Phase I trial of a murine antibody to MUC1 in patients with metastatic cancer: evidence for the activation of humoral and cellular antitumor immunity

J. S. de Bono1,*, S. Y. Rha1, J. Stephenson1,2, B. C. Schultes3, P. Monroe1, G. S. Eckhardt1, L. A. Hammond1, T. L. Whiteside4, C. F. Nicodemus5, J. M. Cermak5, E. K. Rowinsky1 and A. W. Tolcher1

1 Institute for Drug Development, Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX; 2 Brooke Army Medical Center, Fort Sam, Houston, TX; 3 AltaRex, Waltham, MA; 4 University of Pittsburgh Cancer Institute, Pittsburgh, PA; 5 Unither Pharmaceuticals, Wellesley, MA, USA

* Correspondence to: Dr J. S. de Bono, Centre for Cancer Therapeutics, Institute for Cancer Research, Royal Marsden Hospital, Downs Road, Sutton, Surrey SM2 5PT, UK. Tel: +44-20-8722-4302; Fax: +44-20-8642-7979; Email: jdebono{at}icr.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background: BrevaRex® mAb-AR20.5 is a murine anti-MUC1 monoclonal antibody generated to induce MUC1 antigen-specific immune responses through the formation of immune complexes with circulating MUC1 and/or MUC1-expressing tumor cells that may target these immune complexes (IC) to receptors on dendritic cells (DCs).

Patients and methods: A phase I study focusing on safety and immunology evaluated 1, 2 and 4-mg doses. Seventeen patients with MUC1-positive cancers received intravenous infusions of the antibody over 30 min on weeks 1, 3, 5, 9, 13 and 17 of treatment.

Results: mAb-AR20.5 was well-tolerated, not associated with dose-limiting toxicity, and did not induce hypersensitivity reactions. Overall, five of 15 evaluable patients developed human anti-mouse antibodies (HAMA), five developed anti-idiotypic antibodies (Ab2) and seven developed anti-MUC1 antibodies. Immune responses were most prominent in the 2-mg dose cohort for all parameters tested, and treatment-emergent MUC1-specific T-cell responses were detected in five of 10 evaluable patients treated with mAb-AR20.5.

Conclusions: The injection of a murine antibody to MUC1 induces MUC1-specific immune responses in advanced cancer patients. Anti-MUC1 antibody increases correlated with decrease or stabilization of CA15.3 levels (P=0.03). The 2-mg dose of mAb-AR20.5 showed strongest biological activity, and will be evaluated in future efficacy trials.

Key words: antibody, BrevaRex®, mAb-AR20.5, MUC1, phase I trial


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Mucins are highly glycosylated proteins of high molecular weight [1Go] that are present on the membrane surface of normal and malignant epithelial cells and secreted into the plasma. The structure and pattern of expression of MUC1 proteins on cells indicate the diverse roles in both normal epithelial function and carcinogenesis [2Go]. Mucins provide protection and lubrication to the tissue surface, modulate the immune response, regulate cellular adhesion and cellular motility, and transmit transmembrane signals through tyrosine and serine phosphorylation in their cytoplasmic tail domains. Studies indicate that MUC1 binds growth factor receptor binding protein-2 (Grb2) and son of sevenless (SOS), modulates the function of ß-catenin and E-cadherin-mediated cellular adhesion, and potentiates the downstream signaling of growth factor receptors [3Go–6Go]. Evidence implicating MUC1 in the process of carcinogenesis has been derived from MUC1 knockout mice studies. These animals develop normally, but when mammary tumors are induced by the polyoma middle T antigen under the control of the mouse mammary tumor virus (MMTV) promoter, they develop tumors that grow substantially more slowly, and with a lower incidence of tumor metastases, than wild-type mice [7Go]. Clinical studies analyzing the incidence of naturally occurring MUC1 antibodies in early breast cancer patients also indicate that MUC1 may play a role in breast cancer progression. Stage I and II breast carcinoma patients that possess intrinsic antibodies to MUC1 have significantly improved survival, whereas stage II patients who exhibited anti-MUC1 antibodies had predominantly local recurrences, with a statistically significant reduction in distant metastatic disease recurrence [8Go]. Similar results have been observed in other tumor types including pancreatic cancer, suggesting that the generation of an immune response to MUC1 may represent an important therapeutic strategy [9Go].

