Purpose: RAV12 is a high affinity, internalizing, chimeric IgG1 monoclonal antibody that binds RAAG12, a novel primate-restricted N-linked carbohydrate epitope present on multiple cell surface proteins. RAAG12 is highly expressed on many adenocarcinomas, particularly those of gastrointestinal origin. A phase 1 dose-escalation safety and pharmacokinetics trial was conducted in patients with metastatic or recurrent adenocarcinomas.

Experimental Design: RAV12 was initially given i.v. weekly ×4, then by fractionated dosing twice or thrice weekly. Thirty-three patients were treated in the dose escalation segment of the trial in the following cohorts: 0.3 mg/kg qw (6), 1.0 mg/kg qw (8), 1.5 mg/kg qw (7); and 0.5 mg/kg biw (3), 0.75 mg/kg biw (3), and 0.5 mg/kg tiw (6). Twenty patients were enrolled in a maximum tolerated dose cohort expansion at 0.75 mg/kg biw.

Results: Two clinical syndromes were associated with drug administration: abdominal cramping pain with diarrhea, and asymptomatic, self-limited increases of liver function tests. These effects were partially ameliorated with fractionated dosing. Pharmacokinetics was dose dependent. Maximum concentration was reduced, whereas area under the concentration versus time curve was maintained with fractionated dosing. One patient with colorectal cancer experienced a durable partial remission, with a time to progression (TTP) of >8 months. Three additional patients experienced a TTP of >4 months.

Conclusions: RAV12 has activity in recurrent adenocarcinomas. However, the safety profile of the antibody seems to preclude the delivery of highly efficacious doses. Re-engineering the molecule to remove FcRn binding (while maintaining FcγR binding) and to humanize it may improve the toxicity profile and efficacy. Clin Cancer Res; 16(5); 1673–81

Translational Relevance

An oncofetal antigen, RAAG12, discovered as monoclonal antibodies, resulting from the immunization of mice with a fetal kidney progenitor cell line, were screened. The antigen was determined to be a glycotope that may result from aberrant glycosylation associated with the neoplastic state. The antigen was shown to be highly expressed on adenocarcinomas particularly those of gastrointestinal origin. The antibody, RAV12, directed against the novel antigen was shown to induce cell death through oncosis, mediate antibody-dependent cellular cytotoxicity and complement mediated cell lysis, and modulate cellular signaling involving certain growth factor receptors. The study presented here represents the translation of those laboratory findings to the clinic in a first-in-humans dose escalation study designed to evaluate the safety profile (including immunogenicity), determine an maximum tolerated dose, define pharmacokinetics, and evaluate, in preliminary fashion, the efficacy of RAV12 in patients with recurrent adenocarcinomas.

Oncofetal antigens have a long and storied history in oncology. They have been used as diagnostic and prognostic factors, as indicators of therapeutic effect and recurrence, and as therapeutic targets (1, 2). RAV12 is a monoclonal antibody derived from the immunization of mice with a normal fetal kidney progenitor cell line. Resulting hybridomas were screened for the production of antibodies that bound to the immunizing line, tumor cell lines, and tumor tissues, with relative lack of binding to normal tissue (3). RAV12 is a high-affinity, internalizing, chimeric IgG1 monoclonal antibody that binds to RAAG12, a novel primate-specific N-linked carbohydrate epitope (3). The minimal RAV12 binding site on RAAG12 is defined by the trisaccharide, Galβ1-3GlcNacβ1-3Gal. The trisaccharide is a precursor to the Lewis blood group antigens, but RAAG12 is distinct from those antigen structures and is not one of the previously described mucin antigens (3).

RAAG12 is variably expressed (<10% to uniformly) on normal nonkeratinizing epithelia and is not expressed on human cardiovascular, endocrine, lymphohematopoietic, neuromuscular, or central nervous system tissues. In normal polarized epithelia such as those in the gastrointestinal tract, RAAG12 is expressed in the cytoplasm and on the apical membrane surface. The pattern of expression in tumors is distinct in that RAAG12 expression becomes circumferential on the tumor cell surface. This pattern of expression made RAAG12 an attractive tumor target for antibody therapy (4). In vitro binding of RAV12 to RAAG12 on tumor target cells was associated with the induction of oncotic cell death (3), induction of complement mediated cell killing and antibody-dependent cellular cytotoxicity (5), and modulation of cellular signaling in which RAV12 has been shown to accelerate insulin-like growth factor I–dependent insulin-like growth factor I receptor phosphorylation and desensitization (6).

Safety of RAV12 was evaluated in two independent multiple dose studies in cynomolgus monkeys. Transient and sporadic, self-limited elevations in liver function tests and pancreatic enzyme blood levels, as well as transient vomiting, were observed in cynomolgus monkeys treated with RAV12, primarily at doses ≥20 mg/kg. No significant gastrointestinal or hepatic histopathologic abnormalities were observed in the multiple dose i.v. infusion toxicity studies with RAV12.

We conducted a first-in-human, phase 1, dose escalation study to evaluate the safety profile (including immunogenicity), determine a maximum tolerated dose (MTD), define pharmacokinetics (PK), and evaluate, in preliminary fashion, the efficacy of RAV12 in patients with recurrent adenocarcinomas of gastrointestinal origin or of other origin if the tumor bore the RAAG12 antigen.

