Phase I Study of Abagovomab in Patients with Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

Patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer often achieve complete clinical remission following primary surgical cytoreduction, yet the majority of patients relapse (1, 2). Recent data have shown an association between intratumoral T cells and improved clinical outcome, lending support for immune-mediated disease control (3). Immunotherapies may be particularly suited for consideration in patients with clinical remission (4, 5). However, a major barrier to immunotherapy is that tumor-associated antigens and the corresponding peptides are self-antigens and are likely to be tolerated by the host. Therefore, there is a need to use alternative strategies for presenting the critical antigenic epitope(s) to the host in a different molecular environment (6).

CA125 is a cell-surface high molecular weight mucin (MUC 16) that is expressed in >80% of nonmucinous epithelial ovarian cancers (7, 8). Preliminary data evaluating oregovomab, the modified murine monoclonal antibody (mAb) B43.13 that binds to CA125, have shown a delay in time to disease progression in selected subpopulations (9). However, this passive immunotherapy approach is unlikely to be associated with induction of CTL responses. To induce both a humoral and a CTL response against a self-antigen, such as CA125, the anti-idiotype vaccine approach, based on the “immune network hypothesis,” represents an elegant method of transforming epitope structures into idiotypic determinants, which are expressed on the surface of antibodies (6, 10).

The generation and production of abagovomab for clinical use has been described previously (10). This antibody functionally mimics the CA125 antigen and induces humoral and cellular CA125-specific immunity (11). In previous studies, the anti-idiotypic antibody has been shown to induce immune reactivity against CA125-expressing tumors by induction of specific anti-anti-idiotypic antibodies (also called Ab3; refs. 11–13). A phase Ib/II study with arbitrary and fixed dosing using i.m. administration of abagovomab showed specific anti-anti-idiotypic antibody (Ab3) responses in 68.1% of patients with a statistically significant (P < 0.0001) association of the Ab3 response with longer overall survival (14). A study in patients with colorectal cancer with the anti-idiotypic mAb 105AD7 suggested that using lower doses of anti-idiotypic mAb may increase immunogenicity (15). Other studies have supported differences in immunogenicity of antigens based on route of administration (16). These findings support the purpose of this phase I study to determine the safety and immunogenicity of two routes (i.m. versus s.c.) and doses (0.2 versus 2.0 mg) using standardized product to determine an optimal dose and route for phase II/III evaluation.

Eligibility criteria. Eligible patients in this open-label, three-institution study had epithelial carcinoma arising in the ovary, fallopian tube, or peritoneum of stages II to IV. Primary treatment included cytoreductive surgery and platinum-based chemotherapy. All patients had relapsed and completed chemotherapy for recurrence. They could have small-volume residual measurable disease (<2 cm) on computed tomography imaging and/or may have an elevated CA125 level or may have been in complete clinical remission. Other requirements included Karnofsky performance status ≥70%, WBC count ≥1.5 × 10³ μL, platelets ≥100,000 cells/mm³; serum creatinine ≤1.5 times upper limits of normal; and total bilirubin, aspartate aminotransferase, and alkaline phosphatase ≤2 times upper limits of normal. Patients must have completed prior cytotoxic chemotherapy >3 weeks from entry. Patients were ineligible if they (a) had received prior anticancer vaccines, (b) had a known autoimmune disease, (c) had a murine protein allergy, or (d) had a positive human anti-mouse antibody (HAMA) at baseline defined as 100 ng/mL.

All patients provided written informed consent for this Institutional Review Board–approved protocol.

