Phase I study of the investigational anti-guanylyl cyclase...

1 156699_2_art_file_3450457_h5mfx3.docx

Phase I study of the investigational anti-guanylyl cyclase antibody–

drug conjugate TAK-264 (MLN0264) in adult patients with advanced

gastrointestinal malignancies

Authors:

Khaldoun Almhanna,1 Thea Kalebic,2 Cristina Cruz,3 Jason E. Faris,4 David P.

Ryan,4 JungAh Jung,2 Tim Wyant,2 Adedigbo A. Fasanmade,2 Wells Messersmith,5*

Jordi Rodon3*

Author affiliations:

1Department of Gastrointestinal Oncology, Moffitt Cancer Center, Tampa, FL, USA;

2Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda

Pharmaceutical Company Limited, Cambridge, MA, USA; 3Vall d’Hebron Institute of

Oncology, Vall d’Hebron University Hospital and Universitat Autònoma de Barcelona,

Barcelona, Spain; 4Hematology/Oncology, Massachusetts General Hospital Cancer

Center, Boston, MA, USA; 5Division of Medical Oncology, University of Colorado

Cancer Center, Aurora, CO, USA

*Co-senior authors

AACR member: Jordi Rodon Ahnert #128159

Running title [60 characters max, including spaces; currently 49]:

TAK-264 in advanced gastrointestinal malignancies

Keywords from pull-down list of terms (2-10):

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 13, 2016; DOI: 10.1158/1078-0432.CCR-15-2474

http://clincancerres.aacrjournals.org/


Phase I-III trials_Gastrointestinal cancers: colorectal; Phase I-III

trials_Gastrointestinal cancers: stomach; Phase I-III trials_Gastrointestinal cancers:

other; Phase I-III trials_Pancreatic cancer; Antibody immunotherapy; New targets;

Novel antitumor agents; Antibodies/immunoconjugates

User-defined key words (optional, up to 3):

Guanylyl cyclase C; antibody–drug conjugate

Financial support:

This study was funded by Millennium Pharmaceuticals Inc., a wholly owned

subsidiary of Takeda Pharmaceutical Company Limited.

Corresponding author:

Khaldoun Almhanna

Department of Gastrointestinal Oncology, Moffitt Cancer Center, 12902 Magnolia

Drive, Tampa, FL 33612, USA

E-mail: [email protected]

Tel: (813) 745-3636. Fax: (813) 745-7229

Disclosures of conflicts of interest:

KA has received honoraria from Genentech and Lilly, and served in a consulting or

advisory role for Lilly and on a speakers’ bureau for Genentech.

TK, JJ, TW, and AF are full-time employees of Millennium Pharmaceuticals, Inc., a

wholly owned subsidiary of Takeda Pharmaceutical Company Limited.





CC has received travel, accommodations, or expenses from Millennium

Pharmaceuticals, Inc.

JEF has no conflicts of interest to disclose.

DPR has no conflicts of interest to disclose.

WM has received research funding from Millennium Pharmaceuticals, Inc., a wholly

owned subsidiary of Takeda Pharmaceutical Company Limited.

JR has no conflicts of interest to disclose.

Journal: Clinical Cancer Research. Category of manuscript: Cancer Therapy:

Clinical. Abstract word count: 250 words [max 250]. Statement of translational

relevance: 150 words [Max 120-150]. Word count: 4326 [max 5000].

Tables/figures: 6 (max 6). References: 37 [Max 50].

ClinicalTrials.gov identifier: NCT01577758.

Suggested reviewers (2–5 required):

1. Steven J. Cohen, MD, Fox Chase Cancer Center, Philadelphia, PA, email:

[email protected]

2. Bert O'Neil, MD, Indiana Cancer Pavilion, Indiana University, Indianapolis, IN,

email: [email protected] / [email protected]

3. Tanios Bekaii-Saab, MD, The Ohio State University Comprehensive Cancer

Center, Columbus, OH, email: [email protected] / bekaii-

[email protected]

4. Anthony W. Tolcher, MD, FRCP(C), South Texas Accelerated Research

Therapeutics (START), San Antonio, TX, email: [email protected]





Abstract

Purpose: To assess the safety, tolerability, and preliminary antitumor activity of the

investigational anti-GCC antibody-drug conjugate TAK-264 (formerly MLN0264) in

adult patients with advanced gastrointestinal malignancies.

Experimental Design: Adult patients with GCC-expressing gastrointestinal

malignancies (H-score ≥10) were eligible for inclusion. TAK-264 was administered as

a 30-minute IV infusion once every 3 weeks for up to 17 cycles. Dose escalation

proceeded using a Bayesian continual reassessment method. At the maximum

tolerated dose (MTD), 25 patients with metastatic colorectal cancer (mCRC) were

enrolled in a prespecified dose-expansion cohort.

Results: 41 patients were enrolled, including 35 (85%) with mCRC. During dose

escalation (0.3-2.4 mg/kg), 4 of 19 patients experienced dose-limiting toxicities of

grade 4 neutropenia; the MTD was determined as 1.8 mg/kg. Patients received a

median of 2 cycles of TAK-264 (range 1-12); 9 received ≥4 cycles. Common drug-

related adverse events (AEs) included nausea and decreased appetite (each 41%),

fatigue (32%), diarrhea, anemia, alopecia, and neutropenia (each 27%); grade ≥3

AEs included neutropenia (22%), hypokalemia, and febrile neutropenia (each 7%).

Peripheral neuropathy was reported in 4 (10%) patients. Pharmacokinetic data

showed approximately dose-proportional systemic exposure and a mean plasma

half-life of around 4 days, supporting the dosing schedule. Overall, 39 patients were

response-evaluable; 3 experienced durable stable disease and 1 with gastric

adenocarcinoma had a partial response. GCC expression did not appear to correlate

with treatment duration.





Conclusions: These findings suggest that TAK-264 has a manageable safety

profile, with preliminary evidence of potential antitumor activity in specific

gastrointestinal malignancies. Further investigation is underway.