Patient selection

Patients, 18 y of age or older, with histologically confirmed adenocarcinomas of gastrointestinal origin (gastroesophageal, pancreatic, colorectal) or other origin if proven to be RAAG12 positive [≥10% of cells staining by immunohistochemistry (IHC); historical or contemporaneous, primary, or metastatic pathologic material was considered acceptable because previous studies had shown >80% concordance between matched primary and metastatic specimens; ref. 3] were eligible for this study. Patients had to have metastatic or recurrent disease and have received at least one (or at least two, in the case of breast or colorectal cancer), but no more than six, prior therapies for metastatic or recurrent disease. Concomitant antineoplastic, immunosuppressive, or vaccine therapy was not allowed. Chemotherapy and radiotherapy must have been completed for ≥4 wk and previous monoclonal antibody therapy at of least three half-lives before study entry. Patients had to have an Eastern Cooperative Oncology Group performance status 0 or 1 and adequate organ reserve including cardiovascular, pulmonary, renal, and hepatic functioning sufficient to undergo therapy. Patients with active infection, a history of thrombosis within 3 mo, or prior clinically relevant malignancy were not eligible. Required laboratory parameters at baseline included the following: platelet count of ≥100,000 per mm3, hemoglobin of ≥9.0 g/dl, absolute neutrophil count of ≥1,500 per mm3, alanine aminotransferase/aspartate aminotransferase/alkaline phosphatase/γ-glutamyl transferase of ≤ 2.5 times the upper limit of normal, total bilirubin of ≤1.5 times the upper limit of normal, amylase/lipase of ≤1.0 times the upper limit of normal, and creatinine of <1.5 mg/dl. Patients had to have at least one radiographically measurable site of disease of ≥2.0 cm in the largest diameter. Patients with known hypersensitivity to murine or recombinant proteins were not eligible to participate in this study. Patients had to provide informed consent as documented by signing an Institutional Review Board–approved consent form.

Study design

The study was designed as an open-label, multidose, single-arm, phase 1, dose-escalation trial to define the toxicity profile including immunogenicity, MTD, PK, and potential efficacy of RAV12 in patients with metastatic or recurrent previously treated adenocarcinoma. The study was carried out in accordance with Good Clinical Practices and relevant Federal Regulations after approval by each participating institution's Institutional Review Board.

A standard “3 plus 3” dose escalation scheme was used. Initially, RAV12 was administered i.v. over 2 h, weekly ×4, beginning on day 1, to cohorts of three patients. Because abdominal discomfort was observed, the infusion rate was subsequently changed to 25 mg/h for the first hour, followed by a rate not to exceed 50 mg/h thereafter. This change was made after five patients were treated in the 1.0 mg/kg cohort. All subsequent patients were treated at the fixed dose rates.

Safety was evaluated throughout and blood samples for chemistry determinations were collected on days 1, 2, 4, 5, 8, 15, 22, 36, and 50, and more frequently if abnormalities were noted. Toxicities were evaluated and reported according to the National Cancer Institute Common Terminology Criteria for Adverse Events v 3.0. Dose limiting toxicity (DLT) was defined initially as any National Cancer Institute Common Terminology Criteria for Adverse Events grade 3 or greater event with certain exceptions notably: decreased hemoglobin, alopecia, nausea/vomiting (in the absence of antiemetics), diarrhea (in the absence of antidiarrheals), fever (in the absence of sepsis or antipyretics), grade 3 fatigue unless not resolving in 7 d, rash other than desquamation, generalized urticaria, systemic skin reaction (unless requiring systemic narcotics or corticosteroids), pain associated with tumor, grade 4 or greater hypersensitivity events, and grade 1 or greater ocular/visual events. Tumor measurements by computed tomographic scanning were obtained on day 43 and assessed according to the Response Evaluation Criteria in Solid Tumors (7).

Dose escalation could occur if none of three patients initially treated or one of six patients treated in an expanded cohort experienced DLT. MTD was defined as the highest dose tested at which <33% of patients treated experienced DLT.

The dose escalation scheme began at 0.3 mg/kg and escalated to 1.0 mg/kg and then to 1.5 mg/kg. The initial dose was 1/33 of the no observable adverse effect level determined in the 5-wk toxicology study in cynomolgus monkeys. Reversible changes in liver function tests were observed in each of the three dose escalation cohorts, and the DLT definitions were amended in collaboration with the U.S. Food & Drug Administration such that occurrences of liver function test (alanine aminotransferase, aspartate aminotransferase, bilirubin, and alkaline phosphatase) or pancreatic enzyme (amylase and lipase) abnormalities were considered dose limiting only if they met the criteria for seriousness (e.g., resulted in hospitalization or were considered medically significant) or failed to resolve to ≤grade 2 levels within 7 d and to ≤grade 1 levels within 2 wk of the last therapy date.

At doses of 1.0 and 1.5 mg/kg qw, an antibody-associated syndrome of moderately severe abdominal discomfort and diarrhea was observed. Because the MTD was exceeded at 1.5 mg/kg, fractionation of the dose to deliver antibody twice or thrice qw over 4 wk was evaluated in three additional cohorts. Doses and schedules delivered were 0.5 mg/kg biw, 0.5 mg/kg tiw, and 0.75 mg/kg biw.