These and other studies were the impetus for the development of monoclonal antibodies (mAbs) targeting MUC1. The murine monoclonal antibody BrevaRex® (mAb-AR20.5; AltaRex Corp., Edmonton, AB, Canada; and United Therapeutics, Silver Spring, MD, USA) recognizes the tandem repeat protein backbone of MUC1, targeting the sequence DTRPAP [10Go]. This highly specific anti-MUC1 IgG1 antibody has demonstrated antitumor activity against MUC1-expressing human tumor xenografts (NIH: OVCAR-3; HLA-A2) in severe combined immunodeficiency mice reconstituted with HLA-A2-positive human peripheral blood leukocytes (>75% tumor growth reduction). Furthermore, utilizing a metastatic MUC1 transfected tumor cell line in a CB6F1 mouse model, mAb-AR20.5 prevented tumor generation in >60% of the mice tested, increasing murine median survival from 51 days to >120 days [10Go].

The mechanism of action of mAb-AR20.5 includes the generation of MUC1-specific immune responses through complex formation of the murine antibody with MUC1 in circulation and/or on MUC1-expressing tumor cells. Dendritic cells that acquire antigenic substances through receptor-mediated endocytosis (FcR, C-type lectins, complement receptors) as compared with macropinocytosis show facilitated uptake and T-cell activation [11Go]. Antigens in IC form, taken up by such receptors, have been shown to induce CD4+ and CD8+ T-cell responses [12Go–16Go]. Regarding MUC1-specific T-cell activation, we have found stronger CD4+ and CD8+ T-cell induction with dendritic cells pulsed ex vivo with MUC1-antibody complexes compared with dendritic cells pulsed with MUC1 alone [17Go, 18Go]. Engagement of the activating Fc receptors (CD16, CD64) also induced dendritic cell maturation [19Go]. The results suggest that effective cancer vaccines may be generated in IC form. Intravenously administered low-dose antibodies such as OvaRex® mAb-B43.13 and BrevaRex® mAb-AR20.5 can target circulating antigen (CA125 or MUC1) and form ICs in vivo that are taken up by antigen-presenting cells and thereby promote a more effective presentation of the antigen to the immune system.

In vitro, mAb-AR20.5 did not induce complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity in the presence of human peripheral blood leukocytes [10Go], and the low dose used in the clinical setting is not sufficient to mediate direct antitumor effects in vivo. MUC1-targeted B and T cells are nonetheless generated by the administration of naked DNA or protein from the MUC1 tandem repeat recognized by mAb-AR20.5. This antibody may therefore enhance the processing and immunogenicity of MUC1 as demonstrated for two other murine antibodies to the tumor antigens CA125 and prostate-specific antigen [20Go–22Go]. Furthermore, studies in rabbits and rats showed induction of an idiotypic immune response by mAb-AR20.5. Rats and rabbits were immunized repeatedly intravenously with mAb-AR20.5 at doses 0.65–5.2 mg/m2 (equivalent to 1–8 mg/60 kg patient), and host-anti-mouse and anti-idiotypic antibodies (Ab2) responses were measured after each of five injections. Doses of 1.15–2.3 mg/m2, equivalent to 2–4 mg/patient, resulted in optimal immunological activity in both animal models (unpublished data).

