin based therapy and documentation of progression or intolerance to this therapy

• No prior treatment with irinotecan

• No prior treatment with an EGFR inhibitor

Note, the published dose of nal-IRI 80 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride) was expressed as the irinotecan hydrochloride trihydrate until October 2015. It is now expressed as the irinotecan free base. Converting a dose based on irinotecan hydrochloride trihydrate to a dose based on irinotecan free base is accomplished by substituting the Molecular Weight of irinotecan hydrochloride trihydrate (677.19 g/mole) with the Molecular Weight of irinotecan free base (586.68 g/mole), which results in a conversion factor of 0.866. Therefore, the published expression of the 80 mg/m ² (based on the amount of irinotecan trihydrate hydrochloride) dose based on irinotecan hydrochloride trihydrate is equivalent to a 69.3 mg/m ² dose based on irinotecan free base. This was rounded to 70 mg/m ² to minimize any potential dosing errors.

Example 10: ONIVYDE® (MM-398 irinotecan liposome)

[0001] One preferred example of an irinotecan liposome described herein is the product that will be marketed as ONIVYDE (irinotecan liposome injection). ONIVYDE is a

topoisomerase inhibitor, formulated with irinotecan hydrochloride trihydrate into a liposomal dispersion, for intravenous use. ONIVYDE indicated for the treatment of metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy.

[0002] ONIVYDE is an irinotecan liposome having a pH of about 7.25. The ONIVYDE product contains irinotecan sucrosofate encapsulated in a liposome, obtained from an irinotecan hydrochloride trihydrate starting material. The chemical name of irinotecan is

can be calculated based on the equivalent amount of irinotecan tnhydrate hydrochloride starting material used to prepare the irinotecan liposomes, or based on the amount of irinotecan in the liposome. There are about 866 mg of irinotecan per gram of irinotecan trihydrate hydrochloride. For example, an ONIVYDE dose of 80 mg based on the amount of irinotecan hydrochloride trihydrate starting material actually contains about 0.866x(80mg) of irinotecan in the final product (i.e., a dose of 80 mg/m ² of ONIVYDE based on the weight of irinotecan hydrochloride starting material is equivalent to about 70 mg/m ² of irinotecan in the final product). ONIVYDE is a sterile, white to slightly yellow opaque isotonic liposomal dispersion. Each 10 mL single-dose vial contains 43 mg irinotecan free base at a

concentration of 4.3 mg/mL. The liposome is a unilamellar lipid bilayer vesicle,

approximately 110 nm in diameter, which encapsulates an aqueous space containing irinotecan in a gelated or precipitated state as the sucrose octasulfate salt. The vesicle is composed of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 6.81 mg/mL, cholesterol 2.22 mg/mL, and methoxy-terminated polyethylene glycol (MW 2000)- distearoylphosphatidyl ethanolamine (MPEG-2000-DSPE) 0.12 mg/mL. Each mL also contains 2-[4-(2-hydroxy ethyl) piperazin-l-yl]ethanesulfonic acid (HEPES) as a buffer 4.05 mg/mL and sodium chloride as an isotonicity reagent 8.42 mg/mL. Each vial of ONIVYDE contains 43 mg/10 mL irinotecan free base as a white to slightly yellow, opaque, liposomal dispersion in a single-dose vial.

[0003] The unit dosage form can be an intravenous formulation having a total volume of about 500 mL. ONIVYDE is prepared for administering by diluting the isotonic liposomal dispersion from the vial as follows: withdraw the calculated volume of ONIVYDE from the vial. ONIVYDE is diluted in 500mL 5% Dextrose Injection, USP or 0.9% Sodium Chloride Injection, USP and mix diluted solution by gentle inversion; protect diluted solution from light and administer diluted solution within 4 hours of preparation when stored at room temperature or within 24 hours of preparation when stored under refrigerated conditions [2°C to 8°C (36°F to 46°F)].