When a MTD and schedule was determined, 20 additional patients were enrolled and treated at that dose and schedule (0.75 mg/kg biw) to gain additional information about safety, to further define PK, and to evaluate efficacy. Evaluable data were obtained for 14 of the 20 additional patients enrolled and were used to define the PK of the MTD expansion cohort. Patients with stable disease or better on day 43 were treated until progression of disease. Subsequent cycles of treatment, when given, were administered at the dose and schedule used during days 1 through 22, beginning at day 50 without interruption.

Immunohistochemistry

Contemporaneous or historical, formalin-fixed, paraffin-embedded tumor specimens were sectioned at 4 to 5 μm using the Leica RM2135 microtome and collected on Superfrost Plus slides, then dried in an oven at 55°C for 60 min.

Before the IHC staining, slides were deparaffinized with xylene followed by hydration with serial concentrations of alcohol, washed with 1× wash buffer (DakoCytomation), then pretreated with the HIER method: submerged in 1× citric (pH 6.0) buffer in a pressure cooker at 125°C for 30 s (DakoCytomation). Following HIER pretreatment, slides were incubated in 5% goat serum with 0.1 mg/mL avidin (Vector) for 30 min, followed by incubating 30 μg/mL d-biotin (Sigma) for 30 min to block endogenous biotin. Slides were incubated with biotinylated Rav12 5 μg/mL for 60 min at room temperature, then incubated in 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase. After washing, slides were incubated in the ABC solution (Vectastain ABC Elite kit, Vector) for 30 min, then incubated in 3,3′-diaminobenzidine (VWR) for 10 min to visualize the immunostaining, then counterstained with hematoxylin (Fisher Scientific). Before the coverslip was applied, slides were dehydrated with serial concentrations of alcohol.

Immunogenicity

Blood samples were obtained on day 1 (baseline), day 8 (predose), day 15 (predose), day 22 (predose), and day 50. Serum was prepared for assay.

A precise and reproducible qualitative ELISA technique was developed and validated at the Alta Analytical Laboratory to measure antibodies to RAV12 in human serum (8). As is commonplace in early monoclonal antibody drug development (911), the assay used was a first generation bridging assay for human antichimeric antibodies (HACA) designed to have few false negatives but, possibly, significant numbers of false positives. The assay was confounded by the presence of circulating RAAG12 and circulating RAV12 antibody. Therefore, sera collected farthest from drug administration (day 50 or early termination date) were relied upon to determine the presence of HACA. Samples were incubated with RAV12 that had been immobilized onto a microtiter plate. After an additional incubation with biotin-RAV12, the plates were washed, and the bound antibodies were detected with poly-horseradish peroxidase-streptavidin and visualized using the SureBlueTMB peroxidase substrate. Absorbance were determined using a plate reader at 450 nm. A normal human serum panel and a positive anti-RAV12 cynomolgus monkey serum sample were included on each plate as negative and positive controls, respectively. These served to monitor the assay. The samples were serially diluted at 1:2 and analyzed in duplicate to determine the presence or absence of anti-RAV12 antibody compared with predose samples.

Pharmacokinetics

During the initial weekly (qw) infusion phase of the dose escalation segment of the study, blood samples were obtained on day 1 (before infusion, and at 1 and 4 h after completion of infusion), 2, 4, 5, 8 (predose and 1 h after completion of infusion), 15 (predose and postdose), 22 (predose and postdose), 29, 36, 43, and 50. Serum was prepared for assay.

During the fractionated (biw and tiw) phase of the dose escalation segment of the study, blood samples were obtained on day 1 (before infusion, and at 1 and 4 h after completion of infusion), 2, 8 (predose and 1 h after completion of infusion), 15 (predose and postdose), 22 (predose and postdose), 29, 36, 43, and 50. Serum was prepared for assay.

During the MTD Cohort Expansion segment of the study, blood samples were obtained on day 1 (predose, 1 and 4 h after completion of infusion), 2, 4 or 5 (predose and 1 h after completion of infusion), 8 (predose and postdose), 11 or 12 (predose and postdose), 15 (predose and postdose), 18 or 19 (predose and postdose), 22 (predose and postdose), 25 (predose and postdose), 26, 29, 36, 43, and 50. Serum was prepared for assay.

A quantitative sandwich enzyme immunoassay technique was developed and validated at the Alta Analytical Laboratory to measure RAV12 in human serum. Control and test samples were incubated with Lacto-N-fucopentose-II-BSA (LNFP-II-BSA established as a surrogate antigen for RAV12 at Raven biotechnologies, Inc.) that had been immobilized onto a microtiter plate. After incubation, unbound material was washed away, and any bound RAV12 was detected using goat anti-human κ-horseradish peroxidase and visualized using the SureBlueTMB peroxidase substrate. Absorbance were determined using a plate reader at 450 nm, and sample concentrations were determined by interpolation of absorbance values on a standard curve.

PK analyses of the individual serum concentration versus time data for RAV12 were done using compartmental methods (WinNonlin v. 5.2; Pharsight Corp.). The modeling generated estimates for the apparent volume of distribution of the central compartment (V1), the elimination rate constant (k10), and the intercompartmental rate constants (k12 and k21). Based on the primary parameters, maximum concentration (Cmax), half-life, area under the concentration versus time curve (AUC), clearance, and steady-state volume of distribution were calculated.

