Evaluation of nimotuzumab Fab2 as an optical imaging agent in EGFR positive cancers.

Molecular-targeted imaging probes can be used with a variety of imaging modalities to detect diseased tissues and guide their removal. EGFR is a useful biomarker for a variety of cancers, because it is expressed at high levels relative to normal tissues. Previously, we showed the anti-EGFR antibody nimotuzumab can be used as a positron emission tomography and fluorescent imaging probe for EGFR positive cancers in mice. These imaging probes are currently in clinical trials for PET imaging and image-guided surgery, respectively. One issue with using antibody probes for imaging is their long circulation time and slow tissue penetration, which requires patients to wait a few days after injection before imaging or surgery, multiple visits and longer radiation exposure. Here, we generated a Fab2 fragment of nimotuzumab, by pepsin digestion and labeled it with IRDye800CW to evaluate its optical imaging properties. The Fab2 had faster tumor accumulation and clearance in mice relative to the nimotuzumab IgG. The fluorescent signal peaked at 2 h post injection and remained high until 6 h post injection. The properties of the Fab2 allow a higher signal to background to be obtained in a shorter time frame, reducing the wait time for imaging after probe infusion.

Pre-clinical study of IRDye800CW-nimotuzumab formulation, stability, pharmacokinetics, and safety.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a target for cancer therapy as it is overexpressed in a wide variety of cancers. Therapeutic antibodies that bind EGFR are being evaluated in clinical trials as imaging agents for positron emission tomography and image-guided surgery. However, some of these antibodies have safety concerns such as infusion reactions, limiting their use in imaging applications. Nimotuzumab is a therapeutic monoclonal antibody that is specific for EGFR and has been used as a therapy in a number of countries. METHODS: Formulation of IRDye800CW-nimotuzumab for a clinical trial application was prepared. The physical, chemical, and pharmaceutical properties were tested to develop the specifications to determine stability of the product. The acute and delayed toxicities were tested and IRDye800CW-nimotuzumab was determined to be non-toxic. Non-compartmental pharmacokinetics analysis was used to determine the half-life of IRDye800CW-nimotuzumab. RESULTS: IRDye800CW-nimotuzumab was determined to be non-toxic from the acute and delayed toxicity study. The half-life of IRDye800CW-nimotuzumab was determined to be 38 ± 1.5 h. A bi-exponential analysis was also used which gave a t1/2 alpha of 1.5 h and t1/2 beta of 40.8 h. CONCLUSIONS: Here, we show preclinical studies demonstrating that nimotuzumab conjugated to IRDye800CW is safe and does not exhibit toxicities commonly associated with EGFR targeting antibodies.

89Zr-Labeled Domain II-Specific scFv-Fc ImmunoPET Probe for Imaging Epidermal Growth Factor Receptor In Vivo.

Epidermal growth factor receptor I (EGFR) is overexpressed in many cancers. The extracellular domain of EGFR has four binding epitopes (domains I- IV). All clinically approved anti-EGFR antibodies bind to domain III. Imaging agents that bind to domains other than domain III of EGFR are needed for accurate quantification of EGFR, patient selection for anti-EGFR therapeutics and monitoring of response to therapies. We recently developed a domain II-specific antibody fragment 8709. In this study, we have evaluated the in vitro and in vivo properties of 89Zr-8709-scFv-Fc (105 kDa). We conjugated 8709-scFv-Fc with the deferoxamine (DFO) chelator and radiolabeled the DFO-8970-scFv with 89Zr. We evaluated the binding of 89Zr-DFO-8709-scFv-Fc in EGFR positive and negative cell lines DLD-1, MDA-MB-231 and MDA-MB-435, respectively, and in mouse xenograft models. Simultaneously, we have compared the binding of 89Zr-8709-scFv-Fc with 111In-nimotuzumab, a domain III anti-EGFR antibody. DFO-8709-scFv-Fc displayed similar cell binding specificity as 8709-scFv-Fc. Saturation cell binding assay and immunoreactive fraction showed that radiolabeling did not alter the binding of 8709-scFv-Fc. Biodistribution and microPET showed good uptake of 89Zr-8709-scFv-Fc in xenografts after 120 h post injection (p.i). and was domain-specific to EGFR domain II. 89Zr-8709-scFv-Fc did not compete for binding in vitro and in vivo with a known domain III binder nimotuzumab. The results show that 89Zr-8709-scFv-Fc is specific to domain II of EGFR making it favorable for quantification of EGFR in vivo, hence, patient selection and monitoring of response to treatment with anti-EGFR antibodies.

Safety and Immunogenicity of a Human Epidermal Growth Factor Receptor 1 (HER1)-Based Vaccine in Prostate Castration-Resistant Carcinoma Patients: A Dose-Escalation Phase I Study Trial.

