Purpose:

Patients with metastatic colorectal cancer refractory to chemotherapy have limited treatment options. Ensituximab (NEO-102) is a novel chimeric mAb targeting a variant of MUC5AC with specificity to colorectal cancer.

Patients and Methods:

Single-arm, phase II trial assessed the efficacy and safety of ensituximab in patients with advanced, refractory cancer who expressed MUC5AC antigen in tumor tissue. Ensituximab was administered intravenously every 2 weeks with 3 mg/kg as recommended phase II dose (RP2D). A minimum sample size of 43 patients was required on the basis of the assumption that ensituximab would improve median overall survival (OS) by 7 months using a one-sided significance level of 10% and 80% power. Written informed consent was obtained from all patients.

Results:

Sixty-three patients with advanced, refractory colorectal cancer were enrolled and 53 subjects were treated in phase II arm. Median age was 58 years and 46% of the patients were female. Among 57 evaluable patients, median OS was 6.8 months. No responses were observed, and stable disease was achieved in 21% of the patients. The most common treatment-related adverse events (AE) at RP2D included fatigue (38%), anemia (30%), nausea (15%), vomiting (11%), increased bilirubin (9%), constipation (8%), decreased appetite (6%), and diarrhea (6%). Serious AEs at least possibly related to ensituximab occurred in 4 patients and included anemia, nausea, increased bilirubin, and hypoxia. No patients discontinued treatment due to drug-related AEs.

Conclusions:

Ensituximab was well tolerated and demonstrated modest antitumor activity in patients with heavily pretreated refractory colorectal cancer.

Translational Relevance

Patients with chemotherapy refractory, advanced colorectal cancer have poor prognosis with limited systemic therapeutic options. MUC5AC is overexpressed in malignant colorectal cancer tissue compared with normal colon mucosa. Ensituximab is a novel chimeric mAb that targets MUC5AC. The antitumor activity is primarily through antibody-dependent cellular cytotoxicity. In this trial, ensituximab demonstrated promising antitumor activity in patients with chemotherapy refractory colorectal cancer with median overall survival (OS) comparable with available therapeutic agents including regorafenib and TAS-102. The study drug was well tolerated with manageable adverse events. No correlation between MUC5AC expression level in tumor tissue and OS was observed. The role of ensituximab could be further explored in future trials utilizing combination therapies.

Despite significant improvement in the survival of patients with colorectal cancer over the past decade, almost all patients with metastatic disease will succumb to the disease, resulting in a significant number of deaths every year (1). Modest progress has been made recently with approval of regorafenib and TAS-102 for advanced, refractory colorectal cancer based on the results of large phase III studies (2, 3). However, median overall survival (OS) for patients with chemotherapy refractory colorectal cancer remains less than a year (4). Therefore, there is an urgent need for development of additional novel therapeutic options for patients with colorectal cancer who have disease progression through standard therapies.

Ensituximab (NEO-102) is a novel chimeric mAb targeting a glycosylated variant of MUC5AC with specificity to colorectal and pancreatic cancer. This mAb was uniquely developed from a colon cancer tumor-associated antigen vaccine preparation tested in Hollinshead's laboratory and an early-phase clinical trial (5). Its mechanism of action is primarily through antibody-dependent cellular cytotoxicity (ADCC; ref. 6). The development of the drug ensituximab has been published previously (6).

MUC5AC is a mucin which contains cysteine regions and participates in the formation of extracellular gels (7). It is typically expressed in the stomach and respiratory tract (8). However, MUC5AC can potentially be an important biomarker as the protein is expressed in the fetal and precancerous colon mucosa but to a lesser extent in normal colon (9). Overexpression of MUC5AC antigen has been associated with pancreatic cancer and colorectal cancer (10). Interestingly, MUC5AC is expressed in colon cancer in an aberrantly glycosylated form and ensituximab is able to discriminate the aberrantly glycosylated form from native MUC5AC (6). Preclinical data demonstrates ensituximab targets only MUC5AC-positive tumors and spares MUC5AC antigen in nonmalignant tissue (6).

An earlier phase I study established the MTD or recommended phase II dose (RP2D) of ensituximab at 3.0 mg/kg i.v. every 2 weeks with encouraging early signs of clinical activity (11). Although there were no partial responses, median OS of patients heavily pretreated with advanced colorectal cancer was 12 months (n = 15). Furthermore, the drug was very well tolerated with few grade 3 or 4 adverse events (AE). In the study, an IHC-based companion diagnostic assay was developed to ensure that patients' tumors expressed the NEO-102 target, which correlated preclinically to antitumor responses. The study screened 75 patients and NEO-102 antigen expression was seen in 47% of the colon cancer specimens. On the basis of encouraging preliminary results of the phase I trial, which included 15 patients with colon cancer, an expansion cohort in the original phase 1 clinical trial was added in an amendment to evaluate efficacy (OS) in patients with refractory colorectal cancer who had progressive disease during or relapse after at least two lines of standard regimens. Thus, we now report the results of the phase II study of ensituximab in patients with refractory colorectal cancer whose tumors tested at least ≥20% positive for NEO-102 target antigen by IHC.

Patient population

Patients at least 18 years of age, diagnosed with advanced refractory colorectal cancer for whom standard treatment was no longer effective or did not offer curative or life-prolonging potential, were eligible to participate in this study. Patients were required to have recurrent or progressive disease after at least two standard chemotherapy regimens, and tumor sections were required to stain ≥20% positive for NEO-102 antigen target as determined by the department of pathology at a central laboratory or in a Clinical Laboratory Improvement Amendments (CLIA) laboratory of a participating site. Patients were required to have measurable disease per RECISTv1.1, adequate organ function, Karnofsky performance score (PS) of ≥70%, and to have completed chemotherapy ≥2 weeks and immunotherapy ≥4 weeks prior to enrollment. Patients with uncontrolled brain metastases or uncontrolled concomitant illness were excluded. Medications associated with prolongation of QT/QTc interval were not allowed. Written informed consent was obtained from all patients.