These preclinical data led to the initiation of a dose-escalation phase I trial of this murine anti-MUC1 antibody in patients with advanced carcinomas expressing MUC1. Three dose levels around the suggested biologically active dose from preclinical models were prospectively planned. Dose escalation to the maximum tolerated dose was not planned, since preclinical studies indicated little evidence of toxicity at doses 100-fold larger than the biologically active dose (unpublished data). The principal objectives of this study were to: (i) characterize the toxicities of mAb-AR20.5 administered as a 30-min intravenous infusion on weeks 1, 3, 5, 9, 13 and 17 in patients with advanced solid malignancies at 1-, 2- and 4-mg doses; (ii) determine the most immunogenic dose with acceptable toxicity and recommend a safe starting dose on this schedule for phase II studies; (iii) characterize the humoral and cellular immunological responses induced by mAb-AR20.5; and (iv) seek preliminary evidence of antitumor activity.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Eligibility
Patients with pathologically documented advanced solid tumors with detectable (>10 U/ml) secreted MUC1 antigen (CA15.3 or CA27.29) refractory to conventional therapy or for whom no effective therapy existed were candidates for this study. Eligibility criteria also included: age ≥18 years; Eastern Cooperative Oncology Group (ECOG) performance status ≤2; a life expectancy of at least 12 weeks; up to four prior chemotherapy regimens; no chemotherapy, wide-field radiotherapy, or other experimental therapy within 4 weeks of treatment; no biological response modifiers within 6 weeks; no prior therapy with murine monoclonal antibodies; no known allergies to murine proteins or prior anaphylactic drug reaction; adequate haematopoietic (absolute neutrophil count ≥1500/µl; lymphocyte count ≥600/mm3; platelet count ≥75 000/µl; hemoglobin ≥9 g/dl), hepatic [total bilirubin level ≤1.5x the upper normal limit (UNL); aspartate aminotransferase and alanine aminotransferase ≤3x UNL] and renal (creatinine ≤1.6 mg/dl) function; no co-existing medical condition of sufficient severity to limit full compliance with the study; no evidence of brain metastases; and no active serious infection. Patients with active autoimmune diseases, known immunodeficiency disease, prior splenomegaly, and/or treatment with immunosuppressive agents including cyclosporin and steroids were excluded. All patients gave written informed consent according to federal and institutional guidelines before starting treatment. The protocol was reviewed and approved by the Institutional Review Board.

Drug administration and dosage
mAb-AR20.5 was supplied as a sterile, lyophilized powder refrigerated at 2–8°C in vials containing 2 mg of antibody, 0.1 mg of stannous chloride dihydrate, 2.7 mg of sodium pyrophosphate and 16.8 mg of sucrose. When required for administration, it was aseptically diluted in 50 ml of normal saline and administered within 4 h of reconstitution. The mAb-AR20.5 dose was administered to the patient by slow intravenous infusion over a 30-min period.

Based on preclinical data showing optimum immunologic activity at doses of 1.15–2.3 mg/m2 in rabbits and rats (equivalent to 2–4 mg/patient), and lack of preclinical toxicity (at doses up to 100 mg/m2), the starting dose for this phase I trial was determined to be 1 mg per dose administered at weeks 1, 3, 5, 9, 13 and 17. Dose escalation by 100% increments to 2 mg and 4 mg was then planned. The dose to be recommended for phase II testing was the maximum safely administered dose seen to induce the optimal cellular and humoral immunological responses.

Patients were monitored for a full 1 h after the completion of mAb-AR20.5 administration for symptoms or signs of anaphylaxis. Individuals, who experienced WHO grade 3 or 4 hypersensitivity events were not eligible to receive further treatment. Safety was evaluated based on adverse event reporting, physical examination including vital signs, ECOG performance status, and clinical laboratory results. Patients who received at least one infusion were considered evaluable for safety. Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria (NCI CTC) version 2.

Pretreatment and follow-up studies
Medical history, physical examination and routine laboratory studies were performed prior to treatment and every 2 weeks thereafter. Routine laboratory studies included serum electrolyte levels, chemistries, renal and liver function tests, complete blood cell counts, differential leukocyte counts, prothrombin time, and urinalysis. Pretreatment studies also included relevant radiological studies for evaluation of all measurable or evaluable sites of malignancy, as well as an assessment of relevant tumor markers (serum CA15.3 and CA27.29). Bioactivity was assessed in terms of immune responses and correlated to clinical response. Physical examination and radiological studies for disease status assessments were repeated after every other course, or as needed to confirm response. Tumor response in patients with measurable disease was determined using computed tomography scan or other imaging modalities as defined by WHO criteria. According to the protocol, the key population to be evaluated for efficacy was patients who had received at least three doses of study drug.

Immunological studies
The immune bioactivity of mAb-AR20.5 was evaluated as the patient's ability to mount both humoral and cellular immune responses, which included measurement of human anti-mouse antibodies (HAMA), Ab2 and anti-MUC1 antibodies, and induction of MUC1 peptide-directed cellular immunity. Serum samples for immunological testing were collected at baseline and prior to each mAb-AR20.5 administration; for humoral assays at weeks 1, 3, 5, 9, 13 and 17, and for cellular assays at weeks 1, 7, 13 and 17.