Statement of translational relevance (Max 120-150 words):

The use of antibody–drug conjugates (ADCs) to target specific antigens preferentially

expressed on cancer cells is a feasible and active treatment approach in Hodgkin

lymphoma, anaplastic large cell lymphoma, and HER2-positive breast cancer, with

multiple ADCs under investigation in other lymphomas and solid tumors. ADCs

comprise a monoclonal antibody, a linker, and a cytotoxic small-molecule drug. For

gastrointestinal cancers, the transmembrane cell surface receptor guanylyl cyclase C

(GCC) has been identified as a potential target for a monoclonal antibody, being

expressed in 60-70% of pancreatic, gastric, and esophageal cancers and 95% of

primary and metastatic colorectal cancer. This first-in-human phase 1 study

investigated TAK-264, a novel investigational ADC targeting GCC, in adult patients

with advanced, GCC-expressing gastrointestinal malignancies. Results demonstrated

a manageable safety profile at the maximum tolerated dose and preliminary evidence

of antitumor activity and early signs of clinical benefit in patients with pancreatic,

esophageal, and gastric carcinoma.





Introduction

Monoclonal antibodies are established as anticancer therapies in a number of

different malignancies. Recently, antibody-drug conjugates (ADCs) have been

approved, and several more are being investigated, for various types of cancer.

ADCs are novel targeted agents composed of a monoclonal antibody, a linker, and a

cytotoxic small-molecule drug (1, 2). The monoclonal antibody is targeted to a

specific antigen preferentially expressed on cancer cells; the cytotoxic drug is

released upon internalization (1). There are two ADCs currently approved:

brentuximab vedotin for Hodgkin lymphoma and anaplastic large cell lymphoma (3-

6) and ado-trastuzumab emtansine for HER2-positive breast cancer (7).

Brentuximab vedotin is composed of an anti-CD30 antibody, a valine-citrulline

protease-cleavable linker, and the potent microtubule-disrupting agent monomethyl

auristatin E (MMAE) (5, 6). It has demonstrated substantial efficacy in pivotal phase 2

studies, including overall response rates of 75% (34% complete remission) in

relapsed/refractory Hodgkin lymphoma and 86% (57% complete remission) in

relapsed/refractory systemic anaplastic large cell lymphoma (4, 6). Brentuximab

vedotin received accelerated approval by the FDA in 2011 and conditional approval

in Europe in 2012 for patients with relapsed or refractory Hodgkin lymphoma and

anaplastic large cell lymphoma (8, 9). Other ADCs are in development based on the

same linker/drug technology, including polatuzumab vedotin and pinatuzumab

vedotin (targeting CD79b and CD22, respectively), both of which are in phase 2

development for the treatment of diffuse large B-cell lymphoma and follicular

lymphoma (10). Ado-trastuzumab emtansine is composed of the anti-HER2 antibody

trastuzumab connected via a stable thioether linker to the potent antimicrotubule





agent derivative of maytansine 1, an inhibitor of microtubule dimerization (11, 12). It

was approved in the US and Europe in 2013 for patients with advanced or metastatic

HER2-positive breast cancer (13, 14).

Exploring the therapeutic potential of ADCs in gastrointestinal cancers is of

significant interest, as they are a leading cause of cancer-related deaths; colorectal,

pancreatic, and gastric cancer had the second-, fourth-, and fourteenth-highest

estimated rates of cancer-related deaths in the USA in 2015 (15). One potential

target is the transmembrane cell surface receptor guanylyl cyclase C (GCC), which is

normally expressed on intestinal epithelial cells but not on extragastrointestinal

tissues (16, 17). In primary and metastatic tumors derived from intestinal epithelial

cells, GCC expression is maintained during neoplastic progression (16, 18). GCC is

expressed in 60-70% of pancreatic, gastric, and esophageal cancers (19-21) and

95% of primary and metastatic colorectal cancer (mCRC) (16, 18, 22-24).

Systemically delivered GCC-targeting agents are expected to be preferentially

delivered to GCC receptors in tumor tissue, while leaving normal tissues unaffected,

as GCC is expressed on the apical side of epithelial tight junctions (10, 16, 23, 25,

26). Access to GCC receptors is enabled in tumor tissues as a result of disrupted cell

polarity, altered tight junction architecture, and disruption of its apical localization (16,

18, 22, 24). TAK-264 (formerly MLN0264) is a novel ADC consisting of a fully human

IgG1 monoclonal anti-GCC antibody conjugated via a protease-cleavable linker to

MMAE. Following binding to GCC, TAK-264 is internalized and transported to

lysosomes where MMAE is released and binds to microtubules, leading to cell cycle

arrest and apoptosis. TAK-264 has been investigated in vivo in animal models of

gastrointestinal tumors, including GCC-expressing human colorectal cancer





xenografts and pancreatic cancer xenograft models, demonstrating selective binding

and internalization into GCC-expressing tumor cells and antitumor activity (27, 28).

This phase 1, first-in-human study assessed the safety and tolerability, dose-limiting

toxicities (DLTs), maximum tolerated dose (MTD), pharmacokinetics, and preliminary

antitumor activity of TAK-264 in adult patients with advanced, GCC-expressing

gastrointestinal malignancies.

