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Phase II multicenter, randomized, double-blind study of recombinant mutated human tumor necrosis factor-α in combination with chemotherapies in cancer patients

Authors


To whom correspondence should be addressed.
E-mail: lemon781106@yahoo.com.cn; zhangwei@fmmu.edu.cn

Abstract

We previously prepared a tumor necrosis factor (TNF)-α mutant (rmhTNF-α) that showed higher antitumor activity and lower systemic toxicity compared with native TNF-α. The safety profile and the pharmacokinetic characteristics of rmhTNF-α were suited for clinical use according to biological Investigational New Drug application, a standard guideline for new drug investigation in China. Here, we evaluate the activity and safety of rmhTNF-α combined with chemotherapies in head/neck, lung, colorectal, stomach, and renal cancer patients. Ninety-five eligible patients received i.m. rmhTNF-α treatment combined with standard chemotherapies. Another 95 patients were treated with standard chemotherapies. After two treatment cycles, one patient achieved a complete response and 24 patients had partial response, yielding an overall response rate (complete response + partial response) of 27.47% in the rmhTNF-α plus chemotherapy cohort. The chemotherapy alone group acquired only a 11.39% response rate (< 0.05). When compared between different cancers, a 48.89% response rate was detected in the 45 lung cancer patients of the combination cohort. The most common grade 1–2 adverse events of rmhTNF-α were drug-related fever, allergy, flu-like symptoms, and myalgia. No significant difference was found in grade 3–4 toxicities between the two cohorts. Based on the results of this research, rmhTNF-α can significantly enhance the effectiveness of chemotherapy. An extended phase III trial of rmhTNF-α combined with standard chemotherapy is warranted for evaluating its antitumor activity and toxicity in a larger cohort of tumor patients. The studies in this paper were registered with the State Food and Drug Administration of China (No. 2003S00692). (Cancer Sci 2012; 103: 288–295)

Human TNF-α (cachectin) is a 51 kDa non-glycosylated, non-covalently linked homotrimeric protein,(1) which was initially found to be effective in causing hemorrhagic necrosis of certain tumors in vivo.(2,3) Subsequent studies proved the multifunction of TNF-α in immunomodulation and inflammatory responses.(4) A loss of careful TNF-α balance can lead to severe inflammatory illnesses such as septic shock and arthritis.(5,6) It is therefore not surprising that systemic application of TNF-α as a treatment for tumors in the clinic is hindered due to severe dose-limiting toxicities,(7,8) typically appearing as vasoplegia, a pathological condition leading to multiorgan failure, the “septic shock-like syndrome”.(6,9)

At present, clinical use of TNF-α has been limited to administration through ILP or IHP in melanoma, soft tissue sarcoma, and unresectable metastatic or primary cancers confined to the liver.(10) Systemic toxicity will be abolished depending upon the efficiency of isolation and the resulting extra-corporeal circulation. When combined with the alkylating agent melphalan, near 100% objective response rates can be obtained by a single ILP or IHP containing TNF-α.(11,12) However, both ILP and IHP are one-shot procedures that cannot easily be repeated because of difficult vascular access after previous perfusion, which greatly limits the duration of the response and the impact on overall survival.(13) As the TNF-α dose in ILP or IHP is quite high, the whole procedure needs to be monitored carefully because 10% or more leakage of TNF-α at any time point will result in severe systemic toxicity.(14) The dramatically good response rates and the limitations in TNF-α local applications have renewed interest in this fascinating pleiotropic cytokine as a systemic anticancer agent. Various strategies, such as small molecule inhibitor of apoptosis antagonists synergism, single chain (scFv) recombinant antibodies or tumor-specific binding peptides delivery have been pursued to minimize the systemic toxicities, and therefore to increase the therapeutic index.(15–18) A more promising approach is to design a TNF-α mutant with low systemic toxicity and high efficiency. A successful TNF mutant suitable for systemic application can also be used in ILP, IHP, and targeted delivery.

