Purpose: The attenuated vaccinia virus modified vaccinia ankara (MVA) has been engineered to deliver the tumor antigen 5T4 (TroVax). TroVax has been evaluated in an open-label phase II trial in metastatic renal cell cancer patients in which the vaccine was administered in combination with interleukin-2 (IL-2). The safety, immunologic, and clinical efficacy of TroVax in combination with IL-2 was determined.

Experimental Design: Twenty-five patients with metastatic renal cell cancer were treated with TroVax plus IL-2. 5T4-specific cellular and humoral responses were monitored throughout the study. Clinical responses were assessed by measuring changes in tumor burden by computed tomography or magnetic resonance imaging scan.

Results: TroVax was well tolerated with no serious adverse event attributed to vaccination. Of 25 intention-to-treat patients, 21 mounted 5T4-specific antibody responses. Two patients showed a complete response for >24 months and one a partial response for >12 months. Six patients had disease stabilization from 6 to >21 months. Median progression-free survival (PFS) and overall survival (OS) were >3.37 months (range, 1.50->24.76) and >12.87 months (range, 1.90->24.76), respectively. A statistically significant relationship was detected between the magnitude of 5T4-specific antibody responses and PFS and OS.

Conclusion: TroVax in combination with IL-2 was safe and well tolerated in all patients. The high frequency of 5T4-specific immune responses and good clinical response rate are encouraging and warrant further investigation.

Translational Relevance

This is the first published study in which the safety, clinical, and immunologic efficacy of TroVax (MVA-5T4), administered alongside IL-2, has been tested in renal cell carcinoma patients. TroVax was well tolerated with no related serious adverse events. A high frequency of immune responses was detected and a significant relationship shown between the magnitude of the 5T4-specific antibody response and progression-free survival and overall survival. These encouraging results support the continued testing of TroVax in renal cancer patients.

Cancer immunotherapy approaches have explored the use of recombinant viral vectors to induce potent immune responses against target antigens expressed specifically by tumors (1, 2). We have selected the attenuated vaccinia virus modified vaccinia ankara (MVA) to deliver the 5T4 tumor-associated antigen. The 5T4 oncofetal antigen belongs to the nonmutated self-antigen category of tumor-associated antigens, and is expressed at high levels in the placenta, specifically on trophoblasts (3, 4). In nonpregnant adult tissues, 5T4 is expressed only at very low levels in digestive tissues such as the esophagus. However, 5T4 is highly expressed typically in >80% of carcinomas of the kidney, breast, gastrointestinal tract, colon, prostate, and ovaries (4, 5). Recently, a detailed immunohistochemistry study in renal cancer showed that 5T4 was expressed at high levels in practically all tumors analyzed and, importantly, expression was retained in the metastatic tissues (6), thus validating 5T4 as a target for immunotherapeutic intervention in renal cell cancer.

Although the precise function of 5T4 in tumor development has yet to be fully elucidated, in vitro studies of both human and murine 5T4 have shown that its overexpression causes alterations in cell adhesion, motility, and morphology. Developmentally, 5T4 is associated most frequently with regions containing undifferentiated progenitor cells or differentiating cells engaged in migration (7) Additionally, tumor cells transfected with cDNA encoding for 5T4 display increased motility and reduced adhesion (8). These findings suggest that 5T4 may play a common role in epitheliomesenchymal transition, a critical process that governs morphogenesis in multicellular organisms and reactivates in the progression of carcinoma (7, 9).

Recent advances in understanding the mechanisms underlying tumorigenesis (i.e., inappropriate signal transduction, angiogenesis, and dysfunctional immunoregulation) have led to the development of new agents more specific to renal cell cancer. Examples include molecularly targeted agents such as monoclonal antibodies, tyrosine kinase inhibitors (e.g., sunitinib and sorafenib), and mammalian target of rapamyacin inhibitors (e.g., temsirolimus). Recently, the Food and Drug Administration approved sunitinib, sorafenib, and temsirolimus for use against advanced renal cell cancer.