Human anti-mouse antibodies. Patients treated with murine antibodies produce HAMA in response to the constant region of the murine antibody. In this trial, HAMA levels were measured using a commercially available enzyme-linked immunosorbant assay (HAMA-ELISA; Medac, Hamburg, Germany). The validated assay was performed by MDS Clinical Trial Laboratories (Toronto, Canada). The detection limit for the assay is 5 ng/ml; the upper limit value for normal subjects is 200 ng/ml. A robust response was defined as achieving a HAMA level >5000 µg/ml at any post-injection timepoint. This cut-off was found to be associated with clinical benefit in studies with mAb-AR20.5 [21Go, 23Go].

Anti-idiotypic antibodies. Ab2 are produced to the variable region of an immunogenic antibody. Ab2 to mAb-AR20.5 were measured by an ELISA assay optimized, qualified and performed by AltaRex Corp. Briefly, patient serum diluted 1:25 was incubated in protein G-coated plates along with standards and controls (purified rabbit anti-AR20.5 antibodies). Biotinylated mAb-AR20.5 was used to complex Ab2 bound to the coated protein G and the complex was then developed with streptavidin–horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AB, USA; diluted 1:15 000) using trimethylbenzidine as a substrate. HAMA reactivity was subtracted by running a parallel sample set with biotinylated control IgG1 (MOPC-21) instead of biotin-AR20.5. The color complex was measured optically in an ELISA photometer at 450 nm. Results are reported in U/ml. The detection limit for the assay is 10 U/ml (or 250 U/ml if adjusted for a 1:25 dilution factor); the upper limit value for normal subjects is 250 U/ml. A robust response was defined as achieving Ab2 levels above twice the cut-off limit for normals (>500 U/ml).

Anti-MUC1 antibodies. Antibodies against the tumor-associated antigen MUC1 were measured in an optimized and qualified ELISA performed by AltaRex Corp. Patient serum samples were either assayed untreated, or they were pretreated with a large excess of mAb-AR20.5 (400 µg/ml) for 1 h at room temperature to displace human anti-MUC1 antibodies from circulating immune complexes. mAb-AR20.5-pretreated samples were then incubated with goat anti-mouse IgG-coated agarose overnight at 4°C followed by removal of the agarose-bound mAb-AR20.5 (murine IgG1), MUC1/mAb-AR20.5 or Ab2/mAb-AR20.5 immune complexes by centrifugation, leaving free human anti-MUC1 antibodies in solution. For the assay, streptavidin-coated plates (Biotin-Trap SAV; Intermedico, Markham, ON, Canada) were incubated with a 31mer biotinylated MUC1 peptide (APDTRPAPGSTAPPAHGVTSAPDTRPAPGSC-Biotin; Alberta Peptide Institute, Edmonton, AB, Canada). Untreated and pretreated patient samples (diluted 1:25 in newborn calf serum) were incubated on the streptavidin and biotinylated MUC1 peptide-coated plates along with standards (mAb-AR20.5–human IgG conjugate) and controls (monkey anti-MUC1 serum, normal human serum). The formed immune complex was detected with peroxidase-conjugated goat F(ab)2 anti-human IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; diluted 1:10 000) and revealed by reaction with the peroxidase substrate ABTS 2,2'-azinobis (3-ethylbenzthiazoline-sulfonic acid) and read in an ELISA photometer at 405/490 nm. Values are reported as U/ml. The detection limit for the assay is 10 U/ml (or 250 U/ml if adjusted for a 1:25 dilution factor); the upper limit value for normal subjects is 250 U/ml. A robust response was defined as a three times increase over baseline.

Epitope specificity of human anti-MUC1 antibodies. Anti-MUC1 antibody-positive serum samples were tested for epitope specificity using the anti-MUC1 antibody ELISA with samples incubated in the presence of five sequence-overlapping MUC1-derived 6mer peptides (6 amino acids long: DTRPAP, APGSTA, TAPPAH, AHGVTS and TSAPDT), spanning the tandem repeat region of 20 amino acids and the sequence GSTAPPAHGVTSAPDTRPAP. The long 31mer MUC1 peptide used to coat the plates provided the positive inhibition control; monkey anti-MUC1 serum and normal human serum served as positive and negative assay control, respectively. Both untreated and pretreated serum samples (for disruption of immune complexes; see above) were used in this study.