We previously prepared an rmhTNF-α featuring the deletion of the first seven amino acids and substitution of four amino acids (Arg for Pro at position 8, Lys for Ser at position 9, Arg for Asp at position 10, and Phe for Leu at position 157). The rmhTNF-α has much higher antitumor effects and an LD50 at least 50 times higher than native TNF-α.(19) Data from primate models show a superior safety profile for rmhTNF-α over TNF-α at 4 × 106 units/kg/day for 60 days. No systemic anaphylaxis or genetic toxicities were found in rodent models and the pharmacokinetic characteristics of the rmhTNF-α were suited for clinical use.(20) In a phase I investigation, rmhTNF-α could be well tolerated with mild side-effects at doses ranging from 2.5 × 105 to 4 × 106 units/m2/day for 7 days. When used alone, a 10.81% response rate was obtained following two cycles of i.m. rmhTNF-α (one cycle constituted 4 × 106 units/m2/day for 7 days) in cancer patients failing standard therapies (Data S1). To obtain efficacy and safety data on rmhTNF-α combined with standard chemotherapy in cancer patients, we carried out a multicenter, randomized, double-blind phase II study. We herein report the results, which provide more insight into the development of TNF-α mutants as anticancer reagents.

Patients and Methods

Patient selection.  This study used a phase II, multicenter, randomized design that assessed the safety and efficacy of rmhTNF-α in combination with chemotherapy in lung cancer, head/neck cancer, gastrointestinal cancer, and renal cancer patients. The study was carried out at Sichuan University West China Hospital (Chengdu, China), Fudan University Cancer Center (Shanghai, China), First Affiliated Hospital of Xi’an Jiaotong University (Xi’an, China), Fujian Medical University Union Hospital (Fuzhou, China), and Shandong University Hospital (Jinan, China) after local institutional review board approval at all five locations. Patients were eligible if they had: histologically or cytologically confirmed invasive carcinoma at either stage III or IV disease; at least one unidimensionally measurable lesion according to Response Evaluation Criteria in Solid Tumors guidelines (worldwide); a KPS of at least 60; no chemo-, immuno-, nor any other antitumor therapies within the preceding 4 weeks; a life expectancy of at least 3 months; and age between 18 and 70 years. Patients were required to have adequate bone marrow reserve and organ function. Liver transaminases had to be less than twice the upper limit of normal, bilirubin < 1.5 mg/dL, creatinine < 1.8 mg/dL, total WBCC > 3000/mm3, platelets > 100 000/mm3, international normalized ratio of prothrombin time ≤ 1.2, and activated partial thromboplastin time within 2 s of the upper limit of normal. Patients specifically excluded from this study included those with resectable early malignant disease, clinically significant cardiac disease, nephrosis, hemorrhagic diatheses, and pregnant or breastfeeding women, as well as people that were hyperergic to polypeptide drugs or biologics. All patients provided written informed consent before treatment.

Preparation of rmhTNF-α.  We undertook the molecular cloning and protein characterization and purification of rmhTNF-α, which has been described previously.(19,20) The rmhTNF-α was purified to more than 99% as determined by SDS-PAGE and had a specific activity of approximately 1 × 109 units/mg of protein as defined by the lysis of actinomycin D-treated mouse L929 cells. Sterility, general safety, and purity studies met Office of Biologies, Food and Drug Administration (China) standards.

