Associations among cytokines, EGF and lymphocyte subpopulations in patients diagnosed with advanced lung cancer.

Aging is considered the single most significant risk factor for the majority of common malignances including lung cancer. Together immunosenescence, changes occurring with aging in the immune system, and inflammaging, characterizes by a chronic, subclinical accumulation of pro-inflammatory factors, are suggested to stand at the origin of most of the diseases of the elderly, such as cancer. The aim of this study was to determine associations among lymphocyte subpopulations, pro-inflammatory cytokines and epidermal growth factor (EGF) in patients diagnosed with non-small cell lung cancer (NSCLC). Forty-six advanced NSCLC patients were enrolled. Sixteen patients with newly diagnosed and before treatment and 30 patients after first-line platinum-based chemotherapy. Peripheral blood subpopulations were studied by flow cytometry and serum concentrations of soluble factors by ELISA. The frequency of naïve CD4+ T cells, naïve B cells and central memory CD8+ T cells were significantly lower in NSCLC patients after chemotherapy, while effector memory CD4+ T cells and terminally differentiated CD8+ T cells were significantly higher. IL-1ß and TNFα significantly correlated among them before and after platinum-based chemotherapy. Terminally differentiated T cells expressing CD57+ significantly correlated with TNFα and IL-1ß. For the first time, associations between EGF serum levels and terminally differentiated CD4+ T cells, and memory B cells were detected. This study confirms the association among terminally differentiated lymphocytes and pro-inflammatory cytokines in patients diagnosed with lung cancer, reinforcing the interconnection between terminally differentiated lymphocytes and pro-inflammatory cytokines. Clinical trial registration number: RPCEC00000205, http://registroclinico.sld.cu/.

Anti-EGF antibodies as surrogate biomarkers of clinical efficacy in stage IIIB/IV non-small-cell lung cancer patients treated with an optimized CIMAvax-EGF vaccination schedule.

We previously reported that CIMAvax-EGF vaccine is safe, immunogenic and efficacious to treat advanced non-small-cell lung cancer (NSCLC) patients. A phase III trial was designed using an optimized immunization schedule. It included higher antigen dose and injections at multiple sites. Immune response and circulating biomarkers were studied in a subset of patients. EGF-specific antibody titers, IgG subclasses, peptide immunodominance and circulating biomarkers were assessed by ELISA. In vitro EGF-neutralization capacity of immune sera and EGF-IgG binding kinetics was evaluated by Western Blot and Surface Plasmon Resonance (SPR) technology, respectively. We show that CIMAvax-EGF elicited mainly IgG3/IgG4 antibodies at titers exceeding 1:4000 in 80% of vaccinated patients after 3 months of treatment. The EGF-specific humoral response was directed against the central region of the EGF molecule. For the first time, the kinetic constants of EGF-specific antibodies were measured evidencing affinity maturation of antibody repertoire up to month 12 of vaccination. Notably, the capacity of post-immune sera to inhibit EGFR phosphorylation significantly increased during the course of the immunization scheme and was related to clinical outcome (P = .013, log-rank test). Basal concentrations of EGF and TGFα in the serum were affected by EGF-based immunization. In conclusion, the CIMAvax-EGF vaccine induces an EGF-specific protective humoral response in a high percent of NSCLC vaccinated patients, the quantity and quality of which were associated with clinical benefit (clinical trial registration number: RPCEC00000161, http://registroclinico.sld.cu/). Abbreviations: EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; Ab: antibody; AR: amphiregulin; NSCLC: non-small-cell lung cancer; rhEGF: recombinant human epidermal growth factor; BSC: best supportive care; TGFα: tumor growth factor alpha; IL-8: interleukin 8; MAb: monoclonal antibody; SPR: surface plasmon resonance.

CIMAvax-EGF: Toward long-term survival of advanced NSCLC.