Despite such promising advances in the treatment options available to renal cancer patients, the therapeutic benefit delivered is often short-lived and can be accompanied by significant toxicities. Given the high level of 5T4 expression on renal cell tumors and the lack of durable alternative treatments for patients with progressive metastatic renal cell cancer, TroVax represents a potentially novel therapeutic option. The rationale for the concurrent use of low-dose IL-2 with TroVax is that this “immunotherapy” could act as an adjuvant without the toxicities associated with high-dose IL-2 treatment. Previously, TroVax had been tested in phase I and II studies in colorectal and prostate cancer patients (10, 11).3

3

R.J. Amato, et al. Vaccination of prostate cancer patients with modified vaccinia Ankara. Delivering the tumor antigen 5T4 (TroVax): a phase II trial. J Immunother 2008;31:577–85.

These completed studies showed TroVax to be safe and well tolerated and able to induce 5T4-specific immune responses in the majority of patients. Furthermore, correlations between 5T4-specific immune responses and clinical benefit were reported in three independent studies (10, 11). This phase II clinical trial is the first completed clinical study to investigate the use of TroVax in patients with progressive, metastatic renal cancer and assesses whether the combination of TroVax plus IL-2 induces robust immune responses and shows clinical activity.

Clinical trial design. This phase II open-label study was conducted in accordance with the Declaration of Helsinki and all of its amendments, or with the laws and regulations of the country in which the research was conducted, whichever afforded greater protection to the patient. All patients gave full written informed consent before study entry. The study investigated the use of TroVax, given to patients by i.m. injection, in combination with IL-2. IL-2 was administered in 8-wk cycles as a s.c. injection at a dose of 250,000 units/kg/d for 5 consecutive d in week 1 and 125,000 units/kg/d for 5 consecutive days in weeks 2 to 6 followed by 2 wk off. TroVax was administered into the deltoid muscle at a dose of approximately 5 × 108 plaque-forming units. The first TroVax immunization was done 2 wk before the start of IL-2, whereas subsequent vaccinations occurred on the first day of each IL-2 cycle. In cycle 1, a further TroVax immunization was given 3 wk after the first injection. At the time of entry to the trial, each patient underwent a computed tomography scan of the chest, abdomen, and pelvis; magnetic resonance imaging of the brain; and, if clinically indicated, a bone scan. Patients were treated for a maximum of six cycles (48 wk), and those with stable disease or clinical response could receive further injections of TroVax (without IL-2) every 3 mo. Throughout the study, patients were assessed for safety, induction of 5T4-specific immune responses, and clinical benefit such as tumor response, progression-free survival (PFS), and overall survival (OS).

Inclusion/exclusion criteria. Patients age >18 y with histologically confirmed clear cell or papillary renal cell cancer with progressive disease were eligible for inclusion in this study. Additional inclusion criteria included any previous treatment for renal cell cancer, Karnofsky performance status ≥80%, serum calcium ≤10 mg/dL, total white cell count ≥3 × 109 L, and hemoglobin ≥9 mg/dL. Patients were excluded from this study if they had a history of central nervous system metastases; were not fit to receive IL-2; were pregnant or lactating; had acute changes on electrocardiogram or an ejection fraction <45%; had allergies to egg proteins, neomycin or previous vaccinia vaccination; were known to test positive for HIV or hepatitis B or C; or had IL-2 therapy within 6 mo of study entry.

Clinical monitoring. Objective tumor response was assessed every 8 wk for the first 48 wk and then every 12 wk thereafter, using the Response Evaluation Criteria in Solid Tumors guidelines (12). Tumor responses were reported as complete responses, partial responses, stable disease, or progressive disease.

Safety was assessed in all patients who received at least one dose of the study agents version 3 of the National Cancer Institute common toxicity criteria for adverse events.4

4

National Cancer Institute Common Toxicity Criteria version 3.0. http://ctep.cancer.gov/reporting/ctc.html.

TroVax plus IL-2 treatment was discontinued if any of the following occurred: disease progression, unacceptable toxicity, or withdrawal of informed consent.

Antigens. Purified recombinant 5T4 protein (13) and MVA (IDT; Rosslau) were used to monitor antigen-specific antibody responses (by ELISA) and cellular responses (by IFNγ ELISPOT). In addition, overlapping 5T4 9-mer peptides (PPR) and CEF peptides (a pool of CTL epitopes derived from cytomegalovirus, EBV, and Flu; Mabtech) were used to measure cellular responses.

Measurement of humoral responses. 5T4 and MVA-specific antibody titers were determined by ELISA as described previously (13). Antibody titers were defined as the greatest dilution of plasma at which the mean absorbance of the test plasma was ≥2 times the absorbance of the negative control (normal human plasma) at the same dilution. A positive antibody response due to vaccination was reported if the postinjection titer was ≥2 times the titer determined before TroVax immunization.