[0004] ONIVYDE (irinotecan liposome injection) is indicated, in combination with 5- fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas that has progressed following gemcitabine-based therapy. Administer

ONIVYDE prior to leucovorin and fluorouracil. The recommended dose of ONIVYDE is 70 mg/m ² irinotecan (free base) administered by intravenous infusion over 90 minutes every 2 weeks. The recommended starting dose of ONIVYDE in patients known to be homozygous for the UGT1A1 *28 allele is 50 mg/m ² irinotecan administered by intravenous infusion over 90 minutes. The dose of ONIVYDE can be increased to 70 mg/m ² as tolerated in subsequent cycles. There is no recommended dose of ONIVYDE for patients with serum bilirubin above the upper limit of normal. ONIVYDE is infused as a diluted solution intravenously over 90 minutes.

[0005] Suitable treatment regimens include ONIVYDE 70 mg/m ² with (1+d racemic form) leucovorin 400 mg/m ² (or 200 mg/m ² of the active 1 form of leucovorin) and fluorouracil 2,400 mg/m ² over 46 hours every 2 weeks (ONIVYDE/ 5 -FU/L V; n=l 17), ONIVYDE 100 mg/m ² every 3 weeks (n=147), or leucovorin 200 mg/m ² and fluorouracil 2000 mg/m ² over 24 hours weekly for 4 weeks followed by 2 week rest (5-FU/LV; n=134).

Example 11: Activity of MM-151 in combination with Nal-IRI in LoVo CRC xenograft model

The combination effect of MM-151 TC (MM-151 preclinical tool compound; refer to Example 8) and nal-IRI was studied in a subcutaneous CRC xenograft model (LoVo). MM-151 TC was utilized for cross-reactivity with murine EGFR, and was administered intraperitoneally (IP) at 1 cycle/week comprised of a loading dose (mAb2E5+P2X+P3X) on C1D1, maintenance doses (mAb2E5+P2X+P3X) on Dl of subsequent cycles, and P2X maintenance doses on D3 and D5 every cycle. Nal-IRI is administered intravenously via tail vein injection weekly. LoVo tumor-bearing mice were randomized for efficacy study at Day 16 post-inoculation. A combination of nal-IRI + MM-151 has improved activity compared to the two agents alone. Table 9 illustrates response analysis based on RECIST-like criteria (modified for preclinical tumor volume analyses) on Day 30 post-inoculation. Individual tumors with -30 to -95%, -30 to 30%, and 30% change in volume were classified as partial response (PR), stable disease (SD), and progressive disease (PD), respectively. At the doses tested, nal-IRI + MM-151 resulted in 62.5% PR and 37.5% SD with no PD, while MM-151 and nal-IRI monotherapies resulted in 100% and 75% PD, respectively.

between -95% to -30%, SD = -30% to 30%, PD = tumor volume > 30%.

Example 12: Activity of Nal-IRI is superior to free irinotecan in a KRAS/NRAS/BRAF wild type LIM1215 CRC xenograft at a 5-fold lower dose

Figure 21 demonstrates the anti-tumor activity of nal-IRI at 5 mg/kg compared to free irinotecan at 25 mg/kg. The doses were chosen due to similar SN-38 (active metabolite of irinotecan) exposure in the tumors based on previously published data [Kalra (2014) Cancer Research, 74(23):7003-13]. At 5-fold lower doses administered and presumably equivalent SN-38 exposure, nal-IRI has superior anti -tumor activity compared to free irinotecan. This is believed to be attributed to the prolonged duration of SN-38 present in the tumors for nal-IRI as a result of the liposomal delivery mechanism, in contrast to the free irinotecan in which the small molecules have faster clearance than the liposomal irinotecan (nal-IRI) as shown previously in Figure 2. This differential activity between nal-IRI and IRI at the equivalent SN-38 exposure doses was also previously demonstrated in HT-29 CRC xenografts [Kalra (2014) Cancer Research, 74(23):7003-13].