Treatment plan. Patients were randomized to four cohorts of 11 patients each. Cohorts varied by dose and route: cohort 1, 0.2 mg i.m.; cohort 2, 2.0 mg i.m.; cohort 3, 0.2 mg s.c.; cohort 4, 2.0 mg s.c. Immunizations were administered every 2 weeks for four injections (weeks 0, 2, 4, and 6) and then monthly for two additional injections (weeks 10 and 14). The immunogen was supplied by Goodwin Biotechnology, Inc. (Plantation, FL) and stored in 1 cm³ vials at 2°C to 8°C. It was drawn into a 3 cm³ syringe and administered with a 25-gauge needle (s.c. into buttocks, thighs, abdomen, or arms) or 22-gauge needle (i.m. into buttocks or arms). Patients remained in the treatment clinic for 30 minutes after immunization for observation.

Dose adjustment. Dose modification or delay was not permitted. Patients were to be removed from study for a dose-limiting toxicity defined using National Cancer Institute toxicity criteria (version 3.0) as (a) grade ≥II allergic reaction, (b) grade ≥II autoimmune reaction, (c) grade ≥III hematologic or nonhematologic toxicity, including fever, or (d) grade III injection site reaction.

Evaluation during study. Pretreatment evaluations included a history, physical and radiologic examination, vital signs, Karnofsky performance status assessment, and laboratory tests, including hematologic, biochemistry, CA125 serum level, and immunologic testing for HAMA level. Lymphocyte subpopulations were characterized at baseline and week 16 by flow cytometry using phycoerythrin-labeled anti-human CD4 (CD4⁺ T cells) and PP-labeled anti-human CD8 (CD8⁺ T cells), both purchased from BD PharMingen (San Diego, CA). Phycoerythrin-labeled anti-human CD56 (natural killer cells) and phycocyanin-labeled anti-human CD19 (B cells) were purchased from Beckman Coulter (Fullerton, CA). Patients had repeat complete blood cell count and biochemistry panel at weeks 2, 4, 6, 10, 14, and 20. CA125 serum level was repeated at weeks 4, 10, 14, and 20. Computed tomography imaging was done every 3 months or sooner if progression was suspected. Immune studies for HAMA, Ab3, and CA125-specific T-cell responses were done at weeks 0, 10, 16, and 20.

Molecular typing of HLA. HLA typing was done at the HLA typing laboratory of the Roswell Park Cancer Institute (Buffalo, NY) using sequence-specific primer pairs obtained from GenoVision (West Chester, PA; ref. 17).

Determination of HAMA. Anti-allotypic and anti-isotypic nonspecific HAMA titers (positive if >100 ng/mL) were determined by a commercially available ELISA (Medac, Hamburg, Germany).

Determination of anti-anti-idiotypic (Ab3) antibodies. Ab3 titers were assessed by ELISA as described previously (13, 18). Briefly, HAMAs that may interfere were eliminated using mouse IgG agarose. Absorbed sera were allowed to bind to abagovomab F(ab′)₂-coated microtiter plates. Complete abagovomab was added followed by incubation with horseradish peroxidase–labeled goat anti-mouse IgG antibodies. Ab3 concentrations in patients' sera are given in arbitrary units per mL corresponding to 1 ng/mL Ab1 (mAb OC 125, anti-CA125). Ab3 responses were termed positive if Ab3 concentration exceeded 1,000 arbitrary units/mL (18).

Determination of CA125-specific antibodies (Ab1′). Binding of antibodies in patient sera to CA125-positive OVCAR3 and SKOV8 human ovarian carcinoma cell lines was measured by flow cytometry. Tumor cells (2 × 10⁵) were resuspended in PBS/0.01% NaN₃/5% FCS and incubated for 1 hour at 4°C with postimmune sera (diluted 1:50) from patients immunized with anti-idiotypic mAb ACA125. After washing, FITC-conjugated rabbit anti-human IgG antibodies (DAKO, Hamburg, Germany) or FITC-conjugated rabbit anti-mouse IgG (DAKO) was added and incubation was continued for 30 minutes at 4°C. A negative control included cells that were incubated with conjugated secondary antibodies alone. Flow cytometric analysis was done on FACSCalibur (Becton Dickinson, Heidelberg, Germany). Binding capacity was rated positive if the binding of postimmune sera to CA125-positive cells exceeded 15% after subtracting the negative control (set on 5%), and the difference of the binding capacity between preimmune and postimmune sera was >5%.