Metastatic castration-resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. Activation of the human epidermal growth factor receptor 1 (HER1) in prostate cancer contributes to metastatic progression as well as to disease relapse. Here, we determined the toxicity and immunogenicity of a HER1-based cancer vaccine in CRPC patients included in a phase I clinical trial. CRPC patients (n = 24) were intramuscularly vaccinated with HER1 vaccine consisting of the extracellular domain of HER1 molecule (ECD) and very small size proteoliposome from Neisseria meningitidis (VSSP) and Montanide ISA-51 VG as adjuvants. Patients were included in five groups according to the vaccine dose (100, 200, 400, 600, and 800 µg). The primary endpoints were safety and immunogenicity. The anti-HER1 antibodies were measured by an ELISA, the recognition of an HER1 positive tumor cell line and the inhibition of HER1 phosphorylation by sera were determined by flow cytometry and western blot analysis, respectively. The HER1-specific T cell response was assessed by determination of IFN-γ-producing T cells using ELISpot assay. The vaccine was well tolerated. No grade III or IV adverse events were reported. High titers of anti-HER1 antibodies were observed in most of the evaluated patients. There were no significant differences regarding the geometric means of the anti-HER1 titers among the dose groups except the group of 100 µg in which antibody titers were significantly lower. A Th1-type IgG subclasses pattern was predominant in most patients. Only patients receiving the higher doses of vaccine showed significant tumor cell recognition and HER1 phosphorylation inhibition by hyperimmune sera. Forty two percent of the patients showed a specific T cell response against HER1 peptides pool in post-treatment samples. There was a trend toward survival benefit in those patients showing high anti-HER1 specific antibody titers and a significant association between cellular immune response and clinical outcome.

Effects of an epidermal growth factor receptor-based cancer vaccine on wound healing and inflammation processes in murine experimental models.

Anti-epidermal growth factor receptor (EGFR) therapies have been proven clinically effective for a variety of epithelial tumours. Vaccination of mice with the extracellular domain (ECD) of autologous EGFR overcomes the tolerance to self-EGFR and has antimetastatic effect on EGFR+ tumour. Because EGF/EGFR-signalling plays an important role in the inflammation stage of wound healing, the main objective of this study was to explore the possible role of murine (m) EGFR-ECD vaccine in the croton-oil-induced ear oedema and wound healing process in mice as autologous experimental models, mimicking the possible post-surgical wound complication in patients treated with human EGFR-ECD/VSSP vaccine. Mice were intramuscularly immunised four times; biweekly with the mEGFR-ECD/VSSP/Mont. Seven days later, an 8 mm diameter, full-thickness skin wound was created on the back of each animal. Immunisation induced a strong specific humoral response against the mEGFR-ECD protein and a DTH dose-response curve but interestingly, animals treated with mEGFR-ECD/VSSP/Mont had similar inflammatory and healing speed responses compared to control ones. These data suggest that application of mEGFR-ECD/VSSP vaccine as a therapeutic approach in cancer patients could not elicit a poor healing process after surgery.

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: a case study.

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.

Radiosensitization induced by the anti-epidermal growth factor receptor monoclonal antibodies cetuximab and nimotuzumab in A431 cells.

Epidermal growth factor receptors (EGFR) are overexpressed in a wide range of malignancies including head and neck, colon, and breast cancers. It has been identified that carcinomas with high expression levels of EGFR are more resistant to radiotherapy. Therefore, inhibiting nuclear translocation of EGFR to increase the radiosensitivity of malignant cells expressing EGFR offers the potential for increasing the therapeutic index of radiotherapy. The purpose of the present study was to quantify and to compare the radiosensitizing properties of the well-known anti-EGFR antibodies, cetuximab and nimotuzumab in human epidermoid A431 overexpressing EGFR cells. Cells were treated with two concentrations of the antibodies and then irradiated with a single dose of 4 Gy. The results indicated that the two antibodies induced radiosensitization increasing the percentage of dead/dying cells and the yield of γ-H2AX foci 24 h after irradiation. Whereas cetuximab exhibited a significant increase in radiosensitization at the highest concentration, the effects of nimotuzumab were more modest. A correlation between γ-H2AX foci signals and dead/dying cells was observed. The disparity in modulation of radiation-induced DNA damage by the two antibodies could be associated with the level of their respective intrinsic cytotoxic properties. Overall, the findings highlight the potential therapeutic benefit of combination therapy with anti-EGFR antibodies and radiotherapy for relevant carcinomas.

A clinical exploratory study with itolizumab, an anti-CD6 monoclonal antibody, in patients with rheumatoid arthritis.

T cells are involved in the pathogenesis of rheumatoid arthritis (RA). CD6 is a co-stimulatory molecule, predominantly expressed on lymphocytes, that has been linked to autoreactive responses. The purpose of this study was to evaluate the safety, immunogenicity and preliminary efficacy of itolizumab, a humanized anti-CD6 monoclonal antibody, in patients with active rheumatoid arthritis. Fifteen patients were enrolled in a phase I, open-label, dose-finding study. Five cohorts of patients received a weekly antibody monotherapy with a dose-range from 0.1 to 0.8 mg/kg. Itolizumab showed a good safety profile, with no severe or serious adverse events reported so far. No signs or symptoms associated with immunosuppression were observed in the study. Objective clinical responses were achieved in more than 80% of patients after treatment completion, and these responses tend to be sustained afterwards. This clinical study constitutes the first evidence of the safety and positive clinical effect of a monotherapy using an anti-CD6 antibody in patients with rheumatoid arthritis.