Patient characteristics

Between December 2004 and February 2008, 53 patients were enrolled in this study—33 in the dose escalation segment and 20 in a MTD cohort expansion segment. Table 1 summarizes the patient characteristics. This was a heavily pretreated population of patients with median number of prior therapies for recurrent or metastatic disease of four. Table 1 also summarizes the patients enrolled by cohort, tumor type, and tumor RAAG12 IHC. Colorectal cancer patients predominated in the trial, but patients with other gastrointestinal malignancies were represented, as well as a few patients with diseases of nongastrointestinal origin.

Table 1.

Patient characteristics

No. of patients treated  53 
No. of patients enrolled and not dosed  
Enrollment by cohort   
    Cohort 1 0.3 mg/kg qw 
    Cohort 2 1.0 mg/kg qw 
    Cohort 3 1.5 Mg/kg qw 
    Cohort 4 0.5 mg/kg biw 
    Cohort 5 0.75 mg/kg biw 
    Cohort 6 0.5 mg/kg tiw 
    Cohort 7 (MTD cohort) 0.75 mg/kg biw 20 
Median age (range)  62 (36-81) 
Gender Male 28 (53%) 
Female 25 (47%) 
Race Caucasian 49 (92%) 
Black 3 (6%) 
Asian 1 (2%) 
ECOG performance status 31 (58%) 
22 (42%) 
Median number of prior therapies (range)  4 (1-8) 
Histologic diagnosis Colorectal 33 (62.3%) 
Pancreatic 8 (15.1%) 
Gastroesophageal 7 (13.2%) 
Lung 2 (3.8%) 
Breast 1 (1.9%) 
Biliary tract 1 (1.9%) 
Ovarian 1 (1.9%) 
Tumor IHC (% of cells RAAG12 positive) <10% 4 (7.8%) 
10-49% 7 (13.7%) 
50-74% 14 (27.5%) 
75-100% 26 (51%) 
Missing 2 (3.9%) 
No. of patients treated  53 
No. of patients enrolled and not dosed  
Enrollment by cohort   
    Cohort 1 0.3 mg/kg qw 
    Cohort 2 1.0 mg/kg qw 
    Cohort 3 1.5 Mg/kg qw 
    Cohort 4 0.5 mg/kg biw 
    Cohort 5 0.75 mg/kg biw 
    Cohort 6 0.5 mg/kg tiw 
    Cohort 7 (MTD cohort) 0.75 mg/kg biw 20 
Median age (range)  62 (36-81) 
Gender Male 28 (53%) 
Female 25 (47%) 
Race Caucasian 49 (92%) 
Black 3 (6%) 
Asian 1 (2%) 
ECOG performance status 31 (58%) 
22 (42%) 
Median number of prior therapies (range)  4 (1-8) 
Histologic diagnosis Colorectal 33 (62.3%) 
Pancreatic 8 (15.1%) 
Gastroesophageal 7 (13.2%) 
Lung 2 (3.8%) 
Breast 1 (1.9%) 
Biliary tract 1 (1.9%) 
Ovarian 1 (1.9%) 
Tumor IHC (% of cells RAAG12 positive) <10% 4 (7.8%) 
10-49% 7 (13.7%) 
50-74% 14 (27.5%) 
75-100% 26 (51%) 
Missing 2 (3.9%) 

Abbreviation: ECOG, Eastern Cooperative Oncology Group.

Safety profile

Table 2 summarizes the treatment-emergent adverse events observed following administration of RAV12. The antibody was moderately well tolerated in this patient population. However, two antibody-associated syndromes were recognized.

Table 2.