Analysis of CA125-specific T-cell immune response. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque centrifugation of heparinized blood and cryopreserved in AIM-V, 10% human serum albumin, and 10% DMSO. In vitro sensitization of cells was done by using modifications of procedures described by others (19–21). Effector cells for enzyme-linked immunospot (ELISPOT) were generated on day 1 from nonadherent cells activated with phytohemagglutinin (10 m g/mL) and interleukin (IL)-7 (20 ng/mL). Concurrently, target cells (plastic-adherent PBMCS) were incubated separately with abagovomab (50 μg/mL) and granulocyte macrophage colony-stimulating factor (1,000 units/mL). On day 3, target and effector cells were cocultured in the presence of IL-2 (10 units/mL). On day 7, CA125 was added to the cultures at 500 units/mL in fresh medium containing IL-2. Target cells for ELISPOT were generated from nonadherent cells activated with phytohemagglutinin (10 μg/mL) and IL-7 (20 ng/mL; ref. 20). ELISPOT restimulation of effector cells for IFN-γ production was done with medium alone or with phorbol 12-myristate 13-acetate/ionomycin (1 ng/mL and 1 μmol/L) or CA125 (500 units/mL) or abagovomab (20 μg/mL) or a combination of CA125 and abagovomab in duplicate wells to give a final volume of 200 μL. As a control, PBMCs from a normal donor were sensitized in an identical manner and used in ELISPOT. Plates were incubated for 48 hours at 37°C and developed using biotinylated secondary anti-IFN-γ from Mabtech (Mariemont, OH), clone Mab7-B6-1, and the Vectastain Elite Standard avidin-biotin complex method and 3-amino-9-ethylcarbazole substrate kits (Vector Laboratories, Burlingame, CA). Computer-assisted image analysis was used to quantitate the assays using the Zeiss (Thornwood, NY) ELISPOT 4.1.56 system. Specifically, a positive response was defined as at least 20 more spot-forming units per 100,000 PBMCs in antigen-stimulated wells than in control wells and ∼1.5 times the number of spot-forming units in the experimental than in the control (22).

Luminex cytokine assay for detection of IFN-γ and IL-10. IFN-γ and IL-10 were detected in sera as described previously (23). Briefly, capture and detection antibody pairs directed against different noncompeting epitopes of IFN-γ (R&D Systems, Minneapolis, MN) and IL-10 (BD PharMingen) were covalently coupled to multianalyte carboxylated microspheres (Luminex Corp., Austin, TX) according to the manufacturer's directions. The Luminex cytokine assays were done in 96-well microtiter plates (MultiScreen-HV plates, Millipore, Billerica, MA) with polyvinylidene difluoride membranes using a Tecan Genesis liquid handling robot (Tecan, Research Triangle Park, NC) for all dilutions, reagent additions, and manipulations of the microtiter plate. Bead sets coated with capture antibody were diluted in PBS-TBN and pooled, and 1,000 beads from each set were added per well. Recombinant protein standards were titrated from 33,333 to 0.56 pg/mL using 3-fold dilutions in PBS-TBN. Samples and standards were added to wells containing beads. The plates were incubated at ambient temperature for 20 minutes on a rocker and then washed twice with PBS using a vacuum manifold to aspirate. Biotinylated detection antibodies to each cytokine, diluted in PBS-TBN, were added next, and the plates were incubated and washed as before. Finally, phycoerythrin-conjugated streptavidin (Caltag Laboratories, Burlingame, CA) was added to each well and the plates were incubated for 20 minutes and washed. The beads were resuspended in 160 μL PBS-TBN and analyzed on a Luminex 100 (Luminex). Using BeadView Software (Upstate, Charlottesville, VA), a regression was calculated using the bead mean fluorescent intensity values for each concentration of recombinant protein standard. Points deviating from the best fit line (i.e., below detection limits or above saturation) were excluded from the curve. Sample cytokine concentrations were calculated from the mean fluorescent intensities of their beads by interpolating the resulting best fit line.