Measurement of cellular responses. The IFNγ ELISPOT was used to monitor antigen-specific cellular responses throughout the trial as described previously (11). A positive ELISPOT response induced by TroVax was reported if the mean spot forming units per well in response to antigen was ≥3 times the mean spot performing units per well in wells containing medium alone, and if the mean spot performing units per well in response to antigen was ≥10 and the antigen-specific frequency (number of antigen specific cells per 106 total peripheral blood mononuclear cells) after immunization was ≥2 times the frequency before TroVax vaccination.

Statistical analysis. No formal statistical analysis of the data was planned for this phase II open label study. However, a retrospective exploratory analysis was undertaken to identify potential correlates between immunologic and clinical responses. A number of variables were investigated, including PFS, OS, and the antibody responses to MVA and 5T4 induced following vaccination. The log-rank test was used to identify possible relationships between immunologic and clinical response variables. A P value of ≤0.05 was considered to be statistically significant.

Recruitment. From November 2005 to January 2007, 25 eligible patients were recruited. All patients were evaluated for efficacy and safety. Immunologic data are summarized for all patients. A more detailed analysis of immunologic data is presented on the 11 patients who received ≥4 injections, which was defined in the protocol as the criterion by which patients were considered to be evaluable for assessment for immune responses.

Patient characteristics. In general, this patient population was middle-aged (median, 51 years; range, 25-70 years) and relatively active (Zubrod performance status of <1 in all 25). Pathologic distribution consisted of 13 clear cell (52%), 7 papillary (28%), 4 mixed clear cell (16%), and 1 mixed papillary (4%). Twenty-two patients (80%) had received prior treatment with immunotherapy, chemotherapy, or molecular targeted agents. Of these patients, 10 (40%) had received 1; 3 (12%) had received 2; 7 (28%) had received 3; and 2 (8%) had received 4 prior treatments. Five patients (20%) had received IL-2 as part of a previous therapy. All patients had a 28-day washout period before they commenced treatment with TroVax. Patient characteristics are detailed in Table 1.

Table 1.