Example 13: Activity of nal-IRI + 5-FU + MM-151 TC vs. irinotecan + 5-FU + cetuximab

The combination of free irinotecan + 5-FU/LV + cetuximab is one of the standard of care (SOC) regimens for treating metastatic CRC. Activity of the experimental treatment, nal-IRI + 5-FU + MM-151 TC, was compared to the SOC irinotecan + 5-FU + cetuximab in LFM1215 KRAS/NRAS/BRAF wild type CRC xenograft models. Two dose levels of nal-IRI and irinotecan were chosen for combining with constant doses of 5-FU and MM-151 TC or cetuximab. "Low dose" combinations include Nal-IRI (1.25 mg/kg, IV) + 5-FU (50 mg/kg, IP) + MM-151 TC (3 mg/kg, IP) and irinotecan (6.25 mg/kg, IV) + 5-FU (50 mg/kg, IP) + cetuximab (3 mg/kg, IP). "High dose" combinations include Nal-IRI (5 mg/kg, IV) + 5-FU (50 mg/kg, IP) + MM-151 TC (3 mg/kg, IP) and irinotecan (25 mg/kg, IV) + 5-FU (50 mg/kg, IP) + cetuximab (3 mg/kg, IP). The comparative treatment groups were chosen such that the irinotecan and nal-IRI treatments have similar SN-38 tumor exposure. The dose levels of nal-IRI and IRI were below clinical dose, in which the clinical translated dose for nal-IRI (80 mg/m ² hydrochloride trihydrate q2w for patients) is 13 mg/kg qlw in mouse, and for irinotecan (180 mg/m ² q2w, based on the amount of irinotecan trihydrate hydrochloride) is 30 mg/kg qlw in mouse. The dose levels of MM-151 TC and cetuximab were chosen such that the exposure levels in the blood are nearly equivalent in both the first week (with the loading dose) and the subsequent weeks (with the maintenance doses). Exposure was calculated as the area under the concentration curve (AUC) of cetuximab versus the sum total of the AUC of the three antibodies in the MM-151 TC mixture.

Figures 22 A and B illustrate the anti -tumor activities of low and high dose combinations, respectively. At the 2 dose levels tested, nal-IRI + 5-FU + MM-151 TC are superior to the SOC irinotecan + 5-FU + cetuximab. In addition, Table 10 shows that high dose nal-IRI + 5-FU + MM-151 TC treatment results in 3 CR and 6 PR, while both dose levels of irinotecan + 5-FU + cetuximab did not have any CR.

Table 10. Response of LIM1215 CRC xenograft based on RECIST-like criteria.

Note: Response is calculated based on tumor volume change from baseline volume. CR = tumor volume <-95%, PR = between -95% to -30%, SD = -30% to 30%, PD = tumor volume > 30%.

Example 14: Initiation of Phase 1 Study of MM-151 in Combination with the

ONIVYDE® (irinotecan liposome injection) Regimen in Metastatic Colorectal Cancer

This phase I clinical study examines oligoclonal EGFR (epidermal growth factor receptor) inhibitor, MM-151, in combination with ONIVYDE® (irinotecan liposome injection) plus fluorouracil (5-FU) and leucovorin in patients with RAS wild-type metastatic colorectal cancer. Data from a prior phase I study of MM-151 supports further clinical evaluation of the investigational therapy in patients with metastatic colorectal cancer. The initiation of this study advances the development path for ONIVYDE.

This phase I study will assess the safety and tolerability of the combination of MM- 151, a novel antibody mixture of three human antibodies designed to target EGFR which promotes tumor growth, and ONIVYDE, also known as MM-398 or "nal-IRI," plus 5-FU and leucovorin as first or second-line treatment in patients with RAS wild-type metastatic colorectal cancer.

Preclinically, MM-151 has shown superior inhibition of the EGFR pathway compared to FDA approved EGFR inhibitors. It is expected that superior topoisom erase- 1 inhibition plus superior EGFR inhibition will lead to improved efficacy in metastatic colorectal cancer.

The trial will determine the side effect profile of MM-151 in combination with ONIVYDE plus 5-FU and leucovorin and recommended dose for subsequent trials with this combination. Eligible patients for the study must have metastatic disease, have had no prior exposure to irinotecan or an EGFR inhibitor, and have received no more than one prior line of treatment for metastatic disease.

Example 15: Evaluation of MM-151 + Nal-IRI + 5-FU + Leucovorin in RAS/RAF Wild- type Metastatic Colorectal Cancer

An open label, non-randomized phase I/II study can evaluate the combination of MM-151 + nal-IRI + 5-FU + Leucovorin in RAS/RAF wild-type Metastatic Colorectal Cancer. In the dose finding cohorts of this phase Ib/II study, a modified toxicity probability interval approach (mTPI) will be utilized to determine the maximum tolerated dose (MTD) of MM-151 in combination with nal-IRI, 5FU and leucovorin. Phase I: Approximately 8-12 patients will be enrolled to determine the maximum tolerated dose, safety and tolerability of MM-151 + nal-IRI + 5-FU + LV in patients with mCRC that are RAS wildtype. Phase II: Approximately 20-30 patients will be enrolled at the maximum tolerated dose of MM-151 in combination with nal-IRI + 5-FU + LV to characterize initial efficacy in conjunction with levels of irinotecan and SN-38 measured in tissue.