Disease assessment. Standard Response Evaluation Criteria in Solid Tumors criteria were used to characterize response or disease progression. Time to objective disease progression was calculated from time of study entry to time of radiographic or clinical disease progression.

Statistical considerations. The primary exploratory study end point was to confirm the safety of abagovomab administration at the doses of 0.2 and 2.0 mg by the i.m. or the s.c. route. The secondary end point was to characterize the immune response induced by anti-idiotypic mAb ACA125 administration primarily by Ab3 determination and select a dose and route for phase II/III evaluation. Although not the end point of this phase I trial, time to objective tumor progression was recorded.

A standard 2 × 2 factorial design was used with 44 patients being randomly entered into one of the four cells (11 in each). The primary end point was post-baseline peak Ab3 value. The main effects for the dose and route were tested using the Kruskal-Wallis test. The proposed sample size for each factor level would enable the detection of a standardized difference of 1 with 80% power and 2.5% significance level. We made the assumption that if the dose level main effect is close to 0 (i.e., the 0.2 mg dose is not superior), the previously tested 2 mg dose would be chosen for phase II trials given its historical efficacy (15). Similarly, if the route main effect was not significant, the s.c. route would be preferred for ease of administration.

Patient characteristics. A total of 44 patients were enrolled and randomized between January and December 2003, and 2 patients withdrew before immunization. The patient characteristics are listed in Table 1. The median age was 57.5 years, with a range of 39 to 78 years. Ninety-three percent had stage III or IV disease at diagnosis and serous histology predominated. Ninety-one percent had a Karnofsky performance score of ≥90%. Platinum/taxane-based treatment was received by all patients as part of primary therapy. Thirty-three percent of patients entered the study in second complete or partial remission, and 67% of patients were in a third or greater complete or partial remission, defining a heavily pretreated group. Patients had received a median of 4 chemotherapy regimens before study entry, ranging from 2 to 11, and the median time from day of last chemotherapy to first vaccination was 1.5 months (0-12.6 months). At the time of study entry, 24% of patients were in complete clinical remission, whereas 76% had small-volume measurable disease or an elevated CA125 serum level.

Patient flow. Forty-two patients received at least one immunization and were eligible for safety analysis and clinical follow-up. Patients were required to remain on study until week 10 (the first post-baseline Ab3 determination) to be considered for analysis of immunogenicity. Nine patients withdrew from study before week 10, leaving 33 patients for immunologic assessment (progression of disease, 6; withdrawal of consent, 2; adverse event thought unrelated to study drug, 1). Twenty-eight (66%) patients received all six immunizations, with the total number of immunizations ranging from two to six.

Adverse events. Abagovomab administration was well tolerated. The most common drug-related adverse events are listed in Table 2. Self-limited and mild fatigue, fever, myalgia, and localized injection site reactions were most frequent. Leukopenia was seen in four patients (grade 1) and anemia (maximum grade 2) was seen in nine patients, but no clinically relevant hematologic abnormalities were noted. No clinical or laboratory evidence of autoimmunity was seen. There were five serious adverse events about four patients in this study (intestinal obstruction, 3; abdominal pain, 1; venous thrombosis, 1). All were considered unlikely related to the study drug. The patient with thrombosis represents the one patient with an adverse event withdrawn from study. No hypersensitivity reactions were seen.