Intention-to-treat patient characteristics

Patient no.Age (y)SexPrior treatmentsPathologySite of target metastatic lesion(s)No. treatments received
IL-2 cyclesTroVax injections
101 24 IFN + Iressa Papillary with sarcomatoid Lung, soft tissue, nodal 
102 54 Capecitabine + Gemzar; Adriamycin + Gemzar; Thalidomide + IFN Papillary Liver, nodal, bone, ovary, gall bladder 
103 53 Lenalidomide Clear cell Lung, bone, adrenal 12 
104 62 Folate Immune; Motexafin Clear cell Lung, soft tissue, bone, pericardium 12 
105 49 Motexafin Sarcomatoid with clear cell Lung, bone, soft tissue 
106 49 Capecitabine + Gemzar Clear cell Lung 12 
107 59 Sunitinib Tarceva + Gleevac; Sorafenib Papillary Lung, nodal, adrenal 
108 44 Sorafenib; IFN + Gleevec; IL-2 Papillary Lung, liver, nodal 
109 38 IFN + Iressa; ABT-510 Papillary Nodal 
110 38 IFN + Gleevec Chromophobe with clear cell Nodal, bone 
111 59 IL-2+IFN; high-dose IL-2; Thalidomide; Folate Immune Clear cell Lung 
112 44 MG98; RAD001; Thalidomide + IL-2 Clear cell with papillary Nodal, bone 
113 46 IFN + Sunitinib Clear cell with sarcomatoid Bone, nodal, ascites 
114 51 None Papillary Adrenal 
115 55 None Papillary Lung, liver 
116 53 Sorafenib Clear cell Lung, bone, Nodal, kidney 
117 59 Tarceva Papillary Soft tissue, nodal 
118 59 Sunitinib; Avastin; Sorafenib Clear cell Lung, liver 
119 55 IFN; IL-2; Sorafenib Clear cell Lung 
120 68 None Clear cell Nodal, lung 
121 48 Sunitinib Clear cell Lung 
122 39 Sunitinib; RAD001 Clear cell Bone, nodal, soft tissue 
123 70 Sorafenib Clear cell Lung 
124 39 Adriamycin + Avastin; Sunitinib; Avastin + Gemzar + Xeloda + IFN Clear cell Lung, nodal, bone, adrenal 
125 50 IL-2 + IFN; Sorafenib; RAD001 Clear cell Soft tissue, liver, bone 
Patient no.Age (y)SexPrior treatmentsPathologySite of target metastatic lesion(s)No. treatments received
IL-2 cyclesTroVax injections
101 24 IFN + Iressa Papillary with sarcomatoid Lung, soft tissue, nodal 
102 54 Capecitabine + Gemzar; Adriamycin + Gemzar; Thalidomide + IFN Papillary Liver, nodal, bone, ovary, gall bladder 
103 53 Lenalidomide Clear cell Lung, bone, adrenal 12 
104 62 Folate Immune; Motexafin Clear cell Lung, soft tissue, bone, pericardium 12 
105 49 Motexafin Sarcomatoid with clear cell Lung, bone, soft tissue 
106 49 Capecitabine + Gemzar Clear cell Lung 12 
107 59 Sunitinib Tarceva + Gleevac; Sorafenib Papillary Lung, nodal, adrenal 
108 44 Sorafenib; IFN + Gleevec; IL-2 Papillary Lung, liver, nodal 
109 38 IFN + Iressa; ABT-510 Papillary Nodal 
110 38 IFN + Gleevec Chromophobe with clear cell Nodal, bone 
111 59 IL-2+IFN; high-dose IL-2; Thalidomide; Folate Immune Clear cell Lung 
112 44 MG98; RAD001; Thalidomide + IL-2 Clear cell with papillary Nodal, bone 
113 46 IFN + Sunitinib Clear cell with sarcomatoid Bone, nodal, ascites 
114 51 None Papillary Adrenal 
115 55 None Papillary Lung, liver 
116 53 Sorafenib Clear cell Lung, bone, Nodal, kidney 
117 59 Tarceva Papillary Soft tissue, nodal 
118 59 Sunitinib; Avastin; Sorafenib Clear cell Lung, liver 
119 55 IFN; IL-2; Sorafenib Clear cell Lung 
120 68 None Clear cell Nodal, lung 
121 48 Sunitinib Clear cell Lung 
122 39 Sunitinib; RAD001 Clear cell Bone, nodal, soft tissue 
123 70 Sorafenib Clear cell Lung 
124 39 Adriamycin + Avastin; Sunitinib; Avastin + Gemzar + Xeloda + IFN Clear cell Lung, nodal, bone, adrenal 
125 50 IL-2 + IFN; Sorafenib; RAD001 Clear cell Soft tissue, liver, bone 

NOTE: The table details the age, sex, prior treatment history, disease pathology, main sites of metastatic disease and the total number of TroVax injections and IL-2 cycles received. Patients who were evaluable for assessment of immune response are indicated by underlining of the patient number.

Safety. There were no TroVax-related serious adverse events and no dose reductions. Adverse events are summarized in Table 2, including all-grade events occurring in at least 10% of patients and grade 3 to 4 events occurring in at least one patient.

Table 2.