T-cell responses. T-cell responses to MUC1 pre- and post-mAb-AR20.5 treatment were assessed for production of interferon (IFN)-{gamma} by enzyme-linked immunospot (ELISPOT) assay in response to stimulation with 31mer MUC1 peptide. The IFN-{gamma} ELISPOT assay was developed, optimized, validated and conducted by the Immunologic Monitoring and Cellular Products Laboratory (University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA). Peripheral blood mononuclear cells (PBMC) obtained from heparinized blood of patients before and after administration of mAb-AR20.5 were isolated on Ficoll–Hypaque gradients and cryopreserved. Cells from pre- and post-therapy samples were thawed simultaneously, washed and stimulated with the 31mer MUC1 peptide for 1 week, using autologous PBMC as antigen-presenting cells. Cultures were then restimulated for 24 h with MUC1 peptide-pulsed autologous dendritic cells generated in parallel with the stimulated PBMC cultures by a previously described method [24Go]. MUC1-specific T cells were quantified in an IFN-{gamma} ELISPOT assay, as described previously [25Go]. Assays were performed in triplicate and results evaluated for responses over background and baseline by permutation test as described [26Go].

Statistical methods and calculations
A description of the patient population included summaries of patient demographics at baseline, disease characteristics at baseline, prior therapies, previous and concurrent medical conditions by disease type, and concomitant medications by class. Categorical variables were summarized by number and percent, and continuous variables were summarized by sample size, mean, median, standard deviation and range (minimum, maximum). Safety information was characterized using descriptive summary statistics including summaries of the adverse events based on incidence, grade and relation to study drug. Humoral immune response data were evaluated based on cut-off values for normal donors and robust responses (see assay descriptions). T-cell responses were assessed for significant increases over background and baseline using a permutation test [26Go]. All calculations of clinical data were performed using SAS software, version 8.0.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
This was an open-label dose escalation study. Seventeen patients (14 women and three men) with MUC1-expressing tumors were entered in this study and received at least one dose of mAb-AR20.5. The pertinent demographic characteristics for the entire study population are listed in Table 1. Patients received either a 1-, 2- or 4-mg dose of mAb-AR20.5, infused at weeks 1, 3, 5, 9, 13 and 17. Two, three, three, eight and one patient(s) received one, two, three, four and six total doses of mAb-AR20.5, respectively (see Table 2). Fifteen patients with pre- and post-baseline samples were evaluable for humoral responses (HAMA, Ab2 and anti-MUC1 antibodies), and 11 patients could be evaluated for cellular responses.


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Table 1. Patient demographics

 

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Table 2. Summary of post-injection antibody responses during the treatment period [no. positive/no. tested (%)]

 
Immunological response assessments
Humoral responses. Overall, five of 15 patients developed HAMA; four developed Ab2 and five developed detectable anti-MUC1 antibodies, including one patient with pre-existing anti-MUC1 antibodies. Robust immune responses were detected in one patient for HAMA (>5000 ng/ml), four patients for Ab2 (>500 U/ml) and four patients for anti-MUC1 antibodies (more than three-fold increase over baseline). The results are summarized in Table 2. Of the five patients who developed HAMA: one patient was in the 1-mg cohort, two in the 2-mg cohort and two in the 4-mg cohort. HAMA responses were highest in the 2-mg dose cohort; however, values were lower than those previously observed in patients treated with OvaRex® mAb-B43.13 [26Go]. Of the four patients who developed Ab2: one patient was in the 1-mg cohort, two in the 2-mg cohort and one in the 4-mg cohort. Anti-MUC1 antibodies were evaluated in serum samples with and without a pretreatment step to disrupt pre-existing immune complexes of anti-MUC1 antibodies with circulating MUC1. Anti-MUC1 antibodies were below the cut-off value for normals in all but one patient, with high baseline anti-MUC1 antibodies without pretreatment. Human anti-MUC1 antibody became detectable, however, after the pretreatment step. Overall, five patients showed anti-MUC1 antibody levels above the normal range: one in the 1-mg cohort, three in the 2-mg cohort and one in the 4-mg cohort. One patient in the 1-mg cohort, two patients in the 2-mg cohort and one patient in the 4-mg cohort showed more than three-fold increases in anti-MUC1 antibodies over baseline (Table 2). One patient in the 2-mg cohort had high levels of anti-MUC1 antibodies at baseline, which nearly doubled after treatment with mAb-AR20.5. Anti-MUC1 antibody levels were highest in the 2-mg dose cohort. In summary, humoral responses were detectable at all the dose levels evaluated for all the parameters tested, although this was least prominent at the 1-mg dose level.