Patients and Methods

Patients

Patients aged ≥18 years diagnosed with a GCC-expressing gastrointestinal

malignancy (H-score ≥10, derivation described below), for whom standard treatment

was no longer effective or did not offer curative or life-prolonging potential, were

eligible. Eligible malignancies included, but were not limited to, mCRC, gastric

carcinoma, esophageal carcinoma, small intestine cancer, pancreatic cancer, and

unknown primary malignancies. Patients should have progressed to standard-of-care

therapies in all cases. For the prespecified expansion cohort, only patients with

mCRC were eligible. Patients required: measurable disease per Response

Evaluation Criteria in Solid Tumors (RECIST 1.1); Eastern Cooperative Oncology

Group (ECOG) performance status 0 or 1; life expectancy of ≥12 weeks; adequate

bone marrow (absolute neutrophil count [ANC] ≥1500 cells/mm3; platelet count

≥100,000/mm3), hepatic (total bilirubin ≤1.5 X upper limit of normal [ULN]; serum

alanine or aspartate aminotransferase [ALT/AST] ≤2 X ULN; serum albumin ≥3.0

g/dL), and renal (serum creatinine ≤1.5 X ULN and/or calculated creatinine clearance





≥60 mL/min) function. Patients had to have completed prior chemotherapy,

immunotherapy, or radiotherapy ≥4 weeks prior to enrollment, and to have available

archived or fresh tumor tissue. Patients were excluded if they had: any comorbidities

that in the view of the treating physician rendered the patient at high risk from

treatment complications; known infection or inflammatory bowel disease; history of

another primary malignancy not in remission for at least 3 years; New York Heart

Association class III or IV; or grade ≥2 peripheral neuropathy. All patients provided

written informed consent.

Study design

Institutional review boards/ethics committees at the participating investigational

centers approved the study, which was conducted according to the principles set out

in the Declaration of Helsinki, International Conference on Harmonisation Good

Clinical Practice guidelines, and local regulatory requirements.

In this multicenter, open-label, dose-escalation study (NCT01577758), TAK-264 was

administered once every 3 weeks as a 30-minute IV infusion (day 1 of 21-day cycles)

for up to 17 cycles or until disease progression or occurrence of unacceptable TAK-

264-related toxicity. Dose escalation was conducted using a Bayesian continual

reassessment method (CRM) based on 2-patient cohorts, according to observed

DLTs during cycle 1. DLTs were defined as: grade 4 neutropenia (ANC < 500

cells/mm3); grade ≥3 neutropenia with fever (oral temperature ≥38.5°C) and/or

infection; grade 4 thrombocytopenia (platelets <25,000/mm3); grade ≥3

thrombocytopenia with clinically meaningful bleeding at any time; grade ≥3 nausea

and/or emesis that occurred despite anti-emetic prophylaxis; grade ≥3 diarrhea that





occurred despite optimal supportive care measures; any other grade ≥3

nonhematologic toxicity with the exception of brief (<1 week) grade 3 fatigue; inability

to start the next cycle of therapy due to >2 weeks treatment delay because of a lack

of adequate recovery of TAK-264-related hematologic/nonhematologic toxicities;

other TAK-264-related grade ≥2 nonhematologic toxicities that, in the opinion of the

investigator, required a dose reduction or discontinuation of TAK-264 therapy.

The CRM algorithm is shown in Fig. 1. Dose escalation or de-escalation was based

on the observed toxicities in all DLT-evaluable patients. After the first 2 patients had

been dosed and had completed the first cycle of therapy, the CRM algorithm was

updated based on the observed DLTs, and the predicted MTD was calculated. Once

at least 6 patients had been treated at a given dose without the algorithm suggesting

escalation or de-escalation, that dose was considered the MTD. After completion of

dose escalation and MTD determination, additional patients with mCRC were

enrolled to a prespecified dose-expansion cohort to achieve up to 20 response-

evaluable patients. Among these patients, at least 6 were required to have high GCC

expression.

Objectives and Assessments

The primary objectives were to assess the safety profile of IV TAK-264 in patients

with advanced GCC-expressing gastrointestinal malignancies, determine the MTD,

and describe the pharmacokinetic profile of TAK-264, total antibody, and MMAE.

Secondary objectives were to evaluate disease response and evidence of antitumor

activity in TAK-264-treated patients and the immunogenicity of TAK-264

(antitherapeutic antibody [ATA] development).





An immunohistochemistry assay was performed to assess the expression of GCC by

utilizing a fully human antibody specific to GCC (Millennium Pharmaceuticals, Inc.).

Tissue sections 5 to 10 micron thick were dewaxed through 4, 5-minute changes of

xylene and then placed in a series of graded alcohol solutions diluted with distilled

water. Steam heat induced epitope recovery (SHIER) was conducted for 20 minutes

in the capillary gap in the upper chamber of a Black and Decker Steamer. An

automated TechMate 500 or TechMate 1000 (Roche Diagnostics) was used to

perform the staining. Following an overnight primary incubation, the visualization was

achieved using a non-biotin based peroxidase detection kit (Ultra Vision). After

completing the staining, slides were dehydrated and glass coverslips and CytoSeal

were used to permanently cover the slides. Positive staining was shown by the

presence of a brown (DAB-HRP) reaction product. Hematoxylin counter stain was

used to visualize cell and tissue morphology. All slides were stained and assessed

under microscope by two blinded pathologists at the central laboratory to assess

quality of staining and evaluate the GCC levels. Based on this semi-quantitative

method, H-score for GCC expression was calculated using the sum of the

percentage of tumor cells with GCC staining and intensity of 1+, 2+, and 3+, up to a

maximum score of 300 (100% at 3+) (29). Preclinical data suggested that both

cytoplasmic and apical GCC expression may play a role in TAK-264 efficacy (data on

file). Therefore, H-scores were determined for both cytoplasmic and apically oriented

staining and summed, giving a total maximum combined H-score of 600. The staining

was detected on tumor cells; there was no staining in the stroma or infiltrating

inflammatory cells.





Adverse events (AEs) were graded using NCI-CTCAE version 4.03. Disease

responses were assessed at the end of every second cycle by RECIST v1.1. Blood

samples were collected for pharmacokinetic analysis pre-dose and at multiple time

points post-dose. Serum levels of TAK-264 and total antibody were assayed using a

quantitative sandwich enzyme immunoassay; plasma levels of free MMAE were

determined using a liquid chromatography tandem mass spectrometry assay. Blood

samples were taken from patients before dosing and at the end of the study to

evaluate presence of antitherapeutic antibody (ATA), using validated assays to

detect TAK-264-binding antibodies and to verify whether these antibodies had

neutralizing activity.