Treatment-related adverse events

ToxicityEvents
Grade 1
Grade 2
Grade 3
Grade 4
n (%)n (%)n (%)n (%)n (%)
Laboratory      
    Anemia 15 (60) 4 (16) 9 (36) 1 (4) 1 (4) 
    Elevated alkaline phosphatase 11 (44) 8 (32) 1 (4) 2 (8)  
    Lymphopenia 5 (20) 3 (12) 1 (4) 1 (4)  
    Hypokalemia 4 (16) 1 (4)  3 (12)  
    Elevated creatinine 3 (12) 2 (8)  1 (4)  
    Elevated WBC 3 (12) 1 (4) 2 (8)   
    Hyperglycemia 3 (12) 1 (4) 2 (8)   
    Hyponatremia 3 (12)   2 (8) 1 (4) 
    Hypocalcemia 1 (4)   1 (4)  
    Hypophosphatemia 1 (4)   1 (4)  
    Thrombocytopenia 1 (4)   1 (4)  
Nonlaboratory      
    Fatigue 17 (68) 9 (36) 8 (32)   
    Fever 11 (44) 10 (40) 1 (4)   
    Nausea 9 (36) 8 (32) 1 (4)   
    Rigors 8 (32) 5 (20) 3 (12)   
    Dyspnea 6 (24) 4 (16) 1 (4)  1 (4) 
    Anorexia 5 (20) 2 (8) 3 (12)   
    Constipation 5 (20) 3 (12) 2 (8)   
    Pneumonia 4 (16)  2 (8) 1 (4) 1 (4) 
    Diarrhea 3 (12) 2 (8) 1 (4)   
    Edema 3 (12) 1 (4) 1 (4) 1 (4)  
    Pleural effusion 2 (8)  1 (4) 1 (4)  
    S.c. necrotic nodules 1 (4)   1 (4)  
ToxicityEvents
Grade 1
Grade 2
Grade 3
Grade 4
n (%)n (%)n (%)n (%)n (%)
Laboratory      
    Anemia 15 (60) 4 (16) 9 (36) 1 (4) 1 (4) 
    Elevated alkaline phosphatase 11 (44) 8 (32) 1 (4) 2 (8)  
    Lymphopenia 5 (20) 3 (12) 1 (4) 1 (4)  
    Hypokalemia 4 (16) 1 (4)  3 (12)  
    Elevated creatinine 3 (12) 2 (8)  1 (4)  
    Elevated WBC 3 (12) 1 (4) 2 (8)   
    Hyperglycemia 3 (12) 1 (4) 2 (8)   
    Hyponatremia 3 (12)   2 (8) 1 (4) 
    Hypocalcemia 1 (4)   1 (4)  
    Hypophosphatemia 1 (4)   1 (4)  
    Thrombocytopenia 1 (4)   1 (4)  
Nonlaboratory      
    Fatigue 17 (68) 9 (36) 8 (32)   
    Fever 11 (44) 10 (40) 1 (4)   
    Nausea 9 (36) 8 (32) 1 (4)   
    Rigors 8 (32) 5 (20) 3 (12)   
    Dyspnea 6 (24) 4 (16) 1 (4)  1 (4) 
    Anorexia 5 (20) 2 (8) 3 (12)   
    Constipation 5 (20) 3 (12) 2 (8)   
    Pneumonia 4 (16)  2 (8) 1 (4) 1 (4) 
    Diarrhea 3 (12) 2 (8) 1 (4)   
    Edema 3 (12) 1 (4) 1 (4) 1 (4)  
    Pleural effusion 2 (8)  1 (4) 1 (4)  
    S.c. necrotic nodules 1 (4)   1 (4)  

NOTE: The total number (and percentage) of adverse events are listed by toxicity and by toxicity grade.

Immunologic responses. Antigen-specific immune responses were assessed in all 25 patients who received TroVax plus IL-2. The average number of IL-2 cycles and TroVax immunizations administered was 2.6 and 4.9, respectively (range, 1-6 for IL-2 and 2-12 for TroVax), and the median was 1 and 3 respectively. All 25 patients mounted MVA-specific antibody responses, and 21 (84%) developed antibody responses to 5T4 following administration of one or more TroVax doses (Table 3). Table 3 summarizes the peak 5T4-specific cellular response, the incidence of 5T4-specific antibody responses, best tumor response, and PFS and OS for all intention-to-treat patients. ELISPOT responses are tabulated as the peak 5T4-specific polyclonal response detected post-TroVax vaccination (only patient 124 showed a preexisting 5T4 response prior to vaccination). All 11 patients who received ≥4 TroVax injections had a positive 5T4 antibody response, with titers ranging from 20 to 5,120. Mean titers peaked after 3 TroVax injections and remained relatively constant thereafter (Fig. 1A) with no significant difference between the titers after the 2nd injection and at any subsequent time point (data not shown). The magnitude of MVA-specific antibody responses was much higher than for 5T4, ranging from 8,000 to 128,000. Similar to 5T4 antibody responses, mean MVA titers peaked after 3 TroVax injections and remained relatively constant thereafter (Fig. 1B), with no significant difference between the titers after the 2nd injection and at any subsequent time point (data not shown).

Table 3.