Lung cancer remains one of the leading causes of cancer-related deaths. Non-small cell lung cancer (NSCLC) is the most common histologic type of lung cancer. Medical and scientific progress has led to longer survival in an increasing number of patients suffering from cancer. Concerning patients with advanced NSCLC, there is a subgroup with long-term survival. The human epidermal growth factor receptor (EGFR) family plays a key role in tumor development. This cluster of genes is associated with augmented angiogenesis and enhanced proliferation, survival, and migration of tumor cells. The CIMAvax-EGF vaccine consists of a chemical conjugate of the EGF with the P64 protein derived from the Meningitis B bacteria and the Montanide ISA 51, as adjuvant. The vaccine induces antibodies against EGF that results in EGF withdrawal. CIMAvax-EGF has been demonstrated to be safe and immunogenic in advanced NSCLC patients. Here we summarize the current knowledge of the mechanism of action of CIMAvax-EGF, highlighting the impact of this anti-EGF-based vaccine on the long-term survival of advanced NSCLC patients.

Differential effects of two therapeutic cancer vaccines on short- and long-term survival populations among patients with advanced lung cancer.

BACKGROUND: Progress in immunotherapy has revolutionized the treatment landscape for advanced lung cancer, with emerging evidence of patients experiencing long-term survivals. The goal of this study was to explore the existence of short- and long-term survival populations and to assess the effect of immunotherapy on them. METHODS: Data from two randomized, multicenter, controlled clinical trials was used to evaluate the effect of two therapeutic vaccines (anti-idiotypic vaccine VAXIRA and anti-EGF vaccine CIMAVAX) on survival curves in advanced non-small cell lung cancer patients. Data were fitted to Kaplan-Meier, standard Weibull survival, and two-component Weibull mixture models. Bayesian Information Criterion was used for model selection. RESULTS: VAXIRA did not modify, neither the fraction of patients with long-term survivals (0.18 in the control group v 0.19 with VAXIRA, P = .88), nor the median overall survival of the patients in the short-term survival subpopulation (6.8 v 7.8 months, P = .24). However, this vaccine showed great benefit for the patients belonging to the subpopulation of patients with long-term survival (33.8 v 76.6 months, P <.0001). CIMAVAX showed impact in the overall survival of both short- and long-term populations (6.8 v 8.8 months, P = .005 and 33.8 v 61.8 months, P = .007). It also increased the proportion of patients with long-term survival (from 0.18 to 0.28, P = .02). CONCLUSIONS: This study shows that therapeutic vaccines produce differential effects on short- and long-term survival populations and illustrates the application of advanced statistical methods to deal with the long-term evolution of patients with advanced lung cancer in the era of immunotherapy.

Pathway Targeted Immunotherapy: Rationale and Evidence of Durable Clinical Responses with a Novel, EGF-directed Agent for Advanced NSCLC.

Abnormalities in the epidermal growth factor (EGF) and EGFR pathway promote progression of NSCLC. Immunization with EGF vaccine induces specific, neutralizing anti-EGF antibodies that prevent binding of the ligand to its receptor. This concept of pathway targeted immunotherapy (PTI) was validated in vitro by dose-related suppression of EGFR, Akt, and Erk1/2 phosphorylation in cell lines with different mutations. A randomized phase II trial showed improved overall survival (OS) in subgroups with advanced NSCLC showing a clear immunologic response. By per-protocol analysis of the ensuing phase IIb trial, patients receiving EGF PTI survived 3 months longer than controls (12.43 versus 9.43 months; hazard ratio = 0.77 [95% confidence interval, 0.61-0.98]). These data were confirmed in a larger trial showing an OS benefit over control of >3 months. The variable most strongly correlated with efficacy was circulating EGF at enrolment. Patients with serum EGF levels >250 pg/mL benefited most from treatment with EGF PTI. Of 188 patients tested, 94 were above this biomarker threshold. The OS benefit from active versus control treatment was 6.7 months. More than 15% of patients had responses for >5 years. Long-term survivors are seen in all EGF PTI trials. Treatment is well-tolerated, induces high anti-EGF antibody titers, reduces levels of circulating serum EGF, achieves durable responses, and significantly prolongs OS. A threshold of 250 pg/mL has been set to enrich the study population in the ongoing pivotal trial. This biomarker-guided study in an enriched population of patients with both squamous and nonsquamous stage IV NSCLC aims to replicate the favorable efficacy/tolerability balance of earlier studies.