Study design.  In this double-blind study, eligible patients were randomly assigned into one of two cohorts to receive rmhTNF-α plus chemotherapy or chemotherapy alone. Pocock and Simon(21) randomization was used to minimize imbalances between two cohorts for prognostic factors including performance status, age, sex, cancer category, clinical stage, and investigative center. The dosing cohorts, treatment schedule, and chemotherapy plans for different cancer patients are listed in Table 1. On days 1 and 11 of each 21-day treatment cycle, the fixed dose of rmhTNF-α (4 × 106 units/m2/day, i.m.) was given for 7 days consecutively in the rmhTNF-α plus chemotherapy group. The chemotherapy group only received chemotherapy reagents. As clinical indications, chemotherapy plans for different cancer patients for each cycle were as follows. Head/neck cancer patients received 5-FU 500 mg/m2 and cyclophosphamide 100 mg/m2 on days 1–5 and cisplatin 100 mg/m2 on day 1. Lung cancer patients received cyclophosphamide 750 mg/m2, adriamycin 40 mg/m2, and cisplatin 80–100 mg/m2 on day 1. Colorectal cancer or renal cancer patients received 5-FU 500 mg/m2 on days 1–4 and cyclophosphamide 100 mg/m2 on days 1–5. Stomach cancer patients received 5-FU 500 mg/m2 on days 1–4, adriamycin 40 mg/m2, and mitomycin C 6 mg/m2 on day 1. Chemotherapy plans in the two cohorts were same. A total two-cycle treatment was applied to all patients unless there was earlier evidence of disease progression, unacceptable toxicity, or non-compliance. The patients discontinued from protocol therapy due to unacceptable toxicity were considered unevaluable for efficacy but assessable for toxicity. Patients that had definite inflammation received antibiotics or anti-infective treatments according to clinical indications.

Table 1.   Protocol of one treatment cycle with recombinant mutated human tumor necrosis factor-α (rmhTNF-α) plus chemotherapy or chemotherapy alone for different cancer types
 rmhTNF-α + chemotherapyChemotherapy
  1. 5-FU, 5-fluorouracil; ADM, adriamycin; CF, cyclophosphamide; DDP, cisplatin; MMC, mitomycin C.

Head/neckrmhTNF-α 4 × 106 units/m2/day i.m., days 1–7 and 11–185-FU 500 mg/m2/day i.v., days 1–5
CF 100 mg/m2/day i.v., days 1–5
DDP 100 mg/m2/day i.v., day 1
5-FU 500 mg/m2/day i.v., days 1–5
CF 100 mg/m2/day i.v., days 1–5
DDP 100 mg/m2/day i.v., day 1
LungCF 750 mg/m2/day i.v., day 1
ADM 40 mg/m2/day i.v., day 1
DDP 80–100 mg/m2/day i.v., day 1
CF 750 mg/m2/day i.v., day 1
ADM 40 mg/m2/day i.v., day 1
DDP 80–100 mg/m2/day i.v., day 1
Colorectal5-FU 500 mg/m2/day i.v., days 1–4
CF 100 mg/m2/day i.v., days 1–5
5-FU 500 mg/m2/day i.v., days 1–4
CF 100 mg/m2/day i.v., days 1–5
Renal5-FU 500 mg/m2/day i.v., days 1–4
CF 100 mg/m2/day i.v., days 1–5
5-FU 500 mg/m2/day i.v., days 1–4
CF 100 mg/m2/day i.v., days 1–5
Stomach5-FU 500 mg/m2/day i.v., days 1–4
ADM 40 mg/m2/day i.v., day 1
MMC 6 mg/m2/day i.v., day 1
5-FU 500 mg/m2/day i.v., days 1–4
ADM 40 mg/m2/day i.v., day 1
MMC 6 mg/m2/day i.v., day 1

Treatment assessments.  All the patients were hospitalized for frequent monitoring of clinical signs including body temperature, blood pressure, respirations, pulse, digestive tract reaction, chills, muscle soreness, injection site reaction, and edema. Hematological and biochemical parameters were obtained before and every 3 days during treatment. Overall status evaluation (KPS), renal and liver function tests, urinalyses, and electrocardiograms were repeated every cycle. Before entering the study, all patients underwent physical examination, and measurement of palpable lesions as well as those assessed by computed tomography or MRI. The baseline assessment method was repeated after two treatment cycles, and every 4 weeks following treatment discontinuation until 2 months had elapsed.