Table 11. Experimental Arms

Primary Outcome Measure:

1. Primary Objective of phase lb

[Time Frame: 6 months] [Safety Issue: Yes]

To determine the maximum tolerated dose, safety and tolerability of MM-151 + nal-IRI + 5- FU + LV in patients with mCRC that are RAS wild-type.

2. Primary Objective of phase Ila

[Time Frame: 2 years] [Safety Issue: No]

To characterize initial efficacy in conjunction with levels of irinotecan and SN-38 measured in tissue

Secondary Outcome Measure:

3. Secondary Objectives of phase lb

[Time Frame: 2 years] [Safety Issue: Yes]

To characterize the adverse event profile (including DLT's); To determine the

pharmacokinetic parameters; To determine the immunogenicity parameters

4. Secondary Objectives of phase Ila

[Time Frame: 2 years] [Safety Issue: Yes]

To further characterize the adverse event profile; To describe the observed objective response rate (ORR) and disease control rate; To measure pretreatment and on-treatment levels of EGFR ligands; To determine the pharmacokinetic parameters; To determine the

immunogenicity parameters

Other Pre-specified Outcome Measures: 5. Exploratory Objectives

[Time Frame: 2 years] [Safety Issue: Yes]

To assess the relationship between biomarkers from tissue, blood and/or urine with toxicity and efficacy parameters, including markers of the development of resistance to treatment Eligibility:

Minimum Age: 18 Years; Gender: Both; No Healthy Volunteers. Inclusion Criteria:

• Pathologically documented, definitively diagnosed, colorectal adenocarcinoma that is metastatic and surgically un-resectable

• For phase lb of the study, patients must be either treatment naive or have had no more than one prior line of therapy with an oxaliplatin-based regimen for metastatic disease

• For phase Ila of the study, patients must have had no prior therapy for metastatic disease.

• Wild-type KRAS/NRAS genes as assessed in tumor tissue via extended RAS testing

• Histologic confirmed BRAF wild-type status of primary colorectal cancer or related metastasis

• Measurable disease in accordance with RECIST vl . l

• ECOG Performance Score (PS) of 0 or 1

• Adequate bone marrow reserves as evidenced by:

ANC > 1,500/μ1 (unsupported by growth factors) o Platelet count > 100,000/μ1 o Hemoglobin > 9 g/dL o

• Adequate hepatic function as evidenced by: Serum total bilirubin < ULN o Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Alkaline Phosphatase (ALP) <

2.5 x ULN (< 5 x ULN is acceptable if bone or liver metastases are present)

• Adequate renal function as evidenced by a serum creatinine < 1.5 x ULN

• Adequate cardiac function as determined by:

An LVEF within normal institutional limits o ECG without clinically significant

abnormalities including prolonged QTc interval (> 450 msecs); abnormal findings on interpretation of ECG are acceptable if principle investigator confirms it is not clinically significant.

• Recovered from the effects of any prior surgery, radiotherapy or other antineoplastic therapy to CTCAE v4.0 grade 1, baseline or less.

• Patient must be willing to provide a pre-treatment biopsy as well as an on study biopsy to the Sponsor. In the event that a patient has an archived tumor sample from a previous biopsy and no anti-cancer treatment was given prior to that biopsy then the archived tumor sample may be submitted in place of a fresh biopsy.

• Willing to abstain from sexual intercourse or to use an effective form of contraception during the study and for 120 days following the last dose of any study therapy. This applies to women of childbearing potential as well as fertile men and their partners.