A Phase III Clinical Trial of the Epidermal Growth Factor Vaccine CIMAvax-EGF as Switch Maintenance Therapy in Advanced Non-Small Cell Lung Cancer Patients.

PURPOSE: EGFR is a well-validated target for patients with non-small cell lung cancer (NSCLC). CIMAvax-EGF is a therapeutic cancer vaccine composed of human recombinant EGF conjugated to a carrier protein and Montanide ISA51 as adjuvant. The vaccine is intended to induce antibodies against self EGFs that block EGF-EGFR interaction. EXPERIMENTAL DESIGN: To evaluate overall survival, safety, immunogenicity, and EGF concentration in serum after CIMAvax-EGF, a randomized phase III trial was done in patients with advanced NSCLC. Four to 6 weeks after first-line chemotherapy, 405 patients with stage IIIB/IV NSCLC were randomly assigned to a vaccine group, which received CIMAvax-EGF or a control group, treated with best supportive care. RESULTS: Long-term vaccination was very safe. Most frequent adverse reactions were grade 1 or 2 injection-site pain, fever, vomiting, and headache. Vaccination induced anti-EGF antibodies and decreased serum EGF concentration. In the safety population, median survival time (MST) was 10.83 months in the vaccine arm versus 8.86 months in the control arm. These differences were not significant according the standard log rank (HR, 0.82; P = 0.100), but according a weighted log rank (P = 0.04) that was applied once the nonproportionality of the HR was verified. Survival benefit was significant (HR, 0.77; P = 0.036) in the per-protocol setting (patients receiving at least four vaccine doses): MST was 12.43 months for the vaccine arm versus 9.43 months for the control arm. MST was higher (14.66 months) for vaccinated patients with high EGF concentration at baseline. CONCLUSIONS: Switch maintenance with CIMAvax-EGF was well tolerated and significantly increased MST of patients that completed induction vaccination. Baseline EGF concentration predicted survival benefit. Clin Cancer Res; 22(15); 3782-90. ©2016 AACR.

Biomarkers related to immunosenescence: relationships with therapy and survival in lung cancer patients.

PURPOSE: There are well-known alterations occurring within the immune system with aging. Collectively, these changes are known as immunosenescence. The incidence of malignancies also increases with age. The aim of this study was to determine the presence of immunosenescence biomarkers in non-small cell lung cancer (NSCLC) patients and to evaluate some of them as predictive biomarkers of CIMAvax-EGF cancer vaccine efficacy. METHODS: Sixty-six NSCLC patients, vaccinated or not with CIMAvax-EGF cancer vaccine, and 37 age-matched controls were enrolled. Peripheral blood samples were studied for CD19+, CD4+, CD8+, CD28-, CD57+ and CD45RA+ subpopulations by flow cytometry. RESULTS: Absolute count of CD19+ and the CD4/CD8 ratio were significantly lower in NSCLC patients than in age-paired controls, while highly differentiated T cells increased in NSCLC patients treated with platinum-based chemotherapy. Using Cox regression, we were able to dichotomize the patient population according to biomarkers. Vaccinated patients with frequency <24 % of CD8 + CD28- T cells, >40 % of CD4 T cells and CD4/CD8 ratio higher than two at the beginning of immunotherapy achieved a 20-month increase in median survival regarding control patients. CONCLUSIONS: Distribution of lymphocyte subsets was influenced by cancer and chemotherapy in NSCLC patients. CD19 + B cells decrease by cancer disease and not by chemotherapy, and CD28- subpopulations increase by chemotherapy and not by cancer. The proportion of CD8 + CD28- T cells, CD4+ T cells and CD4/CD8 ratio can be used as predictive biomarkers of CIMAvax-EGF efficacy in NSCLC patients and thereby could, be a useful tool for a personalized treatment.

Safety, immunogenicity and preliminary efficacy of multiple-site vaccination with an Epidermal Growth Factor (EGF) based cancer vaccine in advanced non small cell lung cancer (NSCLC) patients.