Treatment-emergent adverse events occurring at ≥10% incidence

Body system adverse eventAll grades n (%)Grade 3 n (%)Grade 4 n (%)
Gastrointestinal disorders 
    Abdominal pain 31 (58.5%) 0 (0%) 0 (0%) 
    Nausea 27 (50.9%) 0 (0%) 0 (0%) 
    Diarrhea 26 (49.1%) 3 (5.7%) 0 (0%) 
    Constipation 19 (35.8%) 0 (0%) 0 (0%) 
    Vomiting 18 (34%) 0 (0%) 0 (0%) 
    Abdominal pain upper 8 (15.1%) 2 (3.8%) 1 (1.9%) 
Investigations 
    Aspartate aminotransferase increased 28 (52.8%) 14 (26.4%) 0 (0%) 
    Alkaline phosphatase increased 22 (41.5%) 6 (11.3%) 0 (0%) 
    Alanine aminotransferase increased 22 (41.5%) 7 (13.2%) 0 (0%) 
    γ-Glutamyltransferase increased 11 (20.8%) 6 (11.3%) 2 (3.8%) 
    Lactate dehydrogenase increased 10 (18.9%) 1 (1.9%) 0 (0%) 
    Lipase increased 6 (11.3%) 4 (7.5%) 1 (1.9%) 
General disorders and administration site conditions 
    Fatigue 12 (22.6%) 0 (0%) 0 (0%) 
    Pyrexia 6 (11.3%) 0 (0%) 0 (0%) 
Blood and lymphatic system disorders 
    Anemia 11 (20.8%) 0 (0%) 0 (0%) 
Metabolism and nutrition disorders 
    Anorexia 11 (20.8%) 0 (0%) 0 (0%) 
Musculoskeletal and connective tissue disorders 
    Back pain 9 (17%) 0 (0%) 0 (0%) 
Infections and infestations 
    Urinary tract infection 8 (15.1%) 0 (0%) 0 (0%) 
Respiratory, thoracic, and mediastinal disorders 
    Dyspnoea 8 (15.1%) 0 (0%) 0 (0%) 
Nervous system disorders 
    Dizziness 6 (11.3%) 0 (0%) 0 (0%) 
Body system adverse eventAll grades n (%)Grade 3 n (%)Grade 4 n (%)
Gastrointestinal disorders 
    Abdominal pain 31 (58.5%) 0 (0%) 0 (0%) 
    Nausea 27 (50.9%) 0 (0%) 0 (0%) 
    Diarrhea 26 (49.1%) 3 (5.7%) 0 (0%) 
    Constipation 19 (35.8%) 0 (0%) 0 (0%) 
    Vomiting 18 (34%) 0 (0%) 0 (0%) 
    Abdominal pain upper 8 (15.1%) 2 (3.8%) 1 (1.9%) 
Investigations 
    Aspartate aminotransferase increased 28 (52.8%) 14 (26.4%) 0 (0%) 
    Alkaline phosphatase increased 22 (41.5%) 6 (11.3%) 0 (0%) 
    Alanine aminotransferase increased 22 (41.5%) 7 (13.2%) 0 (0%) 
    γ-Glutamyltransferase increased 11 (20.8%) 6 (11.3%) 2 (3.8%) 
    Lactate dehydrogenase increased 10 (18.9%) 1 (1.9%) 0 (0%) 
    Lipase increased 6 (11.3%) 4 (7.5%) 1 (1.9%) 
General disorders and administration site conditions 
    Fatigue 12 (22.6%) 0 (0%) 0 (0%) 
    Pyrexia 6 (11.3%) 0 (0%) 0 (0%) 
Blood and lymphatic system disorders 
    Anemia 11 (20.8%) 0 (0%) 0 (0%) 
Metabolism and nutrition disorders 
    Anorexia 11 (20.8%) 0 (0%) 0 (0%) 
Musculoskeletal and connective tissue disorders 
    Back pain 9 (17%) 0 (0%) 0 (0%) 
Infections and infestations 
    Urinary tract infection 8 (15.1%) 0 (0%) 0 (0%) 
Respiratory, thoracic, and mediastinal disorders 
    Dyspnoea 8 (15.1%) 0 (0%) 0 (0%) 
Nervous system disorders 
    Dizziness 6 (11.3%) 0 (0%) 0 (0%) 

The first syndrome was characterized by abdominal discomfort with or without diarrhea. This syndrome was noted in the second cohort of patients (those treated at a dose of 1.0 mg/kg qw). It was dose limiting in one patient treated at 1.5 mg/kg qw. The onset of the syndrome was characteristically at or near end of infusion. Patients experienced crampy abdominal pain frequently accompanied by diarrhea. Empirical use of anticholinergic antispasmodics and/or opiate analgesics was often, but not always, helpful in relieving symptoms. The syndrome was generally self-limited, lasting 4 to 24 hours from end of infusion. However, one patient experienced severe diarrhea with up to 20 stools per day, required hospitalization and fluid support, and failed to fully resolve symptoms for 5 days.

The second syndrome was elevations of LFTs. These occurred rapidly after infusion, typically noted on day 2. Transaminases were frequently elevated more significantly than alkaline phosphatase. Bilirubin abnormalities were less commonly seen. No coagulation abnormalities were observed. These changes occurred in patients without symptoms referable to the liver and tended to resolve spontaneously. In two patients treated at 1.5 mg/kg qw, these changes were dose limiting. Liver function test abnormalities were most commonly seen with first infusion. Only in two patients, treated at 1.5 mg/kg qw, did the changes occur repetitively with each subsequent dose. The occurrence of the two drug-associated syndromes is summarized by dose cohort in Table 3.

Table 3.

Occurrence of selected adverse events by dose cohort

Adverse eventDosingPatients per cohortGrade 1Grade 2Grade 3Grade 4Any grade
%%%%%
Abdominal pain 0.3 mg/kg qw 33 33 
1.0 mg/kg qw 62 38 100 
1.5 mg/kg qw 57 43 100 
0.5 mg/kg biw 67 67 
0.5 mg/kg tiw 33 33 
0.75 mg/kg biw 23 26 17 43 
Diarrhea 0.3 mg/kg qw 
1.0 mg/kg qw 63 25 88 
1.5 mg/kg qw 57 14* 71 
0.5 mg/kg biw 33 33 67 
0.5 mg/kg tiw 17 17* 33 
0.75 mg/kg biw 23 35 4* 43 
AST increased 0.3 mg/kg qw 17* 17 
1.0 mg/kg qw 13 13 25* 50 
1.5 mg/kg qw 14 29 57* 100 
0.5 mg/kg biw 67 67 
0.5 mg/kg tiw 33 17 17 67 
0.75 mg/kg biw 23 13 13 17* 43 
Alkaline phosphatase increased 0.3 mg/kg qw 17 17 33 
1.0 mg/kg qw 25 25 
1.5 mg/kg qw 14 29* 43 
0.5 mg/kg biw 33 33 
0.5 mg/kg tiw 33 17 50 
0.75 mg/kg biw 23 13 17 17* 48 
Adverse eventDosingPatients per cohortGrade 1Grade 2Grade 3Grade 4Any grade
%%%%%
Abdominal pain 0.3 mg/kg qw 33 33 
1.0 mg/kg qw 62 38 100 
1.5 mg/kg qw 57 43 100 
0.5 mg/kg biw 67 67 
0.5 mg/kg tiw 33 33 
0.75 mg/kg biw 23 26 17 43 
Diarrhea 0.3 mg/kg qw 
1.0 mg/kg qw 63 25 88 
1.5 mg/kg qw 57 14* 71 
0.5 mg/kg biw 33 33 67 
0.5 mg/kg tiw 17 17* 33 
0.75 mg/kg biw 23 35 4* 43 
AST increased 0.3 mg/kg qw 17* 17 
1.0 mg/kg qw 13 13 25* 50 
1.5 mg/kg qw 14 29 57* 100 
0.5 mg/kg biw 67 67 
0.5 mg/kg tiw 33 17 17 67 
0.75 mg/kg biw 23 13 13 17* 43 
Alkaline phosphatase increased 0.3 mg/kg qw 17 17 33 
1.0 mg/kg qw 25 25 
1.5 mg/kg qw 14 29* 43 
0.5 mg/kg biw 33 33 
0.5 mg/kg tiw 33 17 50 
0.75 mg/kg biw 23 13 17 17* 48 