Study design and objectives

The primary objective of this multi-center phase II study was to assess safety and efficacy of ensituximab in patients with refractory colorectal cancer. All patients provided written informed consent prior to participation. Specifically, all patients signed IHC prescreening consent and only signed protocol treatment informed consent if their tumor tissue stained ≥20% positive for NEO-102 antigen target. The study was a single-arm study with a primary endpoint of OS, which was compared with historical control (3). The secondary endpoints included overall response rate as per RECIST criteria 1.1, safety and tolerability, and exploration of select immunologic correlates associated with administration of ensituximab.

Patients who received at least two doses of ensituximab were considered evaluable for response in the study. Patients continued the study drug until disease progression or development of unacceptable drug-related AEs. The study protocol was approved by the institutional review board at participating sites and was conducted in accordance with the Declaration of Helsinki and other Good Practice Guidelines.

Investigational regimen

In the phase II part, all patients with colorectal cancer received ensituximab at 3.0 mg/kg i.v. every 2 weeks. Six weeks of dosing (four doses of ensituximab) plus 2 weeks for evaluation was considered one course (57 days). At the conclusion of the first course if restaging scans showed stable disease (SD) or clinical response per RECIST1.1, patients without unacceptable toxicity were allowed to proceed with additional courses of ensituximab.

Dose reductions were mandated for grade 3 or higher toxicities related to the study drug. The study drug could be resumed at a lower dose once the toxicity resolved to grade 1 or baseline prior to the next scheduled dose of ensituximab. If toxicity did not resolve to grade 1 or baseline parameters within 14 days, ensituximab was discontinued. Dose reescalation was not allowed.

Assessments

Determination of NEO-102 target

An IHC assay was designed and validated to assess the expression of NEO-102 on tumor tissue as described previously (11). Laboratories were trained to perform the IHC testing for NEO-102 antigen on prospective subject's archived unstained tumor slides or tumor block. Clinical sites with CAP/CLIA laboratories capable of conducting the IHC included Johns Hopkins University Hospital (Baltimore, MD) and Duke University (Durham, NC). Clinical sites who did not have the capability or chose to use the Central Laboratory sent slides or tumor block to University of Texas Southwestern (Dallas, TX) Medical Center.

Assessment of response

Patients were evaluated for response serologically with carcinoembryonic antigen (CEA) levels and radiographically with CT of the chest, abdomen, and pelvis and/or MRI at the completion of every course (57 days). Response was determined locally by RECIST1.1 and patients continued courses of ensituximab until disease progression, unacceptable toxicity, withdrawal of consent, or physician's decision to discontinue. Patients were monitored for safety during and after dosing. Safety evaluations included vital signs, physical exam, performance status evaluation, complete blood count, blood chemistries, coagulation studies, and urinalyses. AEs were assessed according to the NCI Common Terminology Criteria for Adverse Events version 4.03.

Correlative assays

Human antibody to chimeric mAb/human anti-mouse antibody

To ensure that human anti-mouse antibodies were not developed against ensituximab, serum of patients receiving the investigational regimen was tested at BioReliance. During cycle 1 only, blood samples (5 mL) were drawn into red-top tubes for HACA (human antibody to chimeric mAb)/HAMA (human anti-mouse antibody) in the first 10 subjects in this study. HACA/HAMA was measured by ELISA using a commercially available Assay Kit (BioReliance) qualified for use in this application. Samples were drawn on days 1, 4, 15, and 57.

Cytokines

Blood samples (5 mL) were drawn for cytokine analysis during cycle 1 in 10-mL red-top tubes (combined with HACA blood draw in same tube) in the first 10 subjects enrolled in the phase II component of this study to evaluate the toxicity risk of cytokine release syndrome (CRS). Samples were drawn on days 1, 4, 15, and 57. Serum was processed via centrifugation within 2 hours of blood draw and samples were frozen and stored at −20°C until assays were performed. Th1 and Th2 cytokines (IL1b, IL2, IL6, IL8, IL10, IL12p70, GM-CSF, TNFα, and IFNγ; ref. 12) were evaluated using the human proinflammatory 9-plex ultra-sensitive kit, measuring nine specific cytokines in a 96-well MULTI-ARRAY or MULTI-SPOT plate, using a sandwich immunoassay format at BioReliance in accordance with FDA Good Laboratory Practice regulations (21 CFR Part 58).

Other research studies

Blood samples were drawn during cycle 1 on days 1, 15, and 57 on all patients enrolled in this study. Blood was collected into two 8-mL blue-top (sodium citrate) and one 10-mL red-top tube and stored at room temperature until processed to recover serum, plasma, and peripheral blood mononuclear cells (PBMC). The recovered specimens were aliquoted and cryopreserved under appropriate conditions according to standard operating procedures. A selection of the research studies planned to be conducted utilizing both the subjects' serum as well as PBMCs included anti-idiotype assay, natural killer (NK) cell assay, ADCC, and regulatory T-cell assay. The following materials and methods were used to complete these assays.

Cell lines and culture:

Human pancreatic carcinoma cell lines ASPC-1 and CFPAC-1 were obtained from the ATCC. Cancer cells were maintained in RPMI1640 (Corning Life Science). Culture medium was supplemented with 10% USA-sourced and heat-inactivated HyClone FBS defined (GE Healthcare Life Sciences), 100 U/mL penicillin, and 100 μg/mL streptomycin (Corning Life Science).

FcγRIIIa (CD16) genotyping:

DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen) and stored at −80°C until use. The polymorphism of CD16 was determined by performing allele-specific droplet digital PCR using the TaqMan array for CD16 (rs396991; Life Technologies), as described previously (13).

Radioactive in vitro ADCC assay:

Patient PBMC effectors were thawed the evening prior to conducting the assay and allowed to rest overnight in RPMI1640 medium containing l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Corning Life Science), and 10% human AB serum (Gemini Bio-Products). On the day of the assay, human pancreatic cancer cell line ASPC-1 was used as target in a 4-hour 111In-release assay. Cancer cell lines were labeled with 20 μCi 111In-oxyquinoline (GE Healthcare) at 37°C for 20 minutes and then seeded at 3,000 cells/well in 96-well round-bottom culture plates. Cancer cells were then treated with 10 μg/mL of human IgG1 isotype control antibody (Thermo Fisher Scientific) or ensituximab and then PBMCs were added as effectors at effector-to-target (E:T) ratios of 50:1 and 25:1.