Analysis populations and statistical analyses

The safety population included all patients who received at least 1 dose of study

drug. The DLT-evaluable population (used to determine MTD) included dose-

escalation patients who either experienced a DLT during cycle 1 or received TAK-

264 and completed all study procedures in cycle 1 without DLTs. The

pharmacokinetics-evaluable population included patients with sufficient dosing and

pharmacokinetic data to estimate pharmacokinetic parameters. The response-

evaluable population included patients with measureable disease who received TAK-

264 and had at least 1 post-baseline response assessment.

Statistical analyses were primarily descriptive and graphical in nature. Summary

tabulations were used to present the number of observations, mean, standard

deviation, median, minimum, and maximum for continuous variables, and the number

and percentage per category for categorical data. Summary statistics were calculated





for baseline characteristics, dosing, safety (including DLTs), AEs, serious AEs,

laboratory values, vital signs, efficacy (including disease response), biomarkers

(including ATA), and pharmacokinetic parameters. Progression-free survival was

estimated using the Kaplan-Meier method.

Results

Patient characteristics

Forty one patients were enrolled at 3 sites in the US and 1 site in Spain between

June 11, 2012 and February 12, 2014 and received at least 1 dose of TAK-264

(Table 1). In 35/41 (85%) patients the primary diagnosis was mCRC, as this was the

only tumor type included in the prespecified expansion cohort. The other patients had

pancreatic, esophageal, gastric, or other type of adenocarcinoma. GCC H-score

ranged from 10 to 550; 13 patients with high GCC expression levels were enrolled in

the expansion cohort.

DLTs and MTD determination

The dose-escalation cohort included 19 patients: 2 patients each at 0.3 mg/kg, 0.6

mg/kg, 1.2 mg/kg, and 1.5 mg/kg, 6 patients at 1.8 mg/kg, 4 patients at 2.1 mg/kg,

and 1 patient at 2.4 mg/kg. There were no DLTs in the low dose groups (0.3-1.5

mg/kg). At higher doses a total of 4 patients experienced DLTs of grade 4

neutropenia. Of the 4 patients with DLTs, 3 were treated at doses above 1.8 mg/kg.

One patient in the 1.8 mg/kg dose group experienced a DLT of grade 4 febrile

neutropenia on day 12 of cycle 1. The patient discontinued the study per protocol and

the neutropenia resolved in 2 days. Two patients in the 2.1 mg/kg dose group





experienced DLTs. One patient experienced grade 4 neutropenia and grade 3 QTc

prolongation on days 10 and 14 of cycle 1, respectively. Per protocol, the patient

discontinued the study and the DLTs resolved in 4 and 7 days, respectively. The

other patient treated at 2.1 mg/kg had grade 4 neutropenia on day 15 of cycle 1,

which resolved 7 days later. At a dose of 2.4 mg/kg, one patient experienced a DLT

of grade 4 neutropenia on day 12 of cycle 1. The patient discontinued the study per

protocol and the DLT resolved in 4 days.

The MTD was determined as 1.8 mg/kg according to the Bayesian CRM method. A

total of 25 patients with mCRC were enrolled at this dose level, which included the

patients from the expansion cohort and 3 patients from the 1.8 mg/kg dose-

escalation cohort.

Treatment exposure and safety

Patients received a median of 2 cycles of TAK-264 (range 1-12). Overall, 9 (22%)

patients received ≥4 cycles and 5 (12%) patients received ≥6 cycles. Reasons for

treatment discontinuation were disease progression (n=28, 68%), symptomatic

deterioration (n=7, 17%), AEs, and subject withdrawal (each n=3, 7%).

All 41 patients reported AEs. A total of 36 patients (88%) reported at least one drug-

related AE (Table 2). The most common drug-related AEs included nausea and

decreased appetite (each n=17, 41%), fatigue (n=13, 32%), diarrhea, anemia,

alopecia, and neutropenia (each n=11, 27%). Grade ≥3 AEs were experienced by 26

patients (63%) treated at all doses (Table 2). Among these, 17 (41%) were drug-

related. The drug-related grade ≥3 AEs, across all doses, included neutropenia (n=9,





22%), hypokalemia, febrile neutropenia (each n=3, 7%), and QTc prolongation (n=2,

5%). Serious AEs were experienced by 16 patients (39%), with 9 (22%) of these

patients experiencing drug-related serious AEs including small intestinal obstruction

(3 [7%] patients), febrile neutropenia (3 [7%] patients), constipation (2 [5%] patients),

intestinal obstruction (2 [5%] patients), and pyrexia (2 [5%]) patients. Grade 1 or 2

peripheral neuropathy was reported by 4 (10%) patients over the course of the study;

3 patients without baseline peripheral neuropathy experienced grade 1 peripheral

neuropathy; two patients reported peripheral neuropathy during cycles 1 and 4,

respectively, which was reported as not resolved by the end-of-study visit

(approximately 30 days after the last dose of study drug or prior to the start of

subsequent antineoplastic therapy), and the third patient reported grade 1 peripheral

neuropathy during cycle 5, which resolved in 27 days. The fourth patient entered the

study with grade 1 peripheral neuropathy and experienced grade 2 peripheral

neuropathy events during cycles 1 and 2, which resolved in 6 and 9 days,

respectively.

A total of 3 (7%) patients had dose reductions due to AEs. There were 3 (7%)

discontinuations due to AEs, and 1 on-study death considered to be related to

disease progression. The 3 patients who discontinued due to AEs were all treated at

doses above the MTD and experienced DLTs. Therefore, per protocol, they

discontinued the study. Two of the 3 patients were treated at 2.1 mg/kg and one at

2.4 mg/kg. The patient who died of disease progression was a 51-year old male with

esophageal carcinoma who received TAK-264 at the MTD (1.8 mg/kg) and initially

discontinued due to a DLT of grade 4 neutropenia. A re-staging CT scan several

months later, without any further therapy, showed a response. The patient re-entered





the study 6 months after the initial discontinuation at a dose reduction of 1.5 mg/kg

TAK-264. Unfortunately, although the patient tolerated the infusion, his clinical status

declined and he died of disease progression 8 days after the second administration

of TAK-264. The treating physician assumed that his disease was the primary cause

of death. In this study there were no confirmed ATA-positive results detected in the

total number of samples.