All patients who received at least one dose of reagents were considered assessable for safety. Patients who finished two cycles of therapy were evaluated for antitumor response. Standard criteria were used to evaluate response. A CR required the disappearance of all clinical, laboratory, and radiography signs and symptoms of cancer for a minimum of 4 weeks. A PR was defined as a 50% or greater reduction in the sum of the products of diameters of all measurable lesions, lasting for a minimum of 4 weeks. No lesion could increase in size and no new lesions could appear during this interval. An MR was defined as a reduction in the size of measurable lesions by 25–50% in the absence of disease progression. Stable disease indicated no change in measurable lesions or a decrease in size that failed to meet the criteria for minor response. Progressive disease was defined as an increase of 25% or more in any measurable lesion. Progressive disease also included those instances in which new lesions appeared during the treatment interval. Overall response rate was defined as the percentage of patients who had CR or PR after two cycles of therapy. This is the endpoint of efficacy measurement.

Statistical analyses.  Patients were enrolled until 95 patients were treated in each cohort. Each treatment cohort would have at least 80% power to accept the alternative hypotheses and reject the null hypothesis at a 0.05 level. Patient characteristics of two cohorts including performance status, age, sex, cancer category, clinical stage, and previous treatments were tested using the chi square-test, the F test, and the Wilcoxon rank sum test. Overall response rate was the proportion of patients who showed a clinical response (CR or PR) and was calculated for all patients evaluable for efficacy (who finished two cycles of treatment). The efficacies and performance score of two cohorts were tested using the chi-square-test. The incidence of adverse effects and supportive care were analyzed using the chi square-test or Fisher’s exact test. Both WBCC and ANC were used to evaluate the function of bone marrow after treatments. The lowest mean of WBCC and ANC nadirs of two cohorts were tested using the paired t-test. Duration (days) of consecutive decline of WBCC and ANC were tested using the Wilcoxon rank sum test.

Results

Patient characteristics.  One hundred and ninety patients were equally distributed between the rmhTNF-α plus chemotherapy group and the chemotherapy group. Figure 1 shows the trial profile. Ninety-one of 95 patients assigned rmhTNF-α plus chemotherapy treatment and 85 of 95 patients assigned chemotherapy treatment finished the scheduled two cycles of therapy. Four patients were excluded from the efficacy analysis in the rmhTNF-α plus chemotherapy group, one for non-compliance and three for discontinuation caused by repeated fever. The three patients who discontinued treatment were still included in the adverse events analysis. In the chemotherapy group, 10 patients were excluded from the efficacy and toxicity analysis, three for non-compliance and seven for progression.

Figure 1.

 Trial profile showing 190 patients equally distributed between the recombinant mutated human tumor necrosis factor-α (rmhTNF-α) plus chemotherapy group and the chemotherapy group. Ninety-one of 95 patients assigned to rmhTNF-α plus chemotherapy treatment and 85 of 95 patients assigned to chemotherapy treatment finished the two scheduled cycles of therapy. Four patients were excluded from the efficacy analysis in the rmhTNF-α plus chemotherapy group, one for non-compliance and three for discontinuation caused by repeated fever. The three patients who discontinued treatment were still included in the adverse events analysis. In the chemotherapy group, 10 patients were excluded from the efficacy and toxicity analysis, three for non-compliance and seven for progression.

The characteristics of the patients who finished the scheduled therapy are summarized in Table 2. No significant differences were observed in gender, age, KPS, cancer categories, prior treatments, or clinical stage between the two cohorts of patients (> 0.05).

Table 2.   Characteristics of patients who participated in this study
 rmhTNF-α + chemotherapy
n = 91
Chemotherapy
n = 85
  1. No significant differences were found between the two patient cohorts (> 0.05). rmhTNF-α, recombinant mutated human tumor necrosis factor-α.