• At least 18 years of age

• Able to provide informed consent, or have a legal representative able and willing to do so Exclusion Criteria:

• Prior pelvic radiation treatment

• Prior treatment with irinotecan (patients in phase lb)

• Prior treatment with an EGFR inhibitor (patients in phase lb)

• Pregnant or lactating

• Untreated (primary) or uncontrolled CNS (primary or metastatic) malignancies; patients with CNS metastases who have undergone surgery or radiotherapy or who have been on a stable dose of corticosteroids for at least 2 weeks and whose disease is stable prior to the first scheduled day of dosing will be eligible for the trial.

• Clinically significant cardiac disease, including: NYHA Class III or IV congestive heart failure, unstable angina, acute myocardial infarction within six months of planned first dose, arrhythmia requiring therapy (including torsades de pointes, with the exception of extra systoles, minor conduction abnormalities, or controlled and well treated chronic atrial fibrillation)

• History of any second malignancy in the last 3 years; patients with prior history of in-situ cancer or basal or squamous cell skin cancer are eligible. Patients with a history of other malignancies are eligible if they have been continuously disease free for at least 3 years.

• Clinically significant gastrointestinal disorder including hepatic disorders, occlusion, diarrhea > grade 1, malabsorption syndrome, ulcerative colitis, inflammatory bowel disease, or partial bowel obstruction

• Known hypersensitivity to any of the components of nal-IRI, other liposomal products, or any components of 5-FU or LV

• Known hypersensitivity to any of the components of MM-151

• Received other recent antitumor therapy including any standard chemotherapy or radiation within 14 days and bevacizumab within 28 days (and having passed the time of any actual or anticipated toxicities) prior to the first scheduled dose of the study treatment • Any other medical condition deemed by the Investigator to be likely to interfere with a patient's ability to provide informed consent, cooperate and participate in the study, or to interfere with the interpretation of the results

Example 16: Fiijal results of a tlrst-m-hwnian study evaluating the safety,

pharmacology and ieitial efficacy of MM-151, an o!igodona! asiti-EGFK antibody in paiienis with refractory solid tumors.

This is a phase I study thai evaluated MM-151 when administered as a monotherapy and in combination with (min- liposomal ) irinotecan. An expansion cohort was also enrolled to evaluate clinical activity in EGFR-refractory metastatic CRC patients (pts). Subset analyses and additional biomarker evaluations were performed in EGFR-driven indications.

Results: A total of 1 1 1 patients were enrolled (87 patients on monotherapy). A summary of the patient demographics are set forth in Table 12. Specific population demographics for patients with HNSCC, NSCLC, or CRC are set forth in Tables 13, 14, and 15, respectively.

Table 12: Patient demographics

The most common tumor types were CRC (43 [39%]), NSCLC (11 [10%]) and SCCHN (14 [13%]). Weekly dose selection was previously reported. Reported here are final safety and biomarker data. Most adverse events (AEs) were CTCAE Grades 1 and 2. The most common Grade 3 (G3) or higher non-infusion related reaction (IRE.) AEs included EGFR-pathway toxicities, such as maeulopapular rash ( 1 1 [9.9%]), Hypomagnesemia (10

[9%]), general rash (8 [7.2%]) and diarrhea (8 [7.2%])

Table 16: Common related, nosi-IRR adverse events grade 3 or higher

G3 infusion related reactions (IRR) occurred in 8/57 (14%) of patients enrolled at the non-optimized dosing guidelines versus 1/57 ( 1.7%) of patients in the optimized dosing cohorts. Frequency of infusion related reactions experienced at first MM-151 infusion are summarized in Table 17.

Table 17: Frequency of infusion related reactions

Example 17: Biomarker Studies Reveal a Low Frequency of Acquired Mutations Following MM-151 Treatment

In the Phase 1 clinical study described in Example 16, tissue and blood samples were collected from treated patients and used in exploratory biomarker analyses. While samples were requested from all patients identified in Table 12, samples were only collected from a subset of these patients. For those patients from whom samples were obtained, blood and tissue samples were assessed for genomic alterations using next-generation sequencing assays from GuardantHealth (Guardant360) and OmniSeq (OmniSeq Comprehensive; OmniSeq PGM), respectively. The Guardant360 and OmniSeq Comprehensive assays identify somatic mutations, copy number variations, and indels/fusions in oncology-focused gene panels. Table 18 summarizes of the collected samples for biomarker analysis, from patients having EGFR-related indications (i.e., patients for whose condition anti-EGFR antibodies are an approved therapy).