Assays listed above were performed for 4 hours following a previously described procedure (13) and specific ADCC lysis was determined using the following equation:

formula
Nonradioactive in vitro ADCC assay:

Purified NK cells were obtained from human donor PBMCs using the EasySep Human NK Cell Isolation Kit (Stemcell Technologies) according to the manufacturer's protocol. Purified NK cells were exposed to vehicle control (RPMI1640 medium supplemented with l-glutamine, 10% FBS, and antibiotics) or IL15 superagonist (25 ng/mL) for 48 hours prior to use as effectors. On the day of the assay, CFPAC-1 cells were used as target cells following a nonradioactive ADCC assay procedure described previously (14).

Target cells were treated with human IgG1 isotype control antibody (Thermo Fisher Scientific) or ensituximab (10 μg/mL), and then purified NK cells were added at E:T ratios of 6.25:1 and 12.5:1.

After 4-hour incubation at 37°C, ADCC activity was analyzed using the Celigo Imaging Cytometer (Nexcelom Bioscence LLC). Specific ADCC lysis was calculated as described previously (15).

Statistical analysis

Grothey and colleagues (3) in the CORRECT study demonstrated that the median OS was 5.0 months in subjects with previously treated metastatic colorectal cancer randomized to placebo. In this phase II design, subjects were assumed to be recruited for a period of 24 months and followed for survival for 12 months. To determine an improvement over 5.0 month survival, estimated sample size was 43 subjects, assuming that treatment with ensituximab would improve the median OS by 40% (7.0 months) using a one-sided significance level of 10% and 80% power for this single-arm phase II trial. The sample size was estimated using the software: http://www.swogstat.org/stat/public/one_survival.htm.

Data from the ADCC assay were analyzed using GraphPad Prism (GraphPad Software). Comparisons between two groups were conducted by t test, and P < 0.05 was considered statistically significant.

Patient disposition, demographics, and baseline characteristics

Out of 238 patients with colorectal cancer that were screened by IHC, 142 (60%) patients had tumor that was positive for the NEO-102 target antigen. Sixty-three patients with advanced, metastatic colorectal cancer were enrolled in the phase I and phase II portions of this study.

The demographics are depicted in Table 1. Of 63 patients (all dose levels) with metastatic colorectal cancer, the median age was 58 years (range 32–79) with 46 (73%) patients younger than 65 years of age and 34 patients (54%) male. The ethnic background of study population was predominantly White (76%) followed by African American (19%), Hispanic or Latino (6%), Asian (3%), and unknown (2%). Eleven (17%) patients had Karnofsky PS of 100, 13 (21%) of 90, 28 (44%) of 80, and 11 (17%) of 70, which was the minimum PS for eligibility. The mean time from initial diagnosis to study entry was 3.79 years (±2.22 years) with a range of 1.0–9.8 years. All patients had undergone prior cancer-directed therapy, from two to nine prior therapies (median: 3).

Table 1.

Demographics for subjects with colorectal cancer receiving ensituximab.

Results
Demographic Age (years of age): mean (SD)3 mg/kg (N = 53)All subjects (1, 2, 3, and 4 mg/kg; N = 63)
Age 
 Median (min/max) 58 (32/79) 58 (32/79) 
 <65 46 (73%) 46 (73%) 
 ≥65 17 (27%) 17 (27%) 
Gender 
 Male 28 (53%) 34 (54%) 
 Female 25 (47%) 29 (46%) 
Ethnicity 
 Hispanic or Latino 4 (8%) 4 (6%) 
 Not Hispanic or Latino 49 (92%) 59 (94%) 
Race 
 Asian 2 (4%) 2 (3%) 
 Black or African American 11 (21%) 12 (19%) 
 White 39 (74%) 48 (76%) 
 Unknown 1 (2%) 1 (2%) 
Weight: mean (SD) 79 (21.5) 80 (21.2) 
 Median (min/max) 78 (43/144) 79 (43/144) 
Karnofsky performance status 
 100% 9 (17%) 11 (17%) 
 90% 12 (23%) 13 (21%) 
 80% 23 (43%) 28 (44%) 
 70% 9 (17%) 11 (17%) 
 ≤60%  
 Mean (SD) 84.0 (9.68) 83.8 (9.7) 
 Median (min/max) 80 (70–100) 80 (70–100) 
Number of prior lines of therapy 
 Median (min/max) 3 (2/9) 3 (2/9) 
Results
Demographic Age (years of age): mean (SD)3 mg/kg (N = 53)All subjects (1, 2, 3, and 4 mg/kg; N = 63)
Age 
 Median (min/max) 58 (32/79) 58 (32/79) 
 <65 46 (73%) 46 (73%) 
 ≥65 17 (27%) 17 (27%) 
Gender 
 Male 28 (53%) 34 (54%) 
 Female 25 (47%) 29 (46%) 
Ethnicity 
 Hispanic or Latino 4 (8%) 4 (6%) 
 Not Hispanic or Latino 49 (92%) 59 (94%) 
Race 
 Asian 2 (4%) 2 (3%) 
 Black or African American 11 (21%) 12 (19%) 
 White 39 (74%) 48 (76%) 
 Unknown 1 (2%) 1 (2%) 
Weight: mean (SD) 79 (21.5) 80 (21.2) 
 Median (min/max) 78 (43/144) 79 (43/144) 
Karnofsky performance status 
 100% 9 (17%) 11 (17%) 
 90% 12 (23%) 13 (21%) 
 80% 23 (43%) 28 (44%) 
 70% 9 (17%) 11 (17%) 
 ≤60%  
 Mean (SD) 84.0 (9.68) 83.8 (9.7) 
 Median (min/max) 80 (70–100) 80 (70–100) 
Number of prior lines of therapy 
 Median (min/max) 3 (2/9) 3 (2/9) 

Three (5%) patients withdrew from the study therapy, 3 (5%) patients were removed by the investigator, 2 (3%) did not meet criteria for further therapy, 2 (3%) developed dose-limiting toxicity, 1 (2%) patient died (unrelated to study therapy), and 3 (5%) came off study for other reasons (intercurrent illness). The remaining patients were removed from the study therapy due to progression of disease. Fifty-seven (90%) were evaluable for the primary endpoint (OS).