Pharmacokinetics

The pharmacokinetics-evaluable population included all 41 patients. PK samples

were collected pre-dose, post dose and on day 2, 3, 4, 8, and 15. Increases in

exposure to TAK-264 and free MMAE were approximately proportional to dose (Fig.

2). Summary statistics for TAK-264, total antibody, and MMAE pharmacokinetic

parameters during cycle 1 for patients treated at the MTD are shown in Table 3.

Median time to maximum concentration occurred immediately after infusion for TAK-

264 and total antibody, and approximately 3 days after infusion for MMAE. Median

half-life was approximately 4 days for TAK-264 and 3 days for free MMAE.

Antitumor activity

Among 39 response-evaluable patients, one partial response was observed in a 53-

year-old white male patient with gastric adenocarcinoma who had a low GCC

expression level, had received three prior therapies, and had been treated with TAK-

264 at the MTD. This response was observed after 2 treatment cycles and sustained

for 81 days. The patient was progression-free for 121 days. In addition, stable

disease was observed in 17 response-evaluable patients (44%). One 57-year-old

white female patient with pancreatic carcinoma who had a low GCC expression level





received 12 cycles of treatment. This patient was treated at 2.1 mg/kg through cycle

3 and at 1.8 mg/kg for cycles 4-12, maintaining stable disease from the first response

assessment (unscheduled) until the end of cycle 12. A 30-year-old white male patient

with pancreatic carcinoma who had a low GCC expression level received 6 cycles of

treatment at the MTD and had a best response of stable disease, which lasted for 6

cycles. Both of these patients had previously received multiple lines of treatment,

including chemotherapy, radiotherapy, and surgery. A 51-year-old white male patient

with esophageal carcinoma who had an intermediate GCC expression level was

treated at the MTD, but experienced a DLT of febrile neutropenia during cycle 1 and

discontinued, per protocol. A follow-up scan showed a decrease in the size and

number of his lung metastases, suggesting a delayed response to treatment.

Overall, progressive disease was observed as the best response in 21 response-

evaluable patients (54%). The number of treatment cycles received by each patient

according to GCC expression level is shown in Fig. 3. GCC expression, as measured

by H-score, did not appear to correlate with treatment duration.

Median PFS for the response-evaluable population was 44 days (95% CI 39-83).

Using a Cox proportional hazard model, there was no association between GCC

expression and PFS (HR 1.002, p=0.6052). There was also no difference in PFS

when dichotomized by median GCC expression (HR 0.806, p=0.6545).

Discussion





TAK-264 appeared generally well tolerated with a manageable safety profile in

patients with advanced, GCC-positive gastrointestinal malignancies. The MTD for

TAK-264, determined using a Bayesian CRM (30-32), was 1.8 mg/kg. A total of 19

patients were treated during the dose-escalation phase in order to establish the MTD,

with 8 of these patients receiving a dose below the MTD. An advantage of the CRM

algorithm used in this study was that it allowed an accurate MTD to be determined

with a minimal number of patients treated at sub-optimal levels, while utilizing

information from all treated patients to guide the dose modifications (33). It also

allowed rapid dose escalation, which was completed over approximately 12 months,

through 7 increasing doses.

DLTs were reported in 4/19 patients in the dose-escalation phase of the study. Three

of the 4 patients with DLTs of grade 4 neutropenia were treated at doses above 1.8

mg/kg (MTD), while 1 patient who experienced grade 4 febrile neutropenia was

treated at the MTD. Of the 3 patients treated at doses above the MTD, 1 patient also

experienced grade 3 QTc prolongation. The DLTs occurred during days 10-15 of

cycle 1, approximately 7-12 days after the MMAE Tmax of approximately 3 days;

MMAE has a plasma half-life of approximately 3 days, and neutrophils have a

circulation life of approximately 5 days (34). The mechanism by which TAK-264

causes neutropenia is not yet understood, but free MMAE is likely the cause as GCC

has not been found to be expressed in the bone marrow compartment; therefore a

role for this receptor in triggering neutrophil depletion seems unlikely. In other studies

using microtubule-targeting agents such as taxanes, hematologic AEs were relatively

common (35). It appears that neutrophils may be more susceptible to this toxicity

than other bone marrow-derived cells.





Other common drug-related AEs included nausea, fatigue, decreased appetite, and

diarrhea, which are often reported in patients with gastrointestinal malignancies.

Interestingly, patients receiving brentuximab vedotin, who did not suffer from

gastrointestinal malignancies, experienced a spectrum of AEs which included

nausea, fatigue, neutropenia, diarrhea, and pyrexia (3-6), which are to some extent

comparable with the AEs reported for TAK-264. The ADCs polatuzumab vedotin and

pinatuzumab vedotin were also associated with similar toxicities, including

neutropenia, peripheral neuropathy, and diarrhea (36). These ADCs incorporate the

same cytotoxic agent as TAK-264, MMAE; it is assumed that the cytotoxicity of

MMAE is related to its ability to inhibit cell division by binding tubulin, which arrests

the target cell in the G2/M stage of the cell cycle and results in apoptosis (3). In

contrast to these studies of other ADCs, peripheral neuropathy was not a commonly

reported AE with TAK-264, occurring at grade 1 or 2 in 10% of patients. It should be

noted, however, that the majority of patients participating in this study received 2

cycles of therapy. No ATA-positive samples were detected during this study and no

infusion site reactions occurred; this was consistent with expectations as TAK-264

utilizes a fully humanized antibody.