Abbreviation: AST, aspartate aminotransferase.

*One or more events were considered dose-limiting toxicities using definitions current at the time of the event.

Because both of these drug-associated syndromes occurred at or soon after infusion and tended to resolve rapidly thereafter, we hypothesized that they were related to the maximum achieved serum concentration, Cmax. PK modeling suggested that fractionating the dose (i.e., delivering a fraction of the dose twice weekly or thrice weekly) could significantly reduce Cmax while maintaining exposure as measured by the AUC. Modeling indicated that fractionation of the dose more efficiently reduced Cmax than did prolongation of the infusion time due to the relatively long half-life of the antibody.

The PK modeling predictions led us to modify the drug administration scheme to evaluate fractionated dosing by administering drug twice or thrice weekly. The initial dose of 0.5 mg/kg administered twice weekly proved to be better tolerated. Subsequently, two cohorts of patients were treated at 0.5 mg/kg administered thrice weekly and 0.75 mg/kg administered twice weekly. Figure 1 shows that both antibody-associated syndromes were reduced in frequency and severity by fractionated dosing. Because both 0.5 mg/kg tiw and 0.75 mg/kg biw were adequately well tolerated, the escalation of fractionated dosing was not considered feasible in light of the toxicities observed when administering the drug at 1.0 mg/kg qw, and a twice weekly schedule was more appealing than a thrice weekly schedule, the dose/schedule of 0.75 mg/kg biw was considered the MTD.

Fig. 1.

Amelioration of side effects with fractionated dosing. Patients were treated with RAV12 1.5 mg/kg qw (solid columns) or 0.75 mg/kg biw (hatched columns). Data shown are percent of patients experiencing selected adverse events at maximum grade obtained.

Fig. 1.

Amelioration of side effects with fractionated dosing. Patients were treated with RAV12 1.5 mg/kg qw (solid columns) or 0.75 mg/kg biw (hatched columns). Data shown are percent of patients experiencing selected adverse events at maximum grade obtained.

Close modal

Immunogenicity of the RAV12 antibody was also examined in this study. Using a simple bridging assay, 22% of patients tested positive for HACA. Non–life-threatening, mild to moderate infusion reactions occurred in three patients.

Pharmacokinetics

The observed PK parameters and those estimated by compartmental modeling are shown in Table 4. The large individual variation and small sample size of the initial cohorts preclude drawing strong conclusions about the observations. The PK did not seem to be strictly dose proportional. Cmax increased with dose but the Cmax for cohort 3 was larger than would be predicted based on Cmax in cohorts 1 and 2. This was reflected in the smaller V1 in cohort 3 (data not shown). The observed AUC for the first week of treatment increased with increased dose. The mean terminal half-life of RAV12 for the cohorts ranged from 58.7 to 150.6 hours. The mean terminal half-life for all patients in the study was 123.9 ± 95.8 hours. Plasma clearance ranged from 0.46 ± 0.30 to 2.02 ± 1.80 mL/h/kg across the cohorts, with a mean clearance of 0.94 ± 0.87 mL/h/kg for all patients. Volume of distribution at steady state ranged from 48.7 ± 27.1 to 85.9 ± 21.2 mL/kg across the cohorts, with a mean Vss of 67.8 ± 34.8 mL/kg for all patients. The terminal half-life, plasma clearance, and volume of distribution at steady state did not differ significantly between cohorts. The accumulation of RAV12 in plasma was observed in a subset of patients in all cohorts, as measured by an increase in Cmax and AUC over the course of treatment, with the highest frequency occurring in the cohorts receiving RAV12 at the 1.5 mg/kg/wk dosing schedules (data not shown).

Table 4.