The prognosis of patients with advanced non small cell lung (NSCLC) cancer remains dismal. Epidermal Growth Factor Receptor is over-expressed in many epithelial derived tumors and its role in the development and progression of NSCLC is widely documented. CimaVax-EGF is a therapeutic cancer vaccine composed by human recombinant Epidermal Growth Factor (EGF) conjugated to a carrier protein, P64K from Neisseria Meningitides. The vaccine is intended to induce antibodies against self EGF that would block EGF-EGFR interaction. CimaVax-EGF has been evaluated so far in more than 1000 advanced NSCLC patients, as second line therapy. Two separate studies were compared to assess the impact of high dose vaccination at multiple anatomic sites in terms of immunogenicity, safety and preliminary efficacy in stage IIIb/IV NSCLC patients. In both clinical trials, patients started vaccination 1 month after finishing first line chemotherapy. Vaccination at 4 sites with 2.4 mg of EGF (high dose) was very safe. The most frequent adverse events were grade 1 or 2 injection site reactions, fever, headache and vomiting. Patients had a trend toward higher antibody response. The percent of very good responders significantly augmented and there was a faster decrease of circulating EGF. All vaccinated patients and those classified as good responders immunized with high dose at 4 sites, had a large tendency to improved survival.

Combining an EGF-based cancer vaccine with chemotherapy in advanced nonsmall cell lung cancer.

An epidermal growth factor (EGF) vaccine was given before and after standard first line chemotherapy to patients with advanced nonsmall cell lung cancer (NSCLC), to investigate the immunologic and clinical results in a phase 1 study. Twenty patients diagnosed with advanced NSCLC were recruited. Two vaccinations were given before the first line of chemotherapy treatment, with subsequent monthly vaccination after concluding chemotherapy. The EGF vaccination dose was increased compared with previous trials; the primary end points were immunogenicity and safety. Anti-EGF antibody titers were more than 20 times higher than those previously obtained, without any increase in adverse events, serum EGF concentration decreased to undetectable levels in all patients. Ninety-two percent of the evaluated patients (n=13) showed an immunodominant antibody response against the central region on the EGF molecule. High percentages of EGF/EGF receptor binding inhibition were observed, which significantly positively correlated with the increased antibody response against the EGF immunodominant region. Survival of the patients in this study correlates positively with antibody titers. This study has shown that combination of EGF vaccination at high dose, with chemotherapy is feasible and well tolerated higher anti-EGF antibody titers and reduction of serum EGF concentration seen; do not entail an increase in severe adverse events. The correlation of survival with antibody titers observed is being confirmed confirmation in a wider and randomized trial currently ongoing.

Effective inhibition of the epidermal growth factor/epidermal growth factor receptor binding by anti-epidermal growth factor antibodies is related to better survival in advanced non-small-cell lung cancer patients treated with the epidermal growth factor cancer vaccine.

PURPOSE: Epidermal growth factor (EGF) might be a suitable immunotherapeutic target in non-small-cell lung cancer (NSCLC). Our approach consists of active immunotherapy with EGF. The aim of the study is to characterize the humoral response and its effects on signal transduction in relation with the clinical outcome. EXPERIMENTAL DESIGN: Eighty NSCLC patients treated with first-line chemotherapy were randomized to receive the EGF vaccine or supportive care. EGF concentration in sera, anti-EGF antibodies and their capacity to inhibit the binding between EGF/EGF receptor (EGFR), and the EGFR phosphorylation were measured. RESULTS: Seventy-three percent of vaccinated patients developed a good antibody response, whereas none of the controls did. In good antibody-responder patients, self EGF in sera was significantly reduced. In 58% of vaccinated patients, the post-immune sera inhibited EGF/EGFR binding; in the control group, no inhibition occurred. Post-immune sera inhibited the EGFR phosphorylation whereas sera from control patients did not have this capacity. Good antibody-responder patients younger than 60 years had a significantly better survival. A high correlation between anti-EGF antibody titers, EGFR phosphorylation inhibition, and EGF/EGFR binding inhibition was found. There was a significantly better survival for vaccinated patients that showed the higher capacity to inhibit EGF/EGFR binding and for those who showed an immunodominance by the central region of EGF molecule. CONCLUSIONS: Immunization with the EGF vaccine induced neutralizing anti-EGF antibodies capable of inhibiting EGFR phosphorylation. There was a significant positive correlation between antibody titers, EGF/EGFR binding inhibition, immunodominance of anti-EGF antibodies, and survival in advanced NSCLC patients.