Immune response. Lymphocyte subpopulations were analyzed at baseline and at week 16; the results are presented in Table 3. Although the median time from day of last chemotherapy to first immunization was 1.5 months, the results indicate normal frequencies of natural killer cells, B cells, CD8⁺, and CD4⁺ T cells, indirectly indicative of an immunocompetent population. HLA-A alleles were assessed in patients (n) with available samples and showed HLA-A-1 (9), HLA-A-2 (15), HLA-A-3 (6), HLA-A-11 (3), HLA-A-24 (4), HLA-A-29 (2), HLA-A-30 (1), and HLA-A-32 (1) There was no association between HLA-A alleles and induction of immune response.

The primary efficacy end point was Ab3 response as presented in Table 4. At baseline, five patients (15.2%) tested were Ab3 positive (range, 1,042-5,405 ng/mL). For all treatment groups, increasing titers of Ab3 were seen with an overall maximum median Ab3 titer post-baseline of 364,522 ng/mL (range 5,453-687,315 ng/mL). Neither the effect of route of administration (i.m. versus s.c.; P = 0.6268) nor the effect of dose (0.2 versus 2.0 mg; P = 0.4602) on maximum post-baseline Ab3 titer was statistically significant nor was the effect of cohort (P = 0.4944). Ab3 and HAMA responses were seen in all patients who reached the week 10 evaluation point as shown by scatter plot in Fig. 1A and B. With regards to HAMA presented in Table 5, the maximum median post-baseline value was 5,700 ng/mL (373-2,300 ng/mL). Values by cohort are listed in Table 4. During the study, 30 patients (90%) tested were HAMA positive at least at one visit.

Induction of Ab1′ was weakly present in two patients, with a stronger response in the third patient, with reactivity to OVCAR3 and SKOV8 by fluorescence-activated cell sorting (Fig. 2).

Induction of IFN-γ-producing T cells. Analysis for IFN-γ-producing T cells was limited to only five patients with sufficient prevaccination and postvaccination PBMCs, and the results are considered exploratory. The results indicate a consistent increase in CA125-specific IFN-γ-producing T cells following immunizations (Fig. 3). None of the prevaccination or healthy donor samples showed a CA125-specific response.

In a subset of 25 patients with sufficient sera, IFN-γ and IL-10 serum levels were determined with the Luminex assay. The difference between preimmunization (week 0) and postimmunization (week 16) levels of IFN-γ and IL-10 was statistically different from 0 (P < 0.0001 and P = 0.0097, respectively) based on a signed rank test. The median increase for IFN-γ was 243 (range 45-946), and the median difference for IL-10 was 0.8 (range, −8.7 to 19.3). As shown in the box plots in Fig. 4, there was a greater magnitude of increase for IFN-γ compared with IL-10, suggesting induction of a predominant Th1 cytokine production by CA125-specific effector cells.

Clinical data. Although not the outcome of this phase I study, time to clinical disease progression was recorded. Twenty-four percent of patients entered the study in complete clinical remission, whereas 76% had radiographic or CA125 evidence of residual active disease. Sixty-six percent of patients entered the study in third or greater complete clinical remission or response. Over the observational period in the study, 12 patients (36.4%) showed stable disease, whereas 21 patients (63.6%) had disease progression. The median time to disease progression of all patients was 4 months (95% confidence interval, 3-5) estimated using the Kaplan-Meier method. Median overall survival has not been reached with median follow-up of 22 months. For patients in complete clinical remission at the start of study, the median time to progression was 9.6 versus 3.4 months in the non-complete clinical remission group (P = 0.004 using the log-rank test). The uniform Ab3 response (all patients had a positive response) does not permit a comparison group of patients without immune response.

A previous phase Ib/II study showed the immunogenicity of abagovomab given at 2.0 mg i.m. with 68.1% of patients generating Ab3 antibodies and described an association of this humoral response with increased survival (13). However, neither the dose nor the route had been optimized, and current data suggest that both dose and route may affect the efficacy of vaccination (15, 16). The purpose of this phase I study was to evaluate the safety and optimize the dose and route of good manufacturing practice-grade abagovomab before phase II to III testing.