Epitope specificity of anti-MUC1 antibodies. Detectable anti-MUC1 antibody concentrations were low in the untreated serum samples and generally much higher after pretreatment of the samples. Nonetheless, both sources were analyzed for epitope specificity using 6mer MUC1 peptides as inhibitors in the anti-MUC1 antibody assay. Inhibition patterns varied from patient to patient, as summarized in Table 3. The results differed between the native and pretreated samples showing that a difference in specificities or affinities toward certain epitopes may exist between free and complexed anti-MUC1 antibodies. In some cases, a shift of recognized epitopes with increasing immunizations was noted. Overall, peptide AHGVTS was the most recognized epitope (eight of 15 samples) in most patients, and was represented in both native and treated samples. In treated samples, representing some free, but mainly displaced, anti-MUC1 antibodies from immune complexes, peptides APGSTA, TSAPDT and TAPPAH were also frequently recognized (four, five and five of 15 samples, respectively). The epitope represented by peptide DTRPAP, which is the minimal epitope recognized by mAb-AR20.5 [10Go], was recognized in only one of 15 samples. Patients AA003 and AA020 produced antibodies to MUC1 that were restricted to a single 6mer region (TAPPAH and APGSTA, respectively). The remaining patients developed a multi-epitopic humoral response, covering most of the tested epitopes. In the untreated samples that represent only free circulating anti-MUC1 antibodies, peptides APGSTA and TAPPAH were frequently recognized (six and five of 15 samples, respectively) in addition to peptide AHGVTS. The epitopes represented by peptides DTRPAP and TSAPDT were recognized in only three of 15 samples. Three patients showed free circulating antibodies to MUC1 that were restricted to a single 6mer region (AHGVTS in patients AA012 and AA016, and APGSTA or TAPPAH in patient AA001). The remaining patients showed antibodies to multiple epitopes, covering most of the tested peptides. Again, the DTRPAP epitope was recognized the least frequently. These results indicate that the majority of human anti-MUC1 antibodies in patients treated with mAb-AR20.5 were not induced via the idiotypic network (Ab3), which, by definition, would bind to the same epitope as the Ab1 (that is, DTRPAP).


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Table 3. Inhibition of anti-MUC1 antibodies with different MUC1-derived peptides

 
MUC1-specific T-cell responses. Adequate samples at baseline and at least at one timepoint post-mAb-AR20.5 injection were evaluable for cellular response assessment in 10 patients (Figure 1). Although a total of four lymphocyte samples were scheduled to be obtained at weeks 1, 7, 13 and 17 for IFN-{gamma} ELISPOT evaluation of T-cell response, fewer samples were collected from many patients due to premature study exit or insufficient cell numbers. In vitro sensitization (IVS) steps and ELISPOT assay conditions were optimized, using samples obtained from three patients with sufficiently large lymphocyte samples. The greatest sensitivity for MUC1-specific T-cell responses was obtained using a 7-day IVS step with the 31mer MUC1 peptide and whole PBMC as antigen-presenting cells, followed by re-stimulation with the MUC1 peptide-pulsed autologous dendritic cells. The optimal peptide concentration was found to be 10 µg/ml. Under these conditions, positive ELISPOT results were detected as early as week 7 in immunological responders.



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Figure 1. Interferon (IFN)-{gamma} enzyme-linked immunospot (ELISPOT) data from patients treated with BrevaRex® mAb-AR20.5. T-cell responses to MUC1 pre- and post-mAb-AR20.5 treatment were assessed for production of IFN-{gamma} by ELISPOT assay in response to stimulation with 31mer MUC1 peptide (APDTRPAPGSTAPPAHGVTSAPDTRPAPGSC). Peripheral blood mononuclear cells (PBMC) obtained from heparinized blood of patients before and after administration of mAb-AR20.5 were isolated on Ficoll–Hypaque gradients and cryopreserved. Cells from pre- and post-therapy samples were thawed simultaneously, washed and stimulated with the 31mer MUC1 peptide for 1 week, using autologous PBMC as antigen-presenting cells. Cultures were then restimulated for 24 h with MUC1 peptide-pulsed autologous dendritic cells generated in parallel with the stimulated PBMC cultures. MUC1-specific T cells were quantified in an IFN-{gamma} ELISPOT.