Effective inhibition of the epidermal growth factor/epidermal growth factor receptor binding by anti-epidermal growth factor antibodies is related to better survival in advanced non-small-cell lung cancer patients treated with the epidermal growth factor cancer vaccine

PURPOSE: Epidermal growth factor (EGF) might be a suitable immunotherapeutic target in non-small-cell lung cancer (NSCLC). Our approach consists of active immunotherapy with EGF. The aim of the study is to characterize the humoral response and its effects on signal transduction in relation with the clinical outcome. EXPERIMENTAL DESIGN: Eighty NSCLC patients treated with first-line chemotherapy were randomized to receive the EGF vaccine or supportive care. EGF concentration in sera, anti-EGF antibodies and their capacity to inhibit the binding between EGF/EGF receptor (EGFR), and the EGFR phosphorylation were measured. RESULTS: Seventy-three percent of vaccinated patients developed a good antibody response, whereas none of the controls did. In good antibody-responder patients, self EGF in sera was significantly reduced. In 58% of vaccinated patients, the post-immune sera inhibited EGF/EGFR binding; in the control group, no inhibition occurred. Post-immune sera inhibited the EGFR phosphorylation whereas sera from control patients did not have this capacity. Good antibody-responder patients younger than 60 years had a significantly better survival. A high correlation between anti-EGF antibody titers, EGFR phosphorylation inhibition, and EGF/EGFR binding inhibition was found. There was a significantly better survival for vaccinated patients that showed the higher capacity to inhibit EGF/EGFR binding and for those who showed an immunodominance by the central region of EGF molecule. CONCLUSIONS: Immunization with the EGF vaccine induced neutralizing anti-EGF antibodies capable of inhibiting EGFR phosphorylation. There was a significant positive correlation between antibody titers, EGF/EGFR binding inhibition, immunodominance of anti-EGF antibodies, and survival in advanced NSCLC patients(AU)

Therapeutic vaccination with epidermal growth factor (EGF) in advanced lung cancer: analysis of pooled data from three clinical trials.

We have undertaken the analysis of pooled data from three pilot clinical trials of vaccination with Epidermal Growth Factor (EGF) in patients with advanced non small cell lung cancer (NSCLC), addressing particularly the issue of the relationship between immunization and survival. Eighty-three patients with advanced disease were included in three pilot clinical trials and vaccinated with the EGF Vaccine. The trials were designed to evaluate the immunogenicity and safety of the vaccine using different adjuvants, cyclophosphamide pretreatment or not, and different dosage levels of the vaccine. The vaccine elicited specific anti-EGF antibody titers in 83% of subjects, and 49% developed a good anti-EGF antibody response. The adjuvant, the vaccine dose, and cyclophosphamide pretreatment significantly influenced immunogenicity. Patients that seroconverted survived significantly longer than patients who did not. Good antibody responders survived significantly longer than poor responders. Pooled results from these trials confirm that vaccination with EGF is safe and immunogenic in advanced NSCLC patients. The association between good antibody responses and survival consistently appeared in every single trial independently of the specific trial designs. Although these were small pilot nonrandomized clinical trials not intended to confirm therapeutic effect, the survival of the pooled patient population was statistically greater compared with 163 control patients receiving standard treatment.

Active immunotherapy with 1E10 anti-idiotype vaccine in patients with small cell lung cancer: report of a phase I trial.

1E10 is an anti-idiotype murine monoclonal antibody (Ab2 MAb) specific to an Ab1 MAb which reacts with NeuGc-containing gangliosides, sulfatides and with antigens expressed in some human tumors. Preparations containing this Ab2 were capable to induce a strong anti-metastatic effect in tumor-bearing mice. We conducted a Phase I clinical trial to evaluate the toxicity and humoral immune response elicited by 1E10 vaccine in patients with small cell lung cancer (SCLC). Eligible patients were those who after received chemotherapy and/or radiotherapy had partial or complete response to treatment. Patients received four biweekly injections with 2 mg of aluminum hydroxide-precipitated 1E10 MAb, then other six doses at 28-day intervals, and later the patients who maintained a good performance status were reimmunized. Six patients with limited-stage disease and three with extensive-stage disease were enrolled in the study. Most of the patients who received at least four doses of 1E10 vaccine developed strong specific antibody responses against 1E10 MAb and NeuGc-GM3 ganglioside. Antibodies able to react with lung carcinoma tissue sections were detected in sera from vaccinated patients. A prolonged survival was observed in several patients treated with the anti-idiotype vaccine. No evidence of serious adverse effects was found.