Efficacy

The primary efficacy evaluation of OS was conducted on all patients who received two or more infusions of NEO-102. Fifty-seven patients with metastatic colorectal cancer were evaluable for OS (all dose levels; Table 2). The median OS was 6.8 months [95% confidence interval (CI), 5.4–8.0 months; Fig. 1] which is significantly larger than the OS of 5.0 months of the historical control (P = 0.007). Univariate Cox regression analyses show that none of the risk factors (CEA, the number of prior therapies, and the number of doses) were significantly associated with OS. The proportion of patients who had SD was 21.5% (12/57) with 95% CI of 11.4%–33.9%. Evaluating only patients who received the RP2D of 3 mg/kg (n = 48), the median OS was 6.4 months (95% CI, 5.2–8.0 months). Thirty-one evaluable patients (65%) were alive at 6 months, and 16 patients (28%) were alive at 1 year, while 3 patients lived beyond 2 years.

Table 2.

Evaluable patients with metastatic colorectal cancer.

1.5 mg/kg2 mg/kg3 mg/kg4 mg/kgAll subjects
Variable(n = 3)(n = 1)(n = 48)(n = 5)(n = 57)
Median survival (months) 12.2 1.4 6.4 7.5 6.8 
95% CI for the median survival time 11.4–26.6 — 5.2–8.0 1.3–23.7 5.4–8.0 
1.5 mg/kg2 mg/kg3 mg/kg4 mg/kgAll subjects
Variable(n = 3)(n = 1)(n = 48)(n = 5)(n = 57)
Median survival (months) 12.2 1.4 6.4 7.5 6.8 
95% CI for the median survival time 11.4–26.6 — 5.2–8.0 1.3–23.7 5.4–8.0 
Figure 1.

OS of patients with metastatic colorectal cancer treated with ensituximab at different dose levels.

Figure 1.

OS of patients with metastatic colorectal cancer treated with ensituximab at different dose levels.

Close modal

Of the 57 patients with metastatic colorectal cancer at all dose levels who were evaluable for response, 12 (21%) achieved SD at the end of the first course (C1D57). There were no clinical responses (complete or partial responses). Out of these 57 patients, 49 patients were evaluable for progression-free survival (data available on date of progressive disease), and 8 patients were removed from the study prior to development of progressive disease (taken off study for toxicity, patient withdrawal of consent, and investigator discretion). The median progression-free survival of these 49 patients is 56 days, a mean progression-free survival of 70.4 days with a 95% CI of 60.1–80.6 days.

There was no statistically significant correlation between OS and the level of NEO-102 IHC antigen expression.

Safety data

The most common treatment-related AEs in patients with colorectal cancer who received ensituximab at the MTD/RP2D (3 mg/kg) included fatigue (38%), anemia (30%), nausea (15%), vomiting (11%), increased bilirubin (9%), constipation (8%), and chills, decreased appetite, headache, and diarrhea (6%). AEs in patients with colorectal cancer at the RP2D and for all subjects regardless of dose level (1.5 mg/kg, n = 3; 2 mg/kg, n = 1; 3 mg/kg, n = 53; and 4 mg/kg, n = 6) are contained in Table 3. Treatment-emergent serious AEs regardless of attribution are listed in Table 4. Treatment-emergent serious AEs at least possibly related to ensituximab occurred in 2 patients at the RP2D (4%) and included nausea (2%) and hypoxia (2%). Study drug administration at the RP2D was interrupted in 11 patients (17%) for treatment-related AEs regardless of attribution and included anemia (2%), hemolysis (2%), abdominal pain (6%), ascites (2%), small intestinal obstruction (2%), bile duct obstruction (2%), sepsis (2%), alanine aminotransferase/aspartate aminotransferase (AST) increase (2%), bilirubin increase (2%), headache (2%), and hypoxia (2%). Ensituximab drug therapy was discontinued in 3 patients (8%) at the RP2D due to following AEs: anemia (2%), hemolysis (2%), and hypoxia (2%). Overall, all AEs were self-limiting, reversible, and most events were mild in severity.

Table 3.

Incidence of treatment-emergent, treatment-related AEs.