 
The anti-MUC1 T-cell response for each patient within the three dosing groups is summarized in Table 4. Three of five evaluable patients in the 2-mg cohort and two of four patients in the 4-mg cohort showed increases in the number of MUC1-specific IFN-{gamma}-producing T cells. From this limited dataset, it appears that induction of T-cell responses to MUC1 was seen as early as week 7 in both the 2- and 4-mg groups. Only one patient in the 1-mg cohort had a baseline plus at least one post-injection sample, and this patient did not show an increase in MUC1-specific T-cell precursor frequency from baseline.


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Table 4. Summary of T cell ELISPOT data from patients treated with BrevaRex® mAb-AR20.5

 
Patients AA014 and AA016 showed both cellular and humoral responses to MUC1; patient AA008 showed a weak B- and T-cell response to MUC1; patient AA009 only a T-cell response; and patient AA012 only anti-MUC1 antibodies, inclusive of the baseline sample. The remaining patient with anti-MUC1 antibodies, AA001, was not assessed for T-cell responses due to lack of sufficient lymphocytes.

Antitumor activity
There were no objective antitumour responses. Of the patients who received three doses of mAb-AR20.5, only one patient met the criteria for stable disease, and 11 discontinued the study due to disease progression. The time to disease progression ranged from 6.5 to 12 weeks. Four of five patients in the 2-mg cohort, and three of five in the 4-mg cohort demonstrated either stable or decreased values of CA15.3 (a secreted MUC1 protein) during the study. A significant inverse correlation was found between anti-MUC1 antibody increase and CA15.3 levels (depicted in Figures 1 and 2) (r=–0.556; P=0.03). The 1-mg cohort showed CA15.3 increases in all five cases, which is consistent with the immunological profile.



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Figure 2. Inverse correlation between anti-MUC1 antibody responses and human CA15.3 levels. CA15.3 serum levels were tested in commercial assays for CA15.3 or CA27.29 at each visit. Patient serum samples at baseline and at several time points after BrevaRex® mAb-AR20.5 injection were pretreated with a large excess of mAb-AR20.5 (400 µg/ml) for 1 h at room temperature to displace anti-MUC1 antibodies from serum MUC1. mAb-AR20.5-reacted samples were then incubated with goat anti-mouse IgG coated-agarose overnight at 4°C followed by removal of the agarose-bound mAb-AR20.5 or mAb-AR20.5 immune complexes by centrifugation. For the assay, streptavidin-coated plates were incubated with a 31mer biotinylated MUC1 peptide (APDTRPAPGSTAPPAHGVTSAPDTRPAPGSC-Biotin) and pretreated patient samples were incubated on the streptavidin and biotinylated MUC1 peptide coated plates along with standards and controls. The formed immune complex was detected with peroxidase-conjugated goat F(ab)2 anti-human IgG and revealed by reaction with the peroxidase substrate ABTS. The change of CA15.3 serum levels from baseline was plotted against the increase in anti-MUC1 antibody concentrations on study and the data correlated using the non-parametric Spearman correlation calculation.

 
Safety
mAb-AR20.5 was well tolerated at all of the dose levels tested, with minimal toxicity being observed during this study (Tables 5 and 6 ). None of the patients discontinued the study due to adverse events, and there was no dose-limiting toxicity. Five patients were reported as having infusion-related adverse events. The majority of reported events were classified as NCI CTC grade 1 or 2. Most adverse events appeared to be transient, non-clinically significant and resolved without medical intervention. The most frequently reported drug-related events were nausea, which was reported by four patients (23.5%), and fatigue, also reported by four patients (23.5%). Only one patient (5.9%) had a grade 3 event of nausea that was assessed as probably related to the study drug. All other drug-related events were reported by only one patient each. There were no clinically meaningful changes in laboratory parameters attributed to treatment with mAb-AR20.5.