PK of RAV12

Dosing SegmentDosing (mg/kg)NCmaxCmaxAUCαβClearanceVss
(FIRST dose) ng/mL(First week) ng/mL(First week) ng*h/mLt1/2 ht1/2 hmL/h/kgmL/kg
   mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD 
Weekly dosing 0.3 qw 8,561 ± 2,414 8,561 ± 2,414 1,101,716 ± 1,047,351 8.8 ± 5.2 130.0 ± 72.7 0.46 ± 0.28 58.1 ± 29.3 
Weekly dosing 1.0 qw 26,095 ± 6,390 26,095 ± 6,390 2,524,685 ± 1,966,709 7.9 ± 4.9 120.1 ± 91.2 0.84 ± 0.97 66.7 ± 39.0 
Weekly dosing 1.5 qw 63,532 ± 188,83 63,532 ± 18,883 3,192,139 ± 2,174,538 2.7 ± 3.6 86.9 ± 78.6 0.79 ± 0.70 48.7 ± 27.1 
Fractionated dosing 0.5 biw 135,84 ± 5,685 15,449 ± 9,601* 1,076,773 ± 1,040,189 12.9 ± 7.9 122.0 ± 108.1 2.02 ± 1.77 69.7 ± 33.0 
Fractionated dosing 0.75 biw 16,548 ± 5,004 24,114 ± 6,177* 3,219,240 ± 219,002 8.5 ± 5.9 138.7 ± 28.5 0.46 ± 0.03 85.9 ± 21.2 
Fractionated dosing 0.5 tiw 9,500 ± 3,182 16,719 ± 511* 1,415,525 ± 601,847 3.5 ± 2.9 58.7 ± 39.9 1.16 ± 0.80 64.3 ± 22.0 
Fractionated dosing (MTD expansion cohort) 0.75 biw 20 16,016 ± 4,256 20,930 ± 7,947 2,047,996 ± 1,438,285 19.5 ± 20.8 150.6 ± 127.1 1.08 ± 0.83 77.1 ± 42.3 
 Pt. 018 (responder) 1.5 mg/kg qw 65,267 65,267 1,675,588 1.2 15.6 0.90 20.2 
Dosing SegmentDosing (mg/kg)NCmaxCmaxAUCαβClearanceVss
(FIRST dose) ng/mL(First week) ng/mL(First week) ng*h/mLt1/2 ht1/2 hmL/h/kgmL/kg
   mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD 
Weekly dosing 0.3 qw 8,561 ± 2,414 8,561 ± 2,414 1,101,716 ± 1,047,351 8.8 ± 5.2 130.0 ± 72.7 0.46 ± 0.28 58.1 ± 29.3 
Weekly dosing 1.0 qw 26,095 ± 6,390 26,095 ± 6,390 2,524,685 ± 1,966,709 7.9 ± 4.9 120.1 ± 91.2 0.84 ± 0.97 66.7 ± 39.0 
Weekly dosing 1.5 qw 63,532 ± 188,83 63,532 ± 18,883 3,192,139 ± 2,174,538 2.7 ± 3.6 86.9 ± 78.6 0.79 ± 0.70 48.7 ± 27.1 
Fractionated dosing 0.5 biw 135,84 ± 5,685 15,449 ± 9,601* 1,076,773 ± 1,040,189 12.9 ± 7.9 122.0 ± 108.1 2.02 ± 1.77 69.7 ± 33.0 
Fractionated dosing 0.75 biw 16,548 ± 5,004 24,114 ± 6,177* 3,219,240 ± 219,002 8.5 ± 5.9 138.7 ± 28.5 0.46 ± 0.03 85.9 ± 21.2 
Fractionated dosing 0.5 tiw 9,500 ± 3,182 16,719 ± 511* 1,415,525 ± 601,847 3.5 ± 2.9 58.7 ± 39.9 1.16 ± 0.80 64.3 ± 22.0 
Fractionated dosing (MTD expansion cohort) 0.75 biw 20 16,016 ± 4,256 20,930 ± 7,947 2,047,996 ± 1,438,285 19.5 ± 20.8 150.6 ± 127.1 1.08 ± 0.83 77.1 ± 42.3 
 Pt. 018 (responder) 1.5 mg/kg qw 65,267 65,267 1,675,588 1.2 15.6 0.90 20.2 

*Estimated from modeling.

We had hypothesized that fractionated dosing would reduce Cmax proportionally, while maintaining overall exposure, as measured by AUC. A comparison of the once-weekly 1.5 mg/kg/dose cohort (cohort 3) and the twice-weekly 0.75 mg/kg/dose cohorts (cohort 5 and the expanded cohort) showed that the first week AUC exposure levels were comparable, whereas the Cmax levels were significantly reduced by fractionating the dose (Table 4).

Data from 14 patients receiving the twice-weekly 0.75 mg/kg/dose (MTD expansion cohort) was analyzed for PK parameters using a two-compartment PK model. The mean terminal half-life of RAV12 in this cohort was 150.6 hours with significant interpatient variability (range, 14.9-468.4 hours). The mean Cmax following the first dose was 16,016 ng/mL (range, 9,830-23,706 ng/mL). Systemic mean plasma clearance was 1.08 mL/h/kg (range, 0.15-2.47 mg/h/kg). The mean steady state volume of distribution was 77.1 mL/kg (range, 31.9-165.8 mL/kg). Accumulation was observed in a subset of patients as evidenced by increased Cmax, increased AUC, and increased plasma trough levels of RAV12 over the course of dose administration.

Previous preclinical studies established that twice-weekly administration of 3 mg/kg RAV12 maximally inhibited the growth of SNU-16 gastric tumor xenografts in mice over 4 weeks of treatment (3). PK analysis estimated the mean serum trough level of RAV12 during the study to be 48 μg/mL (range, 20-69 μg/mL). In vitro studies with COLO 205 colon tumor cells determined that 7 μg/mL was the 50% effective dose, whereas a concentration of 50 μg/mL or greater of RAV12 caused maximal cytotoxicity in vitro (3). Analysis of the serum trough level of RAV12 in the MTD expansion cohort showed that a mean trough level of 6 μg/mL was achieved over the course of treatment (data not shown). Additional preclinical studies may aid in understanding the relationship between the PK parameters of RAV12 (Cmax, AUC, and trough level) and efficacy.