Gender
 Male6359
 Female2826
Age
 Median53.96 ± 10.7454.22 ± 10.58
 Range26–7025–70
Cancer category
 Head/neck1310
 Lung4545
 Gastroenteritic2726
 Renal64
Prior treatments
 Chemotherapy97
 Radiotherapy2724
 Radiotherapy + chemotherapy87
Karnofsky performance status
 Median83.13 ± 8.7883.83 ± 8.67
 Range60–9560–95
Clinical stage
 I64
 II22
 III3129
 IV5250

Efficacy.  In the rmhTNF-α plus chemotherapy group (n = 91), one developed a CR, 24 developed PR, 12 developed MR, and the others developed SD or PD. In the chemotherapy group (n = 85), one developed a CR, eight developed PR, 12 developed MR, and the others developed SD or PD. The overall response rate (CR + PR) of the rmhTNF-α plus chemotherapy group was 27.47% compared to 11.39% for the chemotherapy group (= 0.009) (Table 3). In terms of KPS, the average of the rmhTNF-α plus chemotherapy group (85.02 ± 10.74) indicated a better clinical status compared to the chemotherapy group (81.35 ± 9.63; P = 0.038).

Table 3.   Responses to treatment with recombinant mutated human tumor necrosis factor-α (rmhTNF-α) plus chemotherapy or chemotherapy alone in cancer patients
 CRPRMRSDPDCR + PR (%)
  1. *P < 0.05. CR, complete response; MR, minor response; PD, progressive disease; PR, partial response; SD, stable disease.

Overall response (no. of patients)
 rmhTNF-α + chemotherapy  (n = 91)12412342025 (27.47)
 Chemotherapy  (n = 85)181235299 (10.59)
 Comparison     P = 0.009*
Response by cancer type (no. of patients)
 Head/neck
  rmhTNF-α + chemotherapy    (n = 13)023442 (15.39)
  Chemotherapy    (n = 10)013331 (10.00)
  Comparison     > 0.05
 Lung
  rmhTNF-α + chemotherapy    (n = 45)121414522 (48.72)
  Chemotherapy    (n = 45)081215108 (17.78)
  Comparison     P = 0.002*
 Gastroenteritic
  rmhTNF-α + chemotherapy    (n = 27)0169111 (3.70)
  Chemotherapy    (n = 26)008990 (0.00)
  Comparison     P > 0.05
 Renal
  rmhTNF-α + chemotherapy    (n = 6)000240 (0.00)
  Chemotherapy (n = 4)000310 (0.00)
  Comparison     > 0.05

Patients with head/neck, lung, gastroenteritic (colorectal or stomach), and renal cancer were equally distributed between two groups, and the response rates by cancer category are shown in Table 3. In patients with lung cancer, the response rate of the rmhTNF-α plus chemotherapy group was 48.72% compared to 17.78% for the chemotherapy group (P = 0.002). No significant differences were observed between the two groups in the head/neck, gastroenteritic, and renal cancer patients (P > 0.05).

Toxicity.  For detection of myelosuppressive status, WBCC and ANC were recorded every 3 days until the values of all patients were higher than 4.0 × 109/L and 2.0 × 109/L, respectively. After both treatment cycles, the average durations (days) when WBCC was lower than 1.0 × 109/L, 2.0 × 109/L, 3.0 × 109/L, or 4.0 × 109/L in the rmhTNF-α plus chemotherapy group were not statistically different from the chemotherapy group (> 0.05). The average durations (days) when the ANC was lower than 0.5 × 109/L, 1.0 × 109/L, 1.5 × 109/L, or 2.0 × 109/L were also not statistically different between the two groups (P > 0.05) (Table 4). The lowest means of WBCC and ANC for the rmhTNF-α plus chemotherapy group were 3.332 ± 1.373 and 1.978 ± 1.141, respectively, which were not significantly different to 3.168 ± 1.188 and 1.802 ± 1.334, respectively, for the chemotherapy group (P = 0.83, P = 0.35) during the first treatment cycle. However, significant differences were observed in the second cycle. The lowest means of WBCC and ANC for the rmhTNF-α plus chemotherapy group in the second cycle were 3.660 ± 1.549 and 2.194 ± 1.277, respectively, which were higher than 3.087 ± 1.270 and 1.687 ± 0.941, respectively, for the chemotherapy group (P = 0.01, P = 0.004). We also counted the patients whose WBCC was lower than 4.0 × 109/L or ANC was lower than 2.0 × 109/L. Twenty-one days after the first cycle, 16 of 91 patients (17.6%) had a WBCC below 4.0 × 109/L in the rmhTNF-α plus chemotherapy group compared to 10 of 85 patients (11.8%) in the chemotherapy group (P = 0.374). Fifteen of 91 patients (16.5%) had an ANC below 2.0 × 109/L in the rmhTNF-α plus chemotherapy group compared to four of 85 patients (4.7%) in the chemotherapy group (P = 0.019). Twenty-one days after the second cycle, 19 of 91 patients (20.9%) had a WBCC below 4.0 × 109/L in the rmhTNF-α plus chemotherapy group compared to 13 of 85 patients (15.3%) in the chemotherapy group (P = 0.462). Thirteen 91 patients (14.3%) had an ANC below 2.0 × 109/L in the rmhTNF-α plus chemotherapy group compared 12 of 85 patients (14.1%) for the chemotherapy group (= 0.864) (Table 4). The significant differences only appeared in the patients with an ANC below 2.0 × 109/L, 21 days after the first cycle (< 0.05).