Phase I/II clinical trial of the humanized anti-EGF-r monoclonal antibody h-R3 labelled with 99mTc in patients with tumour of epithelial origin.

AIM: To evaluate the biodistribution, internal radiation dosimetry and toxicity of the humanized MAb h-R3 labelled with Tc in humans. METHODS: Twenty-five patients with suspected epithelial-derived tumours were included in this study and divided into two groups: group I consisted of 10 patients who received 3 mg/1110 MBq (3 mg/30 mCi); and group II consisted of 15 patients who received 6 mg/2220 MBq (6 mg/60 mCi). Single photon emission computed tomography (SPECT) and planar images, and multiple blood and urine samples were collected up to 24 h after injection. Haematological parameters and adverse effects were classified according to the WHO criteria. Biodistribution, human anti-mouse antibody (HAMA) response and absorbed doses were estimated and reported. RESULTS: Liver, spleen, kidneys and heart were identified as source organs. Their higher uptakes were 53.3+/-6.4%ID, 2.0+/-1.4%ID, 9.8+/-4.3%ID and 2.8+/-0.9%ID, respectively. The urinary bladder and large intestine also had a significant uptake. The mean urinary excretion was around 22%ID. The liver received the highest absorbed doses followed by the kidneys and the urinary bladder wall. There were no haematological or biochemical abnormalities with clinical significance related to the product. No patient developed HAMA response. Preliminary analysis of clinical results showed a sensitivity of 76.5% and a specificity of 100%. CONCLUSIONS: The results of this study suggest that Tc-h-R3 could be used in patients in a safe and effective way, for the diagnosis of epithelial-derived tumours at the two evaluated dose levels.

Pharmacological evaluation of humanized anti-epidermal growth factor receptor, monoclonal antibody h-R3, in patients with advanced epithelial-derived cancer.

Epidermal growth factor receptor (EGFR) overexpression has been detected in many tumors of epithelial origin, and it is often associated with tumor growth advantages and poor prognosis. h-R3 is a genetically engineered humanized antibody (mAb) that recognizes an epitope located in the extracellular domain of human EGFR. The antibody exhibited potent in vitro and in vivo antitumor effect on EGFR overexpressing cell lines. To study safety, pharmacokinetics, and biodistribution, 12 patients with advanced epithelial-derived tumors received single intravenous infusion of h-R3 at four dose levels. Safety evaluation was made according to World Health Organization toxicity criteria. For biodistribution, 3 mg of the total dose were labeled with Technetium and then pooled with the rest of the dose. Anterior and posterior whole-body images were acquired using a gamma camera. Blood samples were taken for pharmacokinetics, antiidiotypic response, and for soluble EGFR detection. After hR3 administration, no evidence of severe toxicity was observed. Secondary reactions were mild and moderate and mainly consisted of tremors, fever, and vomiting. No anaphylactic or skin reactions were detected. Qualitative analysis of whole-body images showed that the liver had the highest mAb uptake. Pharmacokinetic analysis revealed that elimination half-lives and the AUC increased linearly with dose, while total body clearance decreased when increasing doses of h-R3. No relation between shed EGFR and mAb clearance was found. No antiidiotypic response against h-R3 was detected. Several phase II trials are now underway to evaluate the efficacy of h-R3 in the treatment of advanced cancer patients.

La gammagrafía cerebral / Cerebral gammagraphy

Se expone la utilidad de la gammagrafía cerebral en el estudio de las enfermedades cerebrovasculares. Sobre la parte de la experiencia del departamento de medicina nuclear del IOR, los autores concluyen sobre las indicaciones en las cuales esta técnica radioisotópica es útil(AU)