Immunologic and clinical responses in the intent-to-treat patient population

Patient no.5T4-specific ELISPOT responseT4-specific antibody responseBest tumor responsePFS (mo)OS (mo)
101 N/A Yes PD 3.37 3.36 
102 N/A No PD 1.9 1.9 
103 1:12,500 Yes CR ≥24.76 ≥24.76 
104 1:19,250 Yes SD ≥24.76 ≥24.76 
105 <1:200,000 Yes SD 10.43 
106 1:2,336 Yes CR ≥24.5 ≥24.5 
107 N/A No PD 1.83 ≥23.33 
108 N/A No PD 1.63 7.6 
109 N/A Yes PD 2.03 ≥19.53 
110 1/13,150 Yes PD 3.93 
111 <1:200,000 Yes SD 15.03 ≥22.2 
112 <1:200,000 Yes PD 3.53 14.7 
113 N/A Yes PD 2.43 4.7 
114 1:18,867 Yes PD 2.23 ≥16.73 
115 1:5,000 Yes SD ≥15.63 ≥15.63 
116 N/A Yes NE 1.5 2.03 
117 <1:200,000 Yes PD 2.23 ≥14.87 
118 <1:200,000 No PD 3.1 3.53 
119 <1:200,000 Yes PR ≥13.3 ≥13.3 
120 1:918 Yes SD 6.16 ≥13.2 
121 <1:200,000 Yes SD 9.86 ≥12.86 
122 <1:200,000 Yes PD 2.53 ≥9.2 
123 <1:200,000 Yes PD 2.33 ≥11.7 
124 1:6,450 Yes PD 4.2 ≥9.23 
125 <1:200,000 Yes PD 2.27 ≥5.3 
Patient no.5T4-specific ELISPOT responseT4-specific antibody responseBest tumor responsePFS (mo)OS (mo)
101 N/A Yes PD 3.37 3.36 
102 N/A No PD 1.9 1.9 
103 1:12,500 Yes CR ≥24.76 ≥24.76 
104 1:19,250 Yes SD ≥24.76 ≥24.76 
105 <1:200,000 Yes SD 10.43 
106 1:2,336 Yes CR ≥24.5 ≥24.5 
107 N/A No PD 1.83 ≥23.33 
108 N/A No PD 1.63 7.6 
109 N/A Yes PD 2.03 ≥19.53 
110 1/13,150 Yes PD 3.93 
111 <1:200,000 Yes SD 15.03 ≥22.2 
112 <1:200,000 Yes PD 3.53 14.7 
113 N/A Yes PD 2.43 4.7 
114 1:18,867 Yes PD 2.23 ≥16.73 
115 1:5,000 Yes SD ≥15.63 ≥15.63 
116 N/A Yes NE 1.5 2.03 
117 <1:200,000 Yes PD 2.23 ≥14.87 
118 <1:200,000 No PD 3.1 3.53 
119 <1:200,000 Yes PR ≥13.3 ≥13.3 
120 1:918 Yes SD 6.16 ≥13.2 
121 <1:200,000 Yes SD 9.86 ≥12.86 
122 <1:200,000 Yes PD 2.53 ≥9.2 
123 <1:200,000 Yes PD 2.33 ≥11.7 
124 1:6,450 Yes PD 4.2 ≥9.23 
125 <1:200,000 Yes PD 2.27 ≥5.3 

NOTE: Patients who were evaluable for assessment of immune response are indicated by underlining of the patient number. N/A indicates that insufficient peripheral blood mononuclear cells were available for analysis of ELISPOT responses.

Abbreviations: CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease.

Fig. 1.

5T4 and MVA-specific antibody responses in evaluable patients before and after TroVax vaccination. Scatter plots of 5T4-specific (A) or MVA-specific (B) antibody responses are illustrated for all evaluable patients following different numbers of TroVax injections. Horizontal bars, mean antibody titer ± SE.

Fig. 1.

5T4 and MVA-specific antibody responses in evaluable patients before and after TroVax vaccination. Scatter plots of 5T4-specific (A) or MVA-specific (B) antibody responses are illustrated for all evaluable patients following different numbers of TroVax injections. Horizontal bars, mean antibody titer ± SE.

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Of the 11 patients evaluable for assessment of immune responses, 6 (54%) showed positive MVA ELISPOT responses (data not shown) and 5 (45%) showed 5T4-specific ELISPOT responses following TroVax immunization (Table 3). Four of these patients showed both positive MVA and 5T4-specific ELISPOT responses. The magnitude of MVA-specific ELISPOT responses following TroVax vaccination ranged from 1 in 4,405 to 1 in 856 peripheral blood mononuclear cells (data not shown), whereas responses to 5T4 ranged from 1 in 19,231 to 1 in 918 peripheral blood mononuclear cells (Table 3).