System organ class preferred term3 mg/kg (N = 53)All subjects (1, 2, 3, and 4 mg/kg; N = 63)
Subjects with at least 1 treatment-related AE 43 (81%) 51 (81%) 
Blood and lymphatic system disorders 16 (30%) 20 (32%) 
 Anemia 16 (30%) 20 (32%) 
 Hemolysis 3 (6%) 3 (5%) 
Cardiac disorders 2 (3%) 
 Tachycardia 2 (3%) 
Gastrointestinal disorders 15 (28%) 16 (25%) 
 Abdominal pain, upper 1 (2%) 
 Anal incontinence 1 (2%) 
 Constipation 4 (8%) 5 (8%) 
 Diarrhea 3 (6%) 4 (6%) 
 Mouth ulceration 1 (2%) 
 Nausea 8 (15%) 8 (13%) 
 Vomiting 6 (11%) 6 (10%) 
General disorders and administration site conditions 24 (45%) 30 (48%) 
 Asthenia 1 (2%) 
 Chills 3 (6%) 5 (8%) 
 Fatigue 20 (38%) 23 (37%) 
 Gait disturbance 1 (2%) 1 (2%) 
 Malaise 1 (2%) 1 (2%) 
 Mucosal inflammation 1 (2%) 1 (2%) 
 Pyrexia 2 (4%) 5 (8%) 
Hepatobiliary disorders 1 (2%) 2 (3%) 
 Hyperbilirubinemia 1 (2%) 2 (3%) 
Immune system disorders 1 (2%) 2 (3%) 
 Allergic transfusion reaction 1 (2%) 1 (2%) 
 CRS 1 (2%) 
Injury, poisoning, and procedural complications 2 (4%) 2 (3%) 
 Incision site rash 1 (2%) 1 (2%) 
 Infusion-related reaction 1 (2%) 1 (2%) 
Investigations 11 (21%) 12 (19%) 
 AST increased 1 (2%) 1 (2%) 
 Blood bilirubin increased 5 (9%) 6 (10%) 
 Blood creatinine increased 1 (2%) 1 (2%) 
 Blood pressure increased 1 (2%) 1 (2%) 
 Haptoglobin decreased 1 (2%) 1 (2%) 
 Neutrophil count decreased 1 (2%) 1 (2%) 
 Platelet count decreased 1 (2%) 1 (2%) 
 Weight decreased 2 (4%) 2 (3%) 
Metabolism and nutrition disorders 5 (9%) 6 (10%) 
 Decreased appetite 3 (6%) 3 (5%) 
 Hyperglycemia 1 (2%) 1 (2%) 
 Hypokalemia 1 (2%) 1 (2%) 
 Hypovolemia 1 (2%) 
 Vitamin B12 deficiency 1 (2%) 1 (2%) 
Musculoskeletal and connective tissue disorders 5 (9%) 6 (10%) 
 Arthralgia 2 (4%) 2 (3%) 
 Back pain 1 (2%) 2 (3%) 
 Musculoskeletal pain 1 (2%) 1 (2%) 
 Myalgia 1 (2%) 1 (2%) 
Nervous system disorders 5 (9%) 6 (10%) 
 Dizziness 2 (4%) 2 (3%) 
 Headache 3 (6%) 4 (6%) 
 Hypoesthesia 1 (2%) 1 (2%) 
Psychiatric disorders 1 (2%) 
 Insomnia 1 (2%) 
Renal and urinary disorders 1 (2%) 1 (2%) 
 Chromaturia 1 (2%) 1 (2%) 
Respiratory, thoracic, and mediastinal disorders 6 (11%) 7 (11%) 
 Dyspnea 4 (8%) 4 (6%) 
 Hypoxia 1 (2%) 1 (2%) 
 Nasal congestion 1 (2%) 
 Pharyngeal edema 1 (2%) 1 (2%) 
 Upper-airway cough syndrome 1 (2%) 1 (2%) 
Skin and subcutaneous tissue disorders 1 (2%) 2 (3%) 
 Night sweats 1 (2%) 1 (2%) 
 Pruritus 1 (2%) 
Vascular disorders 7 (13%) 9 (14%) 
 Flushing 7 (13%) 8 (13%) 
 Hypertension 1 (2%) 
System organ class preferred term3 mg/kg (N = 53)All subjects (1, 2, 3, and 4 mg/kg; N = 63)
Subjects with at least 1 treatment-related AE 43 (81%) 51 (81%) 
Blood and lymphatic system disorders 16 (30%) 20 (32%) 
 Anemia 16 (30%) 20 (32%) 
 Hemolysis 3 (6%) 3 (5%) 
Cardiac disorders 2 (3%) 
 Tachycardia 2 (3%) 
Gastrointestinal disorders 15 (28%) 16 (25%) 
 Abdominal pain, upper 1 (2%) 
 Anal incontinence 1 (2%) 
 Constipation 4 (8%) 5 (8%) 
 Diarrhea 3 (6%) 4 (6%) 
 Mouth ulceration 1 (2%) 
 Nausea 8 (15%) 8 (13%) 
 Vomiting 6 (11%) 6 (10%) 
General disorders and administration site conditions 24 (45%) 30 (48%) 
 Asthenia 1 (2%) 
 Chills 3 (6%) 5 (8%) 
 Fatigue 20 (38%) 23 (37%) 
 Gait disturbance 1 (2%) 1 (2%) 
 Malaise 1 (2%) 1 (2%) 
 Mucosal inflammation 1 (2%) 1 (2%) 
 Pyrexia 2 (4%) 5 (8%) 
Hepatobiliary disorders 1 (2%) 2 (3%) 
 Hyperbilirubinemia 1 (2%) 2 (3%) 
Immune system disorders 1 (2%) 2 (3%) 
 Allergic transfusion reaction 1 (2%) 1 (2%) 
 CRS 1 (2%) 
Injury, poisoning, and procedural complications 2 (4%) 2 (3%) 
 Incision site rash 1 (2%) 1 (2%) 
 Infusion-related reaction 1 (2%) 1 (2%) 
Investigations 11 (21%) 12 (19%) 
 AST increased 1 (2%) 1 (2%) 
 Blood bilirubin increased 5 (9%) 6 (10%) 
 Blood creatinine increased 1 (2%) 1 (2%) 
 Blood pressure increased 1 (2%) 1 (2%) 
 Haptoglobin decreased 1 (2%) 1 (2%) 
 Neutrophil count decreased 1 (2%) 1 (2%) 
 Platelet count decreased 1 (2%) 1 (2%) 
 Weight decreased 2 (4%) 2 (3%) 
Metabolism and nutrition disorders 5 (9%) 6 (10%) 
 Decreased appetite 3 (6%) 3 (5%) 
 Hyperglycemia 1 (2%) 1 (2%) 
 Hypokalemia 1 (2%) 1 (2%) 
 Hypovolemia 1 (2%) 
 Vitamin B12 deficiency 1 (2%) 1 (2%) 
Musculoskeletal and connective tissue disorders 5 (9%) 6 (10%) 
 Arthralgia 2 (4%) 2 (3%) 
 Back pain 1 (2%) 2 (3%) 
 Musculoskeletal pain 1 (2%) 1 (2%) 
 Myalgia 1 (2%) 1 (2%) 
Nervous system disorders 5 (9%) 6 (10%) 
 Dizziness 2 (4%) 2 (3%) 
 Headache 3 (6%) 4 (6%) 
 Hypoesthesia 1 (2%) 1 (2%) 
Psychiatric disorders 1 (2%) 
 Insomnia 1 (2%) 
Renal and urinary disorders 1 (2%) 1 (2%) 
 Chromaturia 1 (2%) 1 (2%) 
Respiratory, thoracic, and mediastinal disorders 6 (11%) 7 (11%) 
 Dyspnea 4 (8%) 4 (6%) 
 Hypoxia 1 (2%) 1 (2%) 
 Nasal congestion 1 (2%) 
 Pharyngeal edema 1 (2%) 1 (2%) 
 Upper-airway cough syndrome 1 (2%) 1 (2%) 
Skin and subcutaneous tissue disorders 1 (2%) 2 (3%) 
 Night sweats 1 (2%) 1 (2%) 
 Pruritus 1 (2%) 
Vascular disorders 7 (13%) 9 (14%) 
 Flushing 7 (13%) 8 (13%) 
 Hypertension 1 (2%) 
Table 4.