The vaccine was well tolerated using both doses and schedules, and no patient was removed from the study because of treatment-related side effects. Adverse events related to vaccine administration were self-limited and typical of similar vaccine constructs, such as myalgia, fever, and local injection site reactions. Despite the administration of vaccination after the development of HAMA, no hypersensitivity reactions were noted.

With regards to the primary end point of a specific anti-anti-idiotypic antibody response (Ab3), all patients had an Ab3 response (median 285,793 ng/mL). This compares favorably with the 68.1% positivity rate in the prior study (13). The reason for the high rate of Ab3 response in this study, even if patient characteristics between the studies were similar, may be related to the immune status of patients at baseline or to the use of good manufacturing practice-grade standardized product. In the current study, the numbers of immune cell populations at baseline and at week 16 were comparable with those of healthy individuals. This implies that immune competence is common in recurrent ovarian cancer patients with prior therapy and supports the use of this population for exploratory studies of immune-based therapy. In addition, all patients developed HAMA. It is possible that the HAMAs bind to the immunizing anti-idiotypic antibody and the entire complex is endocytosed by antigen-presenting cells, leading to the generation of more robust humoral and cellular responses.

Although we were limited by the number of patients with samples sufficient that could be analyzed for IFN-γ ELISPOT reactivity to CA125, all of the five patients tested showed evidence of T-cell immunity to CA125. We adapted an in vitro sensitization step before IFN-γ ELISPOT because it allows detection of low numbers of T-cell precursors specific for self-epitopes (19) without the requirement for knowledge of CD8⁺ and CD4⁺ T-cell epitopes. The relatively lower response obtained with in vitro stimulation using CA125 alone could be due to the production of Ab3 (24) following stimulation, which binds CA125 (18), resulting in a lower effective concentration of CA125 in the assay. A similar induction of antigen-specific T cells by an anti-idiotypic antibody has been reported in colon carcinoma patients (21). However, our technique suffers from the disadvantage that it does not detect the frequency of circulating precursor cells but the frequency of cells proliferating in response to a cognate CA125 epitope in vitro. Therefore, our results can only be interpreted as present or absent and not as a quantitative comparison of precursor frequencies. Despite these limitations, we consistently showed induction of CA125-specific IFN-γ-producing T cells following immunization using CA125 alone, abagovomab alone, or both for in vitro stimulation.

In support of the ELISPOT data is the observation of significant increase in serum IFN-γ when prevaccination samples (week 0) were compared with postvaccination samples (week 16) in a subset of 25 patients. Although there was also an increase in serum IL-10, this was less striking, suggesting a predominant Th1 cytokine differentiation of vaccine-induced effector cells. Taken together, our data indicate that immunization with abagovomab can evoke an Ab3 (Ab1′) as well as a cellular immune response in ovarian cancer patients.

There were no statistical differences with regards to Ab3 production between cohorts of varying route or dose. The s.c. route will be selected for patient convenience in future studies, and the initial 2.0 mg dose will be used. The hypothesis was that the lower dose in this study may have been more immunogenic (15), but because this has not proven to be true, we have confirmed the previously established dose, which showed the strong association between antibody response and improved outcome.

The described progression-free survival of 4 months overall is similar to that seen in other studies following treatment, such as liposomal doxorubicin or topotecan (25). The median 9.6 months progression-free interval for the complete clinical remission population is similar to that previously described in other studies in this patient population (26). Given the uniform Ab3 response in this phase I study, it is not possible to compare patients with Ab3 positivity with those without as in the previous phase Ib/II study by Wagner et al. (13, 18), where a 68.1% Ab3 response was induced (13, 18).

Abagovomab vaccination is well tolerated, and this study has optimized the dose and route (2.0 mg s.c.) for further evaluation. The uniform immune response is encouraging, and previous data showing an association between Ab3 antibody production and improved outcome warrant phase II or III testing of this optimized construct.