Table 18: Summary of collected samples for biomarker analy

Subset analyses were performed for a cohort of colorectal cancer patients (N=29) who received the minimum efficacious dose of > 6 mg/kg on QW or Q2W schedules (i.e., the "CRC efficacy cohort patients," as used herein). 28 of 29 CRC efficacy cohort patients had archival tissue and/or pre-treatment serum samples for DNA sequencing. Figure 24 summarizes the pre-treatment somatic mutations within the CRC efficacy cohort. Mutually exclusive mutations in KRAS/NRAS/BRAF were observed in 39% of these patients (11/28).

Figure 25 is a table listing the cancer type, treatment schedule, duration of treatment and mutation status (wild type, "WT" or mutant, "MT") for 26 patients from whom we collected both pre- and post-treatment serum samples from the clinical trial described in Example 16. In addition, the table in Figure 25 indicates the measured maximum change in solid tumor size as measured by RECIST 1.1 ("Maximum Percent Change") compared to the solid tumor size measured prior to treatment. The data in Figure 25 demonstrates a low frequency of acquired KRAS/NRAS/BRAF mutations following MM-151 treatment (i.e., 1 of 5 CRC patients and 2 of 19 in all indications that were assessed as WT for all measured KRAS, NRAS and BRAF prior to treatment). Notably, no CRC patients acquired an EGFR exon 12 mutation, and only one of 25 wild type patients (from all indications shown in the Table of Figure 25) acquired a variant of unknown significance at a low allelic frequency. Clinically meaningful durations were achieved in patients presenting with multiple resistance markers, including RAS/RAF mutations. This demonstrated a rate of acquired mutations measured in MM-151 -treated patients that was lower than observed with treatment of other EGFR antibodies in the literature.

Figure 26 is a table detailing the patient treatment parameters, the duration of treatment, the best overall response, duration of treatment and mutation status (wild type, "WT" or mutant, "MT") for 24 of 29 patients within the CRC efficacy cohort who were evaluated for RECIST response in the clinical trial described in Example 16. The Table in Figure 26 also includes patient treatment parameters listing the treatment type (MM-151 monotherapy or combination of MM-151 and irinotecan), whether the first two weekly doses of MM-151 were priming doses (225 mg and 450 mg fixed dose, respectively), dose of MM- 151 administered (after the priming doses, if applicable), MM-151 dose frequency (every week or every two weeks), and (if applicable) the irinotecan dose and dose frequency. Of these 24 patients listed in the Table in Figure 26, 13 patients (54%) had a reduction in target lesions and 7 progressed at first scan, including 2 wild type (WT in KRAS, NRAS, BRAF, PIK3CA, EGFR Exon 12), 3 NRAS mutants, 1 co-occurring PIK3C A mutation, and 1 BRAF mutation. Two patients were noted as progressive having progressive disease (PD) on first scan but had <20% increase in target lesions, one patient had a new non-target lesion noted, and one patient was a clinical progression (not RECIST). Of the 15 remaining patients that did not progress outright, 7 patients were WT, 4 KRAS mutant, 3 BRAF mutant, 2 NRAS mutant, and 2 EGFR ECD mutant (including co-occurring).

Further exploratory clinical biomarker analyses were performed on subsets of patients enrolled in the clinical trial described in Example 16. Downregu!ation of EGFR was measured by imimmohistochemistry (IHC) in matched pre-treatment and on-treatment tissue biopsies (FFPE) collected from 4 patients treated with 1.0.5 mg/kg MM-151 once a week after administration of fixed dose priming doses of 225 rng and 450 rng in weeks 1 and 2 of treatment.. Clinical validation of MM-151 mechanistic foundations was observed by EGFR downregulation, high affinity ligand frequency in refractory mCRC patients, and the ability to overcome both upstream and downstream mutations (acquired and de novo). Additionally, a decrease in measurable lesions was observed in 54% of evaluabie patients in the QIC cohort and a measureable decrease in lesions was observed in both WT and mutant patients.

Preliminary indications of objective clinical activity across both the EGFR-refractory and naive populations suggest potential for broad effect. Biomarker profiling also suggests that MM-151 may overcome mechanisms of resistance.

Endnotes

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