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Table 5A. Summary of adverse events

 

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Table 5B. Adverse events by body system

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
mAb-AR20.5 can be safely and feasibly administered at doses of 1, 2 or 4 mg in patients with MUC1-expressing tumors. There was no apparent dose-limiting toxicity, and no patient discontinued the study due to adverse events. While the ability of mAb-AR20.5 to positively affect clinical outcome remains to be determined, these data indicate that this antibody can activate an anti-MUC1, and potentially antitumour, immune response at doses at or above 2 mg. Antibody responses increased in frequency and level with increasing number of injections. Responses generally peaked between weeks 7 and 13, indicating that at least three to four antibody injections are necessary to induce humoral and cellular responses. These data encourage further clinical study of mAb-AR20.5 at the 2- or 4-mg dose level.

Overall, four patients showed more than three-fold increases in anti-MUC1 antibody levels on study: one in the 1-mg, two in the 2-mg and one in the 4-mg cohort. Human anti-MUC1 antibodies were more readily detectable using a pretreatment step, indicating that these antibodies circulate as immune complexes with MUC1, particularly at high serum MUC1 concentrations. Analysis of the epitope specificity of these antibodies showed a multiepitopic profile in the majority of the patients and only very few antibodies to the mAb-AR20.5 epitope DTRPAP. Similar to the observations reported in studies of oregovomab (OvaRex® mAb-B43.13)-treated patients, where induced human anti-CA125 antibodies also bound to multiple epitopes [21Go], these anti-MUC1 antibodies may be induced via complex formation of mAb-AR20.5 with circulating MUC1, rather than via the idiotypic network.

Three of five evaluable patients in the 2-mg cohort and two of four patients in the 4-mg cohort showed increases in the number of MUC1-specific IFN-{gamma}-producing T cells. From this limited data set, it appears that induction of T-cell responses to MUC1 was seen as early as week 7 in both the 2- and 4-mg groups.

There was considerable variation in the time to peak immunological response (HAMA, Ab2, anti-MUC1 antibody, MUC1-specific T cells). This may have been a result of variability in the immune competence of the population studied, previous chemotherapy and differences in the immunogenicity of the antibody in different patients. This may be important, since increased MUC1 expression varies not only by cancer type, but also by stage and localization in the same tissue, as well as by tumor burden [27Go].

In this population of patients with advanced malignancies, no antitumour activity was observed. However, an inverse correlation between anti-MUC1 immune responses and CA15.3 levels was noted, with patients in the 2- and 4-mg cohort showing the highest incidence of CA15.3 stabilization or decrease and also the highest rate of immunological responses. The clinical significance of these findings needs to be further evaluated in efficacy studies. In patients with more extensive disease, studies should explore concomitant chemo-immunotherapy. Strategies for development of such agents are described in a recent review by Whiteside [28Go]. A clinical study utilizing a related murine antibody to CA125 (OvaRex® mAb-B43.13) in advanced ovarian cancer patients has demonstrated that meaningful cellular immune responses can be generated with concomitant chemotherapy, and that these responses can be associated with a favorable clinical outcome [20Go]. The potential for synergic interaction between cytotoxic and immunotherapies has been further demonstrated in experiments where immune-depleting chemotherapy enabled the persistent transfer of tumor reactive T cells and subsequent regression of metastatic melanoma [29Go]. Finally, future studies must examine the sensitivity and specificity of laboratory methods used to monitor patient immune responses and their application as clinical outcome correlates. A recent study comparing T-cell frequency, as measured by ELISPOT, cytokine flow cytometry and tetramer binding, revealed discordant results [30Go]. While these assays are undergoing refinement [26Go], the use of appropriate laboratory clinical correlates will remain crucial, since classical measures such as objective tumor response (i.e. tumor shrinkage) will be of limited use for the development of this type of agent. These issues notwithstanding, the immunological data presented herein give strong preliminary evidence that injection with mAb-AR20.5 can induce MUC1-specific immune responses in cancer patients with advanced disease, and indicate that this agent merits future evaluation in clinical trials at the 2- or 4-mg dose level.

Received for publication April 15, 2004. Revision received July 13, 2004. Accepted for publication July 14, 2004.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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