Pharmacokinetic data, including a median half-life of approximately 4 days for TAK-

264, suggest that steady-state pharmacokinetics for both the ADC and MMAE

occurred by approximately 21 days, supporting the dosing schedule employed.

Concentration-time profiles showed that TAK-264, total antibody, and MMAE

concentrations peaked following infusion then decreased to low pre-infusion





concentrations prior to the next cycle, suggesting a lack of substantial accumulation

in plasma or serum.

Preliminary response data indicated antitumor activity in this population, especially in

non-mCRC patients. The efficacy of ADCs using MMAE in gastrointestinal

malignancies is unproven, although they have been used successfully in Hodgkin

lymphoma and anaplastic large cell lymphoma (3-6). Microtubule-disrupting agents

have been shown to have varying effects in different tumor cell lines and may work

by different mechanisms depending on cell type (26, 37). In this study, duration of

disease stabilization and antitumor activity did not appear to correlate with GCC

expression level. In total, 52% of patients in the expansion cohort had high levels of

GCC expression. It is intriguing that 4 patients for whom there were early signals of

potential clinical benefit, all non-mCRC, had low levels of GCC expression relative to

the overall population. Further investigation is needed to assess the role of GCC

expression in clinical outcomes with TAK-264.

Several factors are involved in the ADC treatment response including the toxin

selection, binding affinity of the antibody, delivering the ADC and cytotoxic agent to

the tumor cells, and linker technology. While the data from this study are preliminary,

there was less than expected clinical efficacy. The selection of GCC as a target

remains experimental and more data are needed to better assess the potential of

targeting GCC in order to develop a successful treatment strategy for gastrointestinal

malignancies. Various characteristics need to be better defined (such as

heterogeneity and specificity of target expression, internalization rate, intracellular

trafficking) to interpret the limitation of clinical efficacy.





Tissues tested for GCC expression were mostly archived tissues, including some

from primary tumor during initial resection.

The relationship between primary and metastatic sites, and the effects of multiple

lines of therapy, on GCC staining characteristics is currently unknown. Further insight

into the optimal design characteristics of effective ADCs will be best gained as

additional clinical data become available. Several ADCs are currently in clinical

development, and TAK-264 is undergoing further investigation in 2 single-arm, phase

2 clinical studies in patients with gastric (NCT 02202759) and pancreatic cancer

(NCT02202785).

In conclusion, TAK-264 is a first-in-class ADC with a novel target, the GCC receptor.

The data from this first-in-human phase 1 study suggest that TAK-264 has a

manageable safety profile at the MTD of 1.8 mg/kg. Preliminary data suggest

antitumour activity and early signs of clinical benefit in patients with pancreatic,

esophageal, and gastric carcinoma. Combination strategies with active

chemotherapy agents could be considered in the future. Antitumor activity did not

appear to correlate with GCC expression levels. Evolving clinical data from the

ongoing phase 2 clinical trials of TAK-264 will provide critical insight into the design

of next generation ADCs.





Acknowledgments

The authors acknowledge Helen Johns of FireKite, an Ashfield company, part of

UDG Healthcare plc, for writing support during the development of this manuscript,

which was funded by Millennium Pharmaceuticals, Inc. ADC technology was licensed

from Seattle Genetics, Inc.

Author contribution statement:

KA, TK, CC, JEF, DPR, TW, AAF, WM, and JR designed and performed the

research. KA, CC, JEF, DPR, WM, and JR collected data. KA, TK, CC, JEF, DPR,

JJ, TW, AAF, WM, and JR analyzed and interpreted data (pharmacokinetics: AAF).

JJ performed statistical analysis. KA, TK, JJ, TW, and AAF wrote the draft

manuscript. All authors contributed to the writing and reviewing of the manuscript,

and approved the final manuscript for submission.





References

1. Leal M, Sapra P, Hurvitz SA, Senter P, Wahl A, Schutten M, et al. Antibody-drug

conjugates: an emerging modality for the treatment of cancer. Ann N Y Acad Sci

2014; 1321:41-54.

2. Teicher BA, Chari RV. Antibody conjugate therapeutics: challenges and potential.

Clin Cancer Res 2011; 17:6389-6397.

3. Fanale MA, Forero-Torres A, Rosenblatt JD, Advani RH, Franklin AR, Kennedy

DA, et al. A phase I weekly dosing study of brentuximab vedotin in patients with

relapsed/refractory CD30-positive hematologic malignancies. Clin Cancer Res

2012; 18:248-255.

4. Pro B, Advani R, Brice P, Bartlett NL, Rosenblatt JD, Illidge T, et al. Brentuximab

vedotin (SGN-35) in patients with relapsed or refractory systemic anaplastic large-

cell lymphoma: results of a phase II study. J Clin Oncol 2012; 30:2190-2196.

5. Younes A, Bartlett NL, Leonard JP, Kennedy DA, Lynch CM, Sievers EL, et al.

Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J

Med 2010; 363:1812-1821.

6. Younes A, Gopal AK, Smith SE, Ansell SM, Rosenblatt JD, Savage KJ, et al.

Results of a pivotal phase II study of brentuximab vedotin for patients with

relapsed or refractory Hodgkin's lymphoma. J Clin Oncol 2012; 30:2183-2189.

7. Lambert JM, Chari RV. Ado-trastuzumab Emtansine (T-DM1): an antibody-drug

conjugate (ADC) for HER2-positive breast cancer. J Med Chem 2014; 57:6949-

6964.

8. FDA approval: Brentuximab Vedotin.

http://www.fda.gov/AboutFDA/CentersOffices/OfficeofMedicalProductsandTobacc

o/CDER/ucm268969.htm. Accessed 11 March 2015.





9. EMA. Adcetris Summary of Product Characteristics.

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-

_Product_Information/human/002455/WC500135055.pdf. Accessed 11 March

2015.

10. Palanca-Wessels MC, Press OW. Advances in the treatment of hematologic

malignancies using immunoconjugates. Blood 2014; 123:2293-2301.