Incidence of treatment-emergent serious AEs regardless of attribution.

System organ class preferred term3 mg/kg (N = 53)All subjects (1, 2, 3, and 4 mg/kg; N = 63)
Subjects with at least 1 serious AE 17 (32%) 23 (37%) 
Blood and lymphatic system disorders 1 (2%) 5 (8%) 
 Anemia 1 (2%) 4 (6%) 
 Febrile neutropenia 1 (2%) 
Cardiac disorders 1 (2%) 
 Left ventricular dysfunction 1 (2%) 
Gastrointestinal disorders 7 (13%) 8 (13%) 
 Abdominal pain 3 (6%) 3 (5%) 
 Dysphagia 1 (2%) 1 (2%) 
 Gastrointestinal hemorrhage 1 (2%) 2 (3%) 
 Nausea 1 (2%) 1 (2%) 
 Small intestinal obstruction 2 (4%) 2 (3%) 
General disorders and administration site conditions 1 (2%) 1 (2%) 
 Pyrexia 1 (2%) 1 (2%) 
Hepatobiliary disorders 2 (4%) 2 (3%) 
 Bile duct obstruction 1 (2%) 1 (2%) 
 Hyperbilirubinemia 1 (2%) 1 (2%) 
 Jaundice 1 (2%) 1 (2%) 
Infections and infestations 4 (8%) 5 (8%) 
 Catheter site infection 1 (2%) 1 (2%) 
 Influenza 1 (2%) 1 (2%) 
 Pneumonia 1 (2%) 1 (2%) 
 Sepsis 1 (2%) 2 (3%) 
Investigations 1 (2%) 
 Blood bilirubin increased 1 (2%) 
Neoplasms benign, malignant, and unspecified (including cysts and polyps) 2 (4%) 2 (3%) 
 Neoplasm 2 (4%) 2 (3%) 
Nervous system disorders 2 (4%) 2 (3%) 
 Headache 1 (2%) 1 (2%) 
 Syncope 1 (2%) 1 (2%) 
Renal and urinary disorders 1 (2%) 2 (3%) 
 Acute kidney injury 1 (2%) 
 Urinary tract obstruction 1 (2%) 1 (2%) 
Respiratory, thoracic, and mediastinal disorders 5 (9%) 6 (10%) 
 Dyspnea 3 (6%) 4 (6%) 
 Hypoxia 1 (2%) 1 (2%) 
 Respiratory failure 2 (4%) 2 (3%) 
Vascular disorders 1 (2%) 1 (2%) 
 Jugular vein thrombosis 1 (2%) 1 (2%) 
System organ class preferred term3 mg/kg (N = 53)All subjects (1, 2, 3, and 4 mg/kg; N = 63)
Subjects with at least 1 serious AE 17 (32%) 23 (37%) 
Blood and lymphatic system disorders 1 (2%) 5 (8%) 
 Anemia 1 (2%) 4 (6%) 
 Febrile neutropenia 1 (2%) 
Cardiac disorders 1 (2%) 
 Left ventricular dysfunction 1 (2%) 
Gastrointestinal disorders 7 (13%) 8 (13%) 
 Abdominal pain 3 (6%) 3 (5%) 
 Dysphagia 1 (2%) 1 (2%) 
 Gastrointestinal hemorrhage 1 (2%) 2 (3%) 
 Nausea 1 (2%) 1 (2%) 
 Small intestinal obstruction 2 (4%) 2 (3%) 
General disorders and administration site conditions 1 (2%) 1 (2%) 
 Pyrexia 1 (2%) 1 (2%) 
Hepatobiliary disorders 2 (4%) 2 (3%) 
 Bile duct obstruction 1 (2%) 1 (2%) 
 Hyperbilirubinemia 1 (2%) 1 (2%) 
 Jaundice 1 (2%) 1 (2%) 
Infections and infestations 4 (8%) 5 (8%) 
 Catheter site infection 1 (2%) 1 (2%) 
 Influenza 1 (2%) 1 (2%) 
 Pneumonia 1 (2%) 1 (2%) 
 Sepsis 1 (2%) 2 (3%) 
Investigations 1 (2%) 
 Blood bilirubin increased 1 (2%) 
Neoplasms benign, malignant, and unspecified (including cysts and polyps) 2 (4%) 2 (3%) 
 Neoplasm 2 (4%) 2 (3%) 
Nervous system disorders 2 (4%) 2 (3%) 
 Headache 1 (2%) 1 (2%) 
 Syncope 1 (2%) 1 (2%) 
Renal and urinary disorders 1 (2%) 2 (3%) 
 Acute kidney injury 1 (2%) 
 Urinary tract obstruction 1 (2%) 1 (2%) 
Respiratory, thoracic, and mediastinal disorders 5 (9%) 6 (10%) 
 Dyspnea 3 (6%) 4 (6%) 
 Hypoxia 1 (2%) 1 (2%) 
 Respiratory failure 2 (4%) 2 (3%) 
Vascular disorders 1 (2%) 1 (2%) 
 Jugular vein thrombosis 1 (2%) 1 (2%) 

Correlative studies

HACA

HACA assay used a validated, ELISA technique. Results are available on 12 patients. The percent analytic recovery fell within 80%–120% of the nominal concentration for all standards between or equal to 250 and 7.8 ng/mL. The percent coefficient of variation (%CV) of the calculated concentrations for all standards between or equal to 250 and 7.8 ng/mL was ≤20% and the %CV of the calculated concentrations for standards at 3.9 ng/mL was ≤25%. The r2 of the standard curves was ≥0.980. All patient samples had concentrations of HACA less than the assays lower limit of detection of 3.9 ng/mL.