Clinical response. Three patients (12%; 95% confidence interval, 4.60-37.10) had objective tumor responses (2 complete response, 1 partial response; Table 3). These three patients had 5T4-specific antibody responses which exceeded the median of the intention-to-treat population during weeks 2 to 5 (i.e., following 3 vaccinations). Furthermore, none of the responding patients showed objective tumor responses on any of their prior therapies. The best response achieved was stable disease and patient 119 had progressive disease when treated previously with high dose IL-2. Changes in each patient's tumor burden over time are plotted in Fig. 2A, and computed tomography scan images from two of the responding patients (103 and 106) are illustrated in Fig. 2B. An additional six patients (24%) had stable disease ≥6 months. Of these nine patients who experienced clinical benefit, eight had clear cell carcinomas and one (patient 115; stable disease) had papillary histology. Patients were restaged after 3 injections and were removed from study if found to have progressive disease. One of the responding patients (patient 119; partial response) had received three prior therapies (IL-2, IFN, and sorafenib). The objective responses to TroVax plus IL-2 were observed at various metastatic sites including bone, lymphatics, liver, and lungs. The median PFS for all patients was 3.37 (1.50->24.76) months and the median OS was 12.87+ (range 1.90->24.76) months. Kaplan-Meier curves for PFS and OS in all patients are illustrated in Fig. 3A and B, respectively. If the patients were subdivided by their tumor histology into those with clear cell (n = 13), papillary (n = 7), or mixed histology tumors (n = 5), differences in PFS and OS were detected (Fig. 3C and D, respectively). Median PFS was 6.17, 2.03, and 3.53 months for patients with clear cell, papillary, or mixed histology tumors, respectively, whereas median OS was 4.7 months for patients with mixed histology tumors and has yet to be reached for patients with clear cell and papillary tumors. OS in patients with clear cell tumors was significantly longer than those with mixed histology tumors (P = 0.0067).

Fig. 2.

Tumor responses. Change in total tumor burden over time expressed as a percentage of the baseline tumor burden per patient (A). Computed tomography scans taken at baseline (before treatment) are illustrated alongside scans taken at 12 mo (patient 103) and 24 mo (patient 106) in two responding patients (B).

Fig. 2.

Tumor responses. Change in total tumor burden over time expressed as a percentage of the baseline tumor burden per patient (A). Computed tomography scans taken at baseline (before treatment) are illustrated alongside scans taken at 12 mo (patient 103) and 24 mo (patient 106) in two responding patients (B).

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Fig. 3.

PFS and OS in all patients (A and B, respectively), subdivided by histology (C and D, respectively) or antibody response category (E and F, respectively). A to D, clear cell (solid line); mixed (dashed line); papillary (dotted line). E and F, above median antibody (solid line); below median antibody (dashed line).

Fig. 3.

PFS and OS in all patients (A and B, respectively), subdivided by histology (C and D, respectively) or antibody response category (E and F, respectively). A to D, clear cell (solid line); mixed (dashed line); papillary (dotted line). E and F, above median antibody (solid line); below median antibody (dashed line).

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Clinical versus immunologic responses. A retrospective analysis of potential relationships between immunologic and clinical responses was undertaken. Mean MVA and 5T4 specific antibody responses were calculated between weeks 2 and 10 (n = 10 patients), 2 and 18 (n = 9 patients), and 2 and 26 (n = 8 patients), and patients were categorized into those who had greater than median and those with below median antibody responses. The PFS of the two categories was plotted by Kaplan-Meier and differences in the curves determined by the log-rank test. No significant differences in PFS curves were detected when antibody responses were quantified between weeks 2 and 10. However, significant differences in PFS were seen in patients who had above median compared with below median 5T4 antibody responses when quantified between weeks 2 and 18 (Fig. 3E; P = 0.015) and weeks 2 and 26 (P = 0.02; data not shown). No significant differences in PFS were detected when MVA antibody responses were analyzed at any time point (data not shown).

Further exploratory analyses investigated possible relationships between OS and 5T4 and MVA antibody responses detected between weeks 2 and 5 in all 25 patients. It should be emphasized that the survival data are immature, but a significant difference was detected in the survival of patients with above median versus below median 5T4 antibody responses (Fig. 3F; P = 0.04). No significant difference was detected when MVA-specific antibody responses were analyzed in a similar way (data not shown).