Table 4.   Average duration (days) of decline in white blood cell count (WBCC) and absolute neutrophil count (ANC) in cancer patients after treatment with recombinant mutated human tumor necrosis factor-α (rmhTNF-α) plus chemotherapy or chemotherapy aloneThumbnail image of

Side-effects.  Adverse events data for the first and second cycles of the trial are listed in Tables 5 and 6, respectively. Three of 95 patients (3.16%) in the combination group who discontinued treatment for repeated fevers were still included in the adverse events analysis. No clinically significant differences in hematologic toxicities, or renal and liver functions between the two groups were reported at the end of the trial. Other adverse events, including nausea, diarrhea, stomatitis, fever, allergy, rash, unconsciousness, flu-like symptoms, myalgia, infection, and arrhythmia, were recorded daily during treatment. Significant differences were only observed in drug-related fever, allergy, flu-like symptoms, and myalgia between the two groups during both the first and second cycles. The common treatment-related grade 3–4 toxicity was nausea, which did not show significant difference between the two groups (> 0.05). In addition, all patients received antibiotics for infection during treatment. In the first cycle, the durations of antibiotic treatment in the combination group and the chemotherapy group were 9 and 39 days, respectively, which was significantly different (P = 0.000). In the second cycle, no difference was observed. The durations in the combination group and chemotherapy group were 25 and 24 days, respectively. No antipyretic drugs were given to prevent drug-related fever except physical cooling as febrile reactions in most patients were moderate.

Table 5.   Toxicity and adverse events in cancer patients during first treatment cycle using recombinant mutated human tumor necrosis factor-α (rmhTNF-α) plus chemotherapy or chemotherapy alone
 rmhTNF-α + chemotherapy
(n = 94)
Chemotherapy
(n = 85)
Comparison (P III + IV)
GradeGrade
0IIIIIIIV0IIIIIIIV
  1. *P < 0.05. **P < 0.01. ALP, alkaline phosphatase; ALT, alanine transaminase; BUN, blood urea nitrogen; UCr, urine creatinine.

Hematologic toxicities
 Anaemia462023114427305>0.05
 Leukocyte decline43211310421291784>0.05
 Granulocyte decline47181112339161275>0.05
 Platelet decline7395225910343>0.05
Renal functions
 BUN elevation901000821100>0.05
 UCr elevation891010821100>0.05
 Albuminuria892000841000>0.05
 Blood in urine892000850000>0.05
Liver functions
 Bilirubin elevation910000841000>0.05
 ALT elevation874000805100>0.05
 ALP elevation845200804100>0.05
Others
 Nausea/vomiting4020283020242881>0.05
 Diarrhea910000830110>0.05
 Stomatitis872200850000>0.05
 Drug fever57161623802400<0.05*
 Allergy880201850000<0.05*
 Rash/desquamation900001850000>0.05
 Unconsciousness901000850000>0.05
 Flu-like symptoms6023710850000<0.01**
 Myalgia6622300831100<0.05*
 Infection, non-neutropenia872200840100>0.05
 Arrhythmia901000841000>0.05
Table 6.   Toxicity and adverse events in cancer patients during second treatment cycle using recombinant mutated human tumor necrosis factor-α (rmhTNF-α) plus chemotherapy or chemotherapy alone
 rmhTNF-α + chemotherapy
(n = 94)
Chemotherapy
(n = 85)
Comparison (P III + IV)
GradeGrade
0IIIIIIIV0IIIIIIIV
  1. *P < 0.05. ALP, alkaline phosphatase; ALT, alanine transaminase; BUN, blood urea nitrogen; UCr, urine creatinine.