Cytokines

The serum samples from 13 subjects were used for this study. Detection range for IL1β, IL2, IL8, IL10, GM-CSF was 2,500–0.61 pg/mL, and TNFα 12p70 and IFNγ was 2,500–2.44 pg/mL. The standards and serum samples were run in triplicate with four time points of each subject in the same plate for avoiding plate variation. There are no elevations of serum IL1β, IL2, IL10, IL12p70, GM-CSF, IFNγ, and TNFα in these 13 subjects at day 4, 15, and 57 after NEO-102 infusion comparing with serum baseline before infusion. There were unrelated significant elevations in IL8 concentrations in three subjects. Overall, the cytokine level after NEO-102 infusion indicates there is no indication of significant cytokine release after NEO-102 infusion.

Research immune studies

Of the 43 patients with samples available, the following genotypes were identified: FCGR3A 158 V/V (n = 7), FCGR3A 158 F/F (n = 14), and FCGR3A 158 V/F (n = 22).

Comparison of the ability of V/V versus F/F PBMCs treated with ensituximab to induce ADCC on human pancreatic cancer cell line, ASPC-1 that expresses the NEO-102 antigen, is shown in Table 5A.

Table 5A.

CD16 polymorphism and ADCC.

% Specific killing (±SEM)
Donor (n = 2)E:T ratioControlEnsituximab
VV1 50:1 1.1 ± 1.66 31.0 ± 1.098 
 25:1 −0.03 ± 1.54 19.1 ± 1.66 
VV2 50:1 −1.7 ± 1.67 28.4 ± 1.40 
 25:1 0.7 ± 0.84 18.1 ± 1.32 
FF1 50:1 −1.3 ± 0.45 22 ± 1.94 
 25:1 0.6 ± 1.11 11.9 ± 0.10 
FF2 50:1 0.3 ± 1.67 19.3 ± 2.51 
 25:1 1.6 ± 2.4 11.8 ± 0.14 
% Specific killing (±SEM)
Donor (n = 2)E:T ratioControlEnsituximab
VV1 50:1 1.1 ± 1.66 31.0 ± 1.098 
 25:1 −0.03 ± 1.54 19.1 ± 1.66 
VV2 50:1 −1.7 ± 1.67 28.4 ± 1.40 
 25:1 0.7 ± 0.84 18.1 ± 1.32 
FF1 50:1 −1.3 ± 0.45 22 ± 1.94 
 25:1 0.6 ± 1.11 11.9 ± 0.10 
FF2 50:1 0.3 ± 1.67 19.3 ± 2.51 
 25:1 1.6 ± 2.4 11.8 ± 0.14 

Note: Data represent the correlation between the polymorphism of the NK FcγRIIIA receptor and the NEO-102 ADCC activity. A SNP of the FCGR3A gene results in two allotypes of the NK FcγRIIIA with valine (V) or phenylalanine (F) at amino acid 158. In this way, FcγRIIIA can have three different genotypes: V/V, V/F, and F/F. Each genotype has a different avidity for the NEO-102 Fc region.

Although PBMCs with CD16 V/V polymorphism showed a stronger ADCC activity in vitro compared with PBMCs with CD16 F/F polymorphism, there was no correlation between CD16 polymorphism as analyzed above and patient clinical outcomes. The lack of correlation between CD16 polymorphism and patient outcomes suggests that other immune-related factors (under investigation) may impact the efficacy of ensituximab in vitro.

Ensituximab-mediated ADCC against human carcinoma cells is enhanced by an IL15 superagonist

To evaluate the ability of an IL15 superagonist to modulate the ADCC activity of ensituximab, NK cells isolated from PBMCs from two healthy donors were treated with an IL15 superagonist prior to being added in the nonradioactive ADCC assay as effectors. As shown in Table 5B, IL-15 superagonist significantly enhanced NEO-102–mediated ADCC against CFPAC-1 cells (high expression of NEO-102 antigen), compared with the IgG1 isotype control, at the highest E:T ratio in both donors. These data demonstrated that treatment of NK cells with IL15 effectively enhances NEO-102–mediated ADCC against NE0-102–positive carcinoma cells in vitro.

Table 5B.

IL15 superagonist enhances NEO-102–mediated ADCC activity.

% Specific lysis (SD)% Specific lysis (SD)
(Donor 1)(Donor 2)
AntibodyE:T ratioUntreatedIL15UntreatedIL15
SuperagonistSuperagonist
IgG1 12.5:1 7.7 (1.21) 9.5 (2.5) 1.9 (0.7) 10.9 (1.3) 
 6.25:1 −1.1 (1.85) 5.8 (5.5) −0.6 (3.8) 1.7 (4.6) 
NEO-102 12.5:1 14.1 (3.9) 22.1 (1.4)a 1.6 (4.2) 19.7 (0.4)a 
 6.25:1 7.2 (4.7) 4.3 (4.1) 2.1 (0.6) 11.4 (1.7)a 
% Specific lysis (SD)% Specific lysis (SD)
(Donor 1)(Donor 2)
AntibodyE:T ratioUntreatedIL15UntreatedIL15
SuperagonistSuperagonist
IgG1 12.5:1 7.7 (1.21) 9.5 (2.5) 1.9 (0.7) 10.9 (1.3) 
 6.25:1 −1.1 (1.85) 5.8 (5.5) −0.6 (3.8) 1.7 (4.6) 
NEO-102 12.5:1 14.1 (3.9) 22.1 (1.4)a 1.6 (4.2) 19.7 (0.4)a 
 6.25:1 7.2 (4.7) 4.3 (4.1) 2.1 (0.6) 11.4 (1.7)a 

Note: NK cells isolated from three normal donors were treated with IL15 superagonist (25 ng/mL) or medium control (untreated) for 48 hours prior to be used as effector cells in a 4-hour nonradioactive ADCC assay using Celigo Imaging Cytometer. CFPAC-1 (human pancreatic cancer cell line) cells were stained with calcein AM and used as targets at 3,000 cells/well. Results are expressed in % specific lysis (SD).

aStatistically significant (P < 0.05) by Student t test (NEO-102 + IL15 superagonist vs. NEO-102 untreated).