This phase II study showed that TroVax, when given in combination with IL-2, was safe and well tolerated in metastatic renal cell cancer patients. Furthermore, 5T4-specific immune responses were induced in the majority of patients (84%) following vaccination and in all 11 patients who received at least 4 injections of TroVax. The magnitude of 5T4-specific antibody responses was encouraging; indeed, five patients developed titers in excess of 500. In general, the 5T4 antibody responses detected in this trial compared favorably with those reported previously in colorectal cancer patients following TroVax vaccination (10). Conversely, the magnitude and frequency of 5T4-specific cellular responses were slightly lower than reported previously, although the frequency of responses was likely impacted by missing blood samples or poor cell recoveries.

The majority of patients recruited into this study were heavily pretreated; indeed, 48% had received two or more prior therapies, and five patients had received prior IL-2 treatment. Furthermore, only 52% of the patients recruited into this study had homogeneous clear cell pathologies. It has been reported previously that patients with clear cell histologies are more responsive to immunotherapy (14, 15). Likewise, based on the data presented here, patients with clear cell compared with papillary or mixed pathologies showed better tumor responses and PFS. Indeed, only 1 of the 9 patients who showed disease control had a nonclear cell histotype.

Previous studies in renal cell carcinoma patients treated with low dose IL-2 regimens in the first line setting have reported response rates ranging from 6% to 23% (16). Despite being heavily pretreated and containing a large proportion of patients with non–clear cell histologies, the objective response rate (12%) and relatively high frequency of complete responses were surprising, and the survival data, although immature, are encouraging.

Although this was a small open-label study in a heterogeneous patient group, the associations between 5T4 (but not MVA) antibody responses and increased PFS and OS were encouraging. The relationship between 5T4 antibody response and PFS was only evident after patients had received 5 TroVax injections (week 18). The absence of a significant correlation when antibody responses were determined between the 2nd and 4th vaccinations (weeks 2 to 10) may be explained by the likely delay which would be expected between the detection of antibodies in the periphery and the time it takes to exert their therapeutic effect at the tumor site. The difference in the survival of patients who had above median versus below median 5T4 antibody responses in the first 5 weeks of the vaccination schedule was surprising for several reasons: (a) the survival data are immature and (b) it was not expected that antibody responses detected at this early stage would predict enhanced survival. Previously, we reported a relationship between 5T4 (but not MVA) antibody responses and enhanced time to progression and OS in colorectal cancer patients treated with TroVax (10). Further analyses of the relationship between 5T4 antibody responses and survival of renal cell cancer patients recruited to the study described here will be undertaken as the survival data mature.

Despite these encouraging observations, caution needs to be exercised when interpreting data from small open-labeled uncontrolled studies in which it is only possible to show association, not causation. This question would need to be addressed prospectively in a randomized controlled trial. Despite such caveats, it is important to note that no relationship was found between enhanced PFS or OS and MVA-specific antibody responses. If improved PFS/OS were simply a function of the general health status and immune competence of these patients, it is likely that the antibody response to both 5T4 and MVA would have shown an association with PFS/OS. Additional work is required to further characterize the 5T4-specific antibody and cellular responses induced by TroVax, including the ability of each to mediate tumor cell killing, growth arrest, and/or induction of apoptosis.

In conclusion, we have shown that TroVax is safe and well tolerated when administered in combination with IL-2. Despite being heavily pretreated, the majority of patients mounted robust 5T4-specific antibody responses. Having established the safety and immunologic potency of TroVax in metastatic renal cell cancer patients, an ongoing phase III clinical trial focuses on combining TroVax with first-line standard-of-care therapy.

R. Amato is on the speakers' bureau of PER Group, Ingenix, Envision Communications, AOI Communications, ENACT, CURE, COGENIX, and MEDACorp. R. Amato has received research support from Abraxis, ARGOS, AVEO, Bayer/Onyx, Berlex, BMS-Imclone, Cellular Therapeutics, Cytogen, Endocyre, Imclone, Novartis, Oxford Biomedica, and Schering.

We thank Peter Treasure for statistical support, Mike McDonald for his valued comments, the clinical teams (in particular Sarah Eastty), Helga Jones for her regulatory support, and, most importantly, the patients who participated in this trial.

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