Hematologic toxicities
 Anaemia4030173138271241>0.05
 Leukocyte decline39232171302218121>0.05
 Granulocyte decline4717188140111972>0.05
 Platelet decline69145035813821>0.05
Renal functions
 BUN elevation883000841000>0.05
 UCr elevation901000841000>0.05
 Albuminuria910000850000>0.05
 Blood in urine910000841000>0.05
Liver functions
 Bilirubin elevation892000831110>0.05
 ALT elevation882100821100>0.05
 ALP elevation864100814100>0.05
Others
 Nausea/vomiting5117203025173291>0.05
 Diarrhea910000801210>0.05
 Stomatitis892000821100>0.05
 Drug fever63161011750300<0.05*
 Allergy881110850000<0.05*
 Rash/desquamation901000850000>0.05
 Unconsciousness910000850000>0.05
 Flu-like symptoms6818500850000<0.05*
 Myalgia7316200850000<0.05*
 Infection, non-neutropenia892000830400>0.05
 Arrhythmia910000841000>0.05

Discussion

The dramatically good response rates in TNF-α ILP and IHP have renewed interest in this pleiotropic cytokine as a systemic anticancer agent. Based on the fact that the increased basicity on the N-terminal can significantly increase the cytotoxicity of TNF-α on tumor cells, and previous studies on TNF-α mutants,(22–24) we prepared an rmhTNF-α that has much greater antitumor effects and an LD50 at least 50 times higher than native TNF-α.(19) Safety evaluation and pharmacokinetics in rodent and primate models, respectively, showed that rmhTNF-α was suitable for clinical use.(20) In a phase I investigation, rmhTNF-α could be well tolerated with mild side-effects at doses ranging from 2.5 × 105 to 4 × 106 units/m2/day (Tables S1 and S2). When used alone, an overall response rate of 10.81% following i.m. injection of rmhTNF-α (4 × 106 units/m2/day) for two cycles was obtained in patients with intransit melanoma metastases, unresectable skin squamous cell carcinoma, or malignant pleural effusion (Tables S3 and S4).

Human TNF-α induces apoptosis in microvascular endothelial cells and triggers endothelial barrier disruption, that parallel the development of transendothelial permeability. Thus, TNF-α can increase the intratumoral accumulation of chemotherapeutic drugs, which makes chemotherapy more effective.(25,26) In addition, many tumor cell types are more sensitive towards TNF-induced cytotoxicity when they are transcriptionally or translationally inhibited by chemotherapy. In ILP or IHP, TNF-α in combination with the DNA cross-linking alkylating agent melphalan obtained overall response rates of 80–95% in patients with intransit melanoma metastases, unresectable soft tissue extremity sarcomas, or unresectable cancers confined to the liver.(10) To determine whether rmhTNF-α can be more effective when combined with chemotherapy agents, or enhance the efficacy of chemotherapies, we designed this phase II trial to observe the antitumor activities, toxicities, and side-effects of rmhTNF-α combined with different chemotherapy treatments in patients with head/neck cancer, lung cancer, colorectal or stomach cancer, and renal cancer.