We performed this clinical trial to assess the clinical efficacy of NEO-102 in patients with refractory metastatic colorectal cancer with expression of the NEO-102 target antigen. The results from our study clearly demonstrated that ensituximab was very well tolerated in heavily pretreated patients with advanced colorectal cancer. Anemia was the only grade 3 or 4 event which occurred in more than 5% of patients. The mechanism of anemia is unclear but could be related to hemolysis. The most frequent treatment-related AEs included fatigue, anemia, nausea, vomiting, increased bilirubin, and constipation. Treatment was interrupted due to AEs in 21% of the patients treated at the RP2D, while only 3 of those patients' (6%) AEs were treatment related. Most of the AEs were grade 1 or 2 and were easily managed with standard supportive measures.

In terms of efficacy, median survival was 6.8 months, which was significantly higher than historical control of 5 months. However, OS is similar to those achieved in clinical trials with regorafenib or TAS-102 in patients with refractory colorectal cancer (2, 3). Patients were heavily pretreated with a median of three lines of prior therapies including regorafenib. Approximately, 60% of the patients had Eastern Cooperative Oncology Group PS of 2, consistent with real-world setting. No clinical responses were observed in this trial, although 21% of the patients experienced SD, some of which were durable. Three patients received treatment for more than 2 years. One-year OS was 28%. The benefit of ensituximab was observed even in patients previously treated with regorafenib. There was no correlation between degree of expression of NEO-102 on IHC with response to ensituximab or OS, suggesting that intensity of staining or percentage of tumor cells positive may not be a good predictive marker for clinical benefit. Better selection of patients with predictive biomarker may lead to improved outcomes with ensituximab.

In this study, patients with refractory colorectal cancer were only eligible based on ≥20% positive on IHC for NEO-102 target antigen on the tumor samples. Preclinical data demonstrated that antibody-staining results with NEO-102 demonstrated specific immunoreactivity with cancer tissues from patients with colon cancer, whereas only weak binding, if at all, was observed in normal pancreas or colon tissues, which will potentially lead to less toxicity. In our study, 60% of the patients with metastatic colorectal cancer were found to be eligible based upon biomarker requirements.

Extensive correlative analyses were performed as part of this study. Ensituximab was previously tested for ADCC activity against several colorectal and pancreatic tumor cell targets in vitro (6). In addition, we performed ADCC on the pancreatic cancer cell line ASPC-1 using PBMCs from patients with cancer and evaluated the impact of the CD16 polymorphism on the ADCC activity mediated by ensituximab. We observed in vitro that patients harboring the FcγRIIIA 158 V/V genotype had a higher ensituximab-mediated ADCC activity compared with F/F genotype. No correlation was observed between CD16 polymorphism and response or survival of patients in this study, suggesting that other mechanisms may be involved in part in the clinical efficacy of ensituximab. In addition, we evaluated the effects of an IL15 superagonist on the ability of human NK cells to perform ADCC mediated by ensituximab and demonstrated that treatment with the IL15 superagonist significantly enhanced the ADCC mediated by ensituximab against the high NEO-102–positive carcinoma cell line (CFPAC-1), compared with the IgG1 isotype control, in both donors tested. A recent study showed that IL15 superagonist is able to modulate the phenotype of human NK cells toward a more active cytotoxic function, increasing expression of NK-activating receptors, antiapoptotic factors, and factors involved in the NK cytotoxicity, as well as downregulating gene expression of NK-inhibiting receptors and factors involved in the NK-cell exhaustion (14). Patients with metastatic malignant melanoma or metastatic renal cell cancer receiving a human recombinant IL15 in a recent clinical trial showed that IL15 can be administered safely and effectively in patients with cancer. IL15 administration modulated the function and proliferation of NK cells in these patients with cancer, rendering the immune system more aggressive against cancer cells (16).

This study showed that ensituximab appeared to have modest activity with good safety profile in patients with refractory colorectal cancer. The study drug was relatively well tolerated with mild and manageable side effects. Previously conducted phase I trial demonstrated predictable pharmacokinetic profile and elevation of IL6 and IL8 posttreatment. These findings provide supporting rationale for investigating combination therapies using ensituximab with IL15 or other agents such as chemotherapy, to enhance efficacy in this patient population.

R.D. Kim is a paid consultant for Bayer, Lilly, and BMS. S.A. Mavroukakis and M. Fantini are employees/paid consultants for Precision Biologics, Inc. J. Torrealba is an employee/paid consultant for Roche. P.M. Arlen is an employee/paid consultant for Precision Biologics. No potential conflicts of interest were disclosed by the other authors.

Conception and design: R.D. Kim, M.A. Morse, K.Y. Tsang, P.M. Arlen, M.S. Beg

Development of methodology: R.D. Kim, M.A. Morse, M. Fantini, K.Y. Tsang, J. Torrealba, P.M. Arlen, M.S. Beg

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.D. Kim, N.S. Azad, M.A. Morse, E. Poplin, A. Mahipal, B. Tan Jr, S.A. Mavroukakis, K.Y. Tsang, J. Torrealba, M.S. Beg

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.D. Kim, N.S. Azad, M.A. Morse, A. Mahipal, M. Fantini, K.Y. Tsang, P.M. Arlen, M.S. Beg

Writing, review, and/or revision of the manuscript: R.D. Kim, N.S. Azad, M.A. Morse, A. Mahipal, B. Tan Jr, S.A. Mavroukakis, M. Fantini, K.Y. Tsang, J. Torrealba, P.M. Arlen, M.S. Beg

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.D. Kim, N.S. Azad, S.A. Mavroukakis, K.Y. Tsang, A. Zaki

Study supervision: R.D. Kim, N.S. Azad, M.A. Morse

Precision Biologics, Inc. provided funding support for this trial.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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