11. Sadeghi S, Olevsky O, Hurvitz SA. Profiling and targeting HER2-positive breast

cancer using trastuzumab emtansine. Pharmgenomics Pers Med 2014; 7:329-

338.

12. LoRusso PM, Weiss D, Guardino E, Girish S, Sliwkowski MX. Trastuzumab

emtansine: a unique antibody-drug conjugate in development for human

epidermal growth factor receptor 2-positive cancer. Clin Cancer Res 2011;

17:6437-6447.

13. FDA approval: Trastuzumab Emtansine.

http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm340913.htm.

Accessed 11 March 2015.

14. EMA. Kadcyla Summary of Product Characteristics.

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-

_Product_Information/human/002389/WC500158593.pdf. Accessed 11 March

2015.

15. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015;

65:5-29.

16. Carrithers SL, Parkinson SJ, Goldstein SD, Park PK, Urbanski RW, Waldman SA.

Escherichia coli heat-stable enterotoxin receptors. A novel marker for colorectal

tumors. Dis Colon Rectum 1996; 39:171-181.





17. Hyslop T, Waldman SA. Guanylyl cyclase C as a biomarker in colorectal cancer.

Biomark Med 2013; 7:159-167.

18. Carrithers SL, Parkinson SJ, Goldstein S, Park P, Robertson DC, Waldman SA.

Escherichia coli heat-stable toxin receptors in human colonic tumors.

Gastroenterology 1994; 107:1653-1661.

19. John M, Wiedenmann B, Kruhoffer M, Adermann K, Ankorina-Stark I, Schlatter E,

et al. Guanylin stimulates regulated secretion from human neuroendocrine

pancreatic cells. Gastroenterology 1998; 114:791-797.

20. Kloeters O, Friess H, Giese N, Buechler MW, Cetin Y, Kulaksiz H. Uroguanylin

inhibits proliferation of pancreatic cancer cells. Scand J Gastroenterol 2008;

43:447-455.

21. Park J, Schulz S, Haaf J, Kairys JC, Waldman SA. Ectopic expression of guanylyl

cyclase C in adenocarcinomas of the esophagus and stomach. Cancer Epidemiol

Biomarkers Prev 2002; 11:739-744.

22. Buc E, Vartanian MD, Darcha C, Dechelotte P, Pezet D. Guanylyl cyclase C as a

reliable immunohistochemical marker and its ligand Escherichia coli heat-stable

enterotoxin as a potential protein-delivering vehicle for colorectal cancer cells. Eur

J Cancer 2005; 41:1618-1627.

23. Camci C, Sahin A, Sevinc A, Kalender ME, Oztuzcu S, Sever ON, et al.

Peripheral blood guanylyl cyclase c (GCC) expressions are associated with

prognostic parameters and response to therapy in colorectal cancer patients.

Tumour Biol 2011; 32:1265-1270.

24. Winn B, Tavares R, Matoso A, Noble L, Fanion J, Waldman SA, et al. Expression

of the intestinal biomarkers Guanylyl cyclase C and CDX2 in poorly differentiated

colorectal carcinomas. Hum Pathol 2010; 41:123-128.





25. Almenoff JS, Williams SI, Scheving LA, Judd AK, Schoolnik GK. Ligand-based

histochemical localization and capture of cells expressing heat-stable enterotoxin

receptors. Mol Microbiol 1993; 8:865-873.

26. Guarino A, Cohen MB, Overmann G, Thompson MR, Giannella RA. Binding of E.

coli heat-stable enterotoxin to rat intestinal brush borders and to basolateral

membranes. Dig Dis Sci 1987; 32:1017-1026.

27. Veiby P, Zhang J, Yang J, McDonald A, Fasanmade A, Wyant T, et al. The

Investigational Drug MLN0264 First-in-human, First in Class ADC Targeting GCC:

Phase I Dose-escalation Study and Supportive Scientific Rationale. Eur.J.Cancer

2012; 48:iv36

28. Zhang J, Gallery M, Wyant T, Stringer B, Manfredi M, Danaee H, et al. MLN0264,

an investigational, first-in-class antibody-drug conjugate (ADC) targeting guanylyl

cyclase C (GCC), demonstrates antitumor activity alone and in combination with

gemcitabine in human pancreatic cancer xenograft models expressing GCC. Mol

Cancer Ther 2013; 12(11 Suppl):PR12

29. Hirsch FR, Varella-Garcia M, Bunn PA, Jr., Di Maria MV, Veve R, Bremmes RM,

et al. Epidermal growth factor receptor in non-small-cell lung carcinomas:

correlation between gene copy number and protein expression and impact on

prognosis. J Clin Oncol 2003; 21:3798-3807.

30. Normolle D, Lawrence T. Designing dose-escalation trials with late-onset

toxicities using the time-to-event continual reassessment method. J Clin Oncol

2006; 24:4426-4433.

31. O'Quigley J, Shen LZ. Continual reassessment method: a likelihood approach.

Biometrics 1996; 52:673-684.





32. He W, Liu J, Binkowitz B, Quan H. A model-based approach in the estimation of

the maximum tolerated dose in phase I cancer clinical trials. Stat Med 2006;

25:2027-2042.

33. Le TC, Lee JJ, Siu LL. Dose escalation methods in phase I cancer clinical trials. J

Natl Cancer Inst 2009; 101:708-720.

34. Pillay J, den B, I, Vrisekoop N, Kwast LM, de Boer RJ, Borghans JA, et al. In vivo

labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days. Blood 2010;

116:625-627.

35. Klute K, Nackos E, Tasaki S, Nguyen DP, Bander NH, Tagawa ST. Microtubule

inhibitor-based antibody-drug conjugates for cancer therapy. Onco Targets Ther

2014; 7:2227-2236.