After two cycles of rmhTNF-α and chemotherapy combination treatment, one patient achieved a CR lasting at least 2 months and an additional 24 patients had PR, yielding an overall response rate (CR + PR) of 27.47% in 91 patients. The chemotherapy only acquired an 11.39% response rate. When calculated by different cancers, one patient achieved a CR (2.22%) and 21 patients achieved PR (46.67%), yielding a 48.89% response rate in a total of 45 lung cancer patients receiving combination treatment. Chemotherapy alone had only eight patients achieve PR, yielding a response rate of 17.78% in 45 lung cancer patients. The rmhTNF-α can significantly increase the efficacy of chemotherapy in lung cancer patients. In general, the rmhTNF-α did not aggravate or relieve the myelosuppression caused by chemotherapies. Although the lowest means of WBCC and ANC in the combination treatment group were significantly higher than that in the chemotherapy group during the second cycle, we cannot say that rmhTNF-α relieved the myelosuppression, as the number of patients with an ANC below 2.0 × 109/L in the combination group was much higher than that of the chemotherapy group after the first cycle. The side-effects of rmhTNF-α resembling the symptoms of native TNF-α were very low and could be well-tolerated without any additional treatment.

Increasing the therapeutic index through reduced toxicity and enhanced antitumor efficiency is key to TNF-α systematic application. Both ILP and IHP abolish systemic toxicity through isolation and extracorporeal circulation. A tumor vascular targeted TNF-α, NGR-hTNF, decreases systemic toxicity and increases antitumor activities through NGR peptide vascular targeted delivery.(27) The rmhTNF-α we prepared has a specific activity of 1 × 109 units/mg of protein as defined by the lysis of actinomycin D-treated mouse L929 cells, which implied at least a 25-fold higher activity than native TNF-α. When tested in mice, the LD50 of rmhTNF-α was 354 × 107 units/kg compared to 6.16 × 107 units/kg for native TNF-α.(19) The successful application of the rmhTNF-α in our studies depends on its enhanced activity and decreased toxicity. The dose of rmhTNF-α adopted in our studies, 4 × 106 units/m2/day, was actually 4 μg/m2/day of protein after calculated by activity. In previous clinic trials of native TNF-α, the effective doses were 100–400 μg/m2/day depending on the cancer type.(7,8,28,29) The superiority of rmhTNF-α is clear and the resulting small side-effects from the effective dose is very low. In our studies, a high response rate (48.89%) was obtained in 90 lung cancer patients. Of these, 85 were patients with non-small-cell lung cancer, including 23 squamous cell carcinomas, 36 large cell carcinomas, and 26 adenocarcinomas; the others were small-cell carcinomas. No significant data were obtained in other cancer patients. We cannot state that rmhTNF-α is only effective in lung cancer as the case numbers of other cancers were small in this trial. More cases are needed to validate the effectiveness of rmhTNF-α in cancers other than lung.

In conclusion, prokaryotic-expressed rmhTNF-α can significantly enhance the effectiveness of chemotherapies. When combined with chemotherapy, rmhTNF-α obtained an overall response rate of 27.47%, significantly higher than 11.39% for?standard chemotherapy in cancer patients. Repeated fevers, flu-like symptoms, and myalgia need to be noted in future studies. The results of this report are promising for an extended phase III trial of rmhTNF-α for evaluating its antitumor activity and toxicity in more tumor patients.

Acknowledgments

We thank Prof. Derong Liang (Sichuan University West China Hospital, Chengdu, China) for instruction regarding the design and general management of this clinical trial. We thank Prof. Zongzan Ni (Department of Statistics, Sichuan University, Chengdu, China) for assistance with the clinical data processing. We thank all participating institutions that recruited and recorded the patient data for almost 2 years. We are also grateful to SaiDa Biotechnology Co. (Shanghai, China) for their financial support.

Disclosure Statement

The authors have no conflicts of interest.

Abbreviations
5-FU

5-fluorouracil

ANC

absolute neutrophil count

CR

complete response

IHP

isolated hepatic perfusion

ILP

isolated limb perfusion

KPS

Karnofsky performance status

LD50

50% lethal dose

MR

minor response

PD

progressive disease

PR

partial response

rmhTNF-α

recombinant mutated human TNF-α

SD

stable disease

TNF

tumor necrosis factor

WBCC

white blood cell count

Ancillary