Efficacy

Table 5 summarizes efficacy observations in the trial. Of note, one patient with refractory metastatic colorectal cancer involving three sites in the right lung who was treated at 1.5 mg/kg qw experienced an objective, confirmed, partial response detected at day 43 and had time to progression exceeding 8 months. One patient with advanced pancreatic cancer treated at 0.3 mg/kg qw was stable radiographically at day 43, and experienced a >50% decline in CA19-9. The patient continued treatment and had time to progression exceeding 5 months. Two patients with advanced colorectal cancer treated in the MTD Cohort Expansion segment at 0.75 mg/kg biw experienced radiographic stability of disease and had times to progression exceeding 4 months.

Table 5.

Efficacy observations

Dosing SegmentDosingnCR/PRStable diseasePDNE
Weekly dosing 0.3 mg/kg qw 
Weekly dosing 1.0 mg/kg qw 
Weekly dosing 1.5 mg/kg qw 
Fractionated dosing 0.5 mg/kg biw 
Fractionated dosing 0.75 mg/kg biw 
Fractionated dosing 0.5 mg/kg tiw 
Fractionated dosing (MTD expansion cohort) 0.75 mg/kg biw 20 12 
Dosing SegmentDosingnCR/PRStable diseasePDNE
Weekly dosing 0.3 mg/kg qw 
Weekly dosing 1.0 mg/kg qw 
Weekly dosing 1.5 mg/kg qw 
Fractionated dosing 0.5 mg/kg biw 
Fractionated dosing 0.75 mg/kg biw 
Fractionated dosing 0.5 mg/kg tiw 
Fractionated dosing (MTD expansion cohort) 0.75 mg/kg biw 20 12 

This study produced preliminary evidence that RAV12 has activity in the treatment of patients with advanced gastrointestinal malignancies. However, the safety profile of the antibody seems to preclude the delivery of highly efficacious doses of the antibody as no responders were observed with fractionated dosing or in the 20-patient MTD Cohort Expansion segment of the trial.

The mechanism(s) by which the unique RAV12-associated adverse events occurs is only partially understood. It was presumed, before the start of this study, that RAV12 would be excluded by tight junctions from the apical side of gastrointestinal epithelia, in which the RAAG12 antigen is demonstrably present by IHC. However, recent evidence in the in vitro T88 model of colonic epithelium suggests that RAV12 can be carried from the basolateral surface through the cellular cytoplasm by a mechanism of transcytosis mediated, at least in part, by the neonatal Fc receptor (FcRn; ref. 12).8

8Liang T, unpublished observation.

How the binding of RAV12 to its cognate antigen on the luminal surface of the colon leads to cramping pain and diarrhea remains unresolved. Because no patient developed bloody stools, chills, fever, rigors, or sepsis, it seems unlikely that RAV12 exerts a direct cytotoxic effect on the epithelial barrier. The effect of RAV12 may be to induce goblet cell degranulation or alter ion channel function leading to fluid shifts in the bowel. Similarly, how the binding of RAV12 to biliary epithelium might cause a picture of liver injury that is more characteristically hepatocellular rather than cholestatic remains unknown. The possibility that RAV12 could directly affect hepatocytes comes from a recent IHC study of “normal” liver tissue. Although RAAG12 antigen was not observed on the original survey of normal liver tissue, in hepatocytes resident in liver sections adjacent to RAAG12 staining metastatic deposits, RAAG12 staining can be seen on “normal hepatocytes.” Whether RAV12 can directly injure these aberrantly expressing RAAG12-positive hepatocytes, and thus produce the observed changes in liver function tests, is conjectural because no correlation of liver toxicity with liver metastases was made in this study.

The immunogenicity of RAV12 seems, at first observation, to be excessive for a chimeric monoclonal antibody infused into patients with advanced cancer. Twenty-two percent of patients tested positive for HACA in this study. Many of these results were confounded by the presence of either circulating RAAG12 antigen or circulating RAV12 antibody. Clinical sequelae of HACA positivity were observed in only three patients who experienced infusion reactions. Nevertheless, improvement in the immunogenicity profile of this antibody would be useful.

Both the safety profile of the antibody and the rate of immunogenicity suggest that RAV12 antibody might benefit from reengineering. Modification of the Fc portion of the antibody to reduce or eliminate FcRn binding (while leaving intact FcγR binding) and humanization of the antibody to reduce immunogenicity should result in an antibody that could be delivered at higher but more tolerable and, presumably, more effective doses. Given the remarkably high affinity of RAV12 for its cognate antigen, this strategy is likely to maintain on-target delivery despite reduction in half-life likely to result from diminution in FcRn binding. We expect to accomplish this reengineering before the reintroduction of the antibody into clinical trials.

L.S. Rosen, commercial research support: Raven Biotechnologies/MacroGenics, Inc.; J. Marshall, consultant/advisory board: Genentech, Roche, Amgen; H.A. Burris, J. Baughman, and D. Loo, employment and ownership interest, Raven Biotechnologies/MacroGenics, Inc. No additional potential conflicts of interest were disclosed.

Grant Support: Raven biotechnologies, Inc., now MacroGenics, Inc.

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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