36. Morschhauser F, Flinn IW, Advani R, Diefenbach CS, Kolibaba K, Press OW, et

al. Updated Results of a Phase II Randomized Study (ROMULUS) of

Polatuzumab Vedotin or Pinatuzumab Vedotin Plus Rituximab in Patients with

Relapsed/Refractory Non-Hodgkin Lymphoma. Blood (ASH abstracts) 2014; 124.

37. Gascoigne KE, Taylor SS. How do anti-mitotic drugs kill cancer cells? J Cell Sci

2009; 122:2579-2585.





TABLES

Table 1. Patient demographics and baseline disease characteristics.

All patients

(n=41)

Median age, years (range) 60 (30–78)

Male, n (%) 27 (66)

White, n (%) 39 (95)

ECOG performance status, n (%)

Class 0 16 (39)

Class I 25 (61)

Disease stage, n (%)

<IV 4 (10)

IV 36 (88)

Not available 1 (2)

Primary diagnosis, n (%)

CRC 35 (85)

Pancreatic cancer 2 (5)

Esophageal cancer 2 (5)

Gastric carcinoma 1 (2)

Other cancer (adenocarcinoma) 1 (2)

Median combined GCC H-score (range) 350 (10–550)

Median time from diagnosis, months (range) 39.0 (7.4–116.6)

Median number of prior therapies, n (range) 6 (1–11)

Patients with >3 prior lines of chemotherapy, n (%) 28 (68)





Table 2. AEs experienced by ≥10% of patients by severity

AE, n (%)

Any-grade

(N=41)

Grade 1

(N=41)

Grade 2

(N=41)

Grade 3 and 4

(N=41)

Any AE 41 (100) 3 (7) 12 (29) 26 (63)

Nausea 21 (51) 16 (39) 5 (12) 0

Decreased appetite 20 (49) 12 (29) 8 (20) 0

Fatigue 17 (41) 8 (20) 8 (20) 1 (2)

Diarrhea 16 (39) 10 (24) 3 (7) 3 (7)

Anemia 14 (34) 4 (10) 6 (15) 4 (10)

Pyrexia 13 (32) 12 (29) 1 (2) 0

Asthenia 12 (29) 5 (12) 6 (15) 1 (2)

Neutropenia 11 (27) 0 2 (5) 9 (22)*

Alopecia 11 (27) 7 (17) 4 (10) 0

Constipation 10 (24) 3 (7) 7 (17) 0

Vomiting 10 (24) 3 (7) 7 (17) 0

Aspartate aminotransferase

increased 9 (22) 9 (22) 0 0

Abdominal pain 9 (22) 5 (12) 3 (7) 1 (2)

Hypokalemia 9 (22) 5 (12) 0 4 (10)

Alanine aminotransferase

increased 7 (17) 7 (17) 0 0

Dehydration 7 (17) 3 (7) 3 (7) 1 (2)

Arthralgia 7 (17) 4 (10) 3 (7) 0

Hyponatremia 6 (15) 4 (10) 0 2 (5)

Thrombocytopenia 6 (15) 5 (12) 1 (2) 0





Musculoskeletal pain 6 (15) 3 (7) 3 (7) 0

Hyperglycemia 5 (12) 3 (7) 1 (2) 1 (2)

Gamma-glutamyltransferase

increased 5 (12) 1 (2) 2 (5) 2 (5)

Back pain 5 (12) 3 (7) 2 (5) 0

Anxiety 5 (12) 4 (10) 1 (2) 0

Insomnia 5 (12) 1 (2) 4 (10) 0

Dyspnea 5 (12) 2 (5) 3 (7) 0

Small intestinal obstruction 4 (10) 0 2 (5) 2 (5)

Hypophosphatemia 4 (10) 1 (2) 2 (5) 1 (2)

Blood alkaline phosphatase

increased 4 (10) 2 (5) 2 (5) 0

Electrocardiogram QT

prolonged 4 (10) 2 (5) 0 2 (5)

Weight decreased 4 (10) 4 (10) 0 0

Leukopenia 4 (10) 1 (2) 3 (7) 0

Headache 4 (10) 4 (10) 0 0

Cough 4 (10) 4 (10) 0 0

*5 (12%) grade 4





Table 3. Summary of serum TAK-264 (anti-GCC human monoclonal antibody

conjugated to MMAE), serum total antibody (anti-GCC human monoclonal antibody

conjugated to MMAE and unconjugated anti-GCC human monoclonal antibody), and

plasma MMAE pharmacokinetic parameters following cycle 1 administration of TAK-

264 at 1.8 mg/kg (MTD)

TAK-264

(N=28)

Total antibody

(N=28)

MMAE

(N=28)

Geometric mean Cmax, μg/mL 48.6 49.7 0.008

Geometric mean AUCinf,

day*μg/mL 68.9 152.1 0.053

Median Tmax, day 0.0278 0.0278 2.97

Median plasma terminal T1/2,

day 4.40 6.17 3.11

Cmax, maximum observed concentration; AUCinf, area under the concentration time

curve from time 0 to infinity; Tmax, time of Cmax; T1/2, half-life





Figure Legends

Fig 1. Schema for the CRM algorithm used for the dose-escalation portion of the

study. (CRM, continual reassessment method; DLT, dose-limiting toxicity; Mid-high,

midpoint between current dose and next dose; Mid-low, midpoint between previous dose

and current dose; MTD, maximum tolerated dose; PMTD, predicted maximum tolerated

dose. *Dosing intervals were pre-specified and not determined by the CRM algorithm.)

Fig 2. Dose-proportionality evaluation, showing cycle 1 AUCinf versus TAK-264 dose

for A) TAK-264, B) total antibody, and C) MMAE.

Fig 3. Number of TAK-264 cycles received by each patient, by GCC expression

level.




Published OnlineFirst May 13, 2016.Clin Cancer Res Khaldoun Almhanna, Thea Kalebic, Cristina Cruz, et al. with advanced gastrointestinal malignanciesantibody-drug conjugate TAK-264 (MLN0264) in adult patients Phase I study of the investigational anti-guanylyl cyclase

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