Hematological, biochemical, respiratory, cardiovascular and electroneurophysiological parameters in African green monkeys (Cercopithecus aethiops sabaeus). Its use in non-clinical toxicological studies.

BACKGROUND: The purpose of this study is to better characterize the hematological, biochemical, respiratory, cardiovascular and electroneurophysiological parameters in young adult Cercopithecus aethiops sabaeus of both sexes. The rhesus and cynomolgus monkeys are widely used as experimental primate models. However, only few articles have been published testing toxicological effects of pharmaceuticals on African green monkey. METHODS: The present study was carried out with the recompilation of all parameters recorded before the first drug administration in five sub-chronic or chronic toxicological studies performed on 66 Cercopithecus aethiops sabaeus, born in Cuba. RESULTS: This study provides hematological, biochemical, respiratory, cardiovascular and electroneurophysiological data for both choosing animals to be included into experiments and monitoring these parameters during the study. CONCLUSIONS: We conclude that this study provides valuable integrated data for determining the health status, including electroneurophysiological parameters, data not previously reported for this species, of the African green monkey.

Phase I single-dose study of intracavitary-administered Nimotuzumab labeled with 188 Re in adult recurrent high-grade glioma.

Radioimmunotherapy (RIT) may improve the management of malignant gliomas. A Phase I clinical trial was performed to evaluate, for the first time, the toxicity and clinical effect of an intracavitary administration of a single dose of Nimotuzumab (h-R3) labeled wit (188)Re. Nimotuzumab is a humanized monoclonal antibody directed against epidermal growth factor receptors. Three patients with anaplastic astrocytoma (AA) and 8 with glioblastoma multiforme (GBM) were intended to be treated with 3 mg of mAb labelled with 10 or 15 mCi of (188)Re. In patients treated with 10 mCi (n=6) transitory worsening of pre-existing neurological symptoms were observed. Two patients treated with 15 mCi (n=4) developed early severe neurological symptoms and one also developed late severe toxicity (radionecrosis). In the group treated with 10 mCi, 1 GBM patient died in progression 6 months after the treatment, 2 patients (1 GBM and 1 AA) developed stable disease during 3 months. One GBM patient had partial response for more than 1 year and 2 patients (1 GBM and 1 AA) were asymptomatic and in complete response after 3 years of treatment. Maximal tolerated dose of the radioimmunoconjugate (188)Re-Nimotuzumab was 3 mg of the h-R3 labelled with 10 mCi of (188)Re. The radioimmunoconjugate showed a high retention in the surgical created resection cavity and the brain adjacent tissues with a mean value of 85.5 % of the injected dose one hour post-administration. This radioimmunoconjugate may be relatively safe and a promising therapeutic approach for treating high grade gliomas.

Biodistribution and internal dosimetry of the 188Re-labelled humanized monoclonal antibody anti-epidemal growth factor receptor, nimotuzumab, in the locoregional treatment of malignant gliomas.

OBJECTIVE: To evaluate the biodistribution, internal radiation dosimetry and safety of the 188Re-labelled humanized monoclonal antibody nimotuzumab in the locoregional treatment of malignant gliomas. METHODS: Single doses of 370 or 555 MBq of 188Re-labelled nimotuzumab were locoregionally administered to nine patients with recurrent high-grade gliomas, according to an approved dose-escalation study. SPECT, planar scintigraphy and magnetic resonance images were combined for dosimetric and pharmacokinetic studies. Blood and urine samples were collected to evaluate clinical laboratory parameters and for absorbed doses calculations. Biodistribution, internal dosimetry, human anti-mouse antibody response and toxicity were evaluated and reported. RESULTS: The 188Re-nimotuzumab showed a high retention in the surgically created resection cavity with a mean value of 85.5+/-10.3%ID 1 h post-injection. It produced mean absorbed doses in the tumour region of approximately 24.1+/-2.9 Gy in group I (patients receiving 370 MBq) and 31.1+/-6.4 Gy in group II (patients receiving 555 MBq); the normal organs receiving the highest absorbed doses were the kidneys, liver and urinary bladder. About 6.2+/-0.8%ID was excreted by the urinary pathway. The maximum tolerated dose was 370 MBq because two patients showed severe adverse effects after they received 555 MBq of 188Re-nimotuzumab. No patient developed human anti-mouse antibody response. CONCLUSIONS: A locoregional single dose of 188Re-labelled nimotuzumab of approximately 370 MBq could be used safely in the routine treatment of patients suffering with high-grade gliomas. The efficacy of this therapy needs to be evaluated in a phase II clinical trial.

Systemic and skin toxicity in Cercopithecus aethiops sabaeus monkeys treated during 26 weeks with a high intravenous dose of the anti- epidermal growth factor receptor monoclonal antibody Nimotuzumab.

Nimotuzumab (h-R3) is a humanized anti-epidermal growth factor receptor monoclonal antibody (mAb) registered for treating head and neck tumours. The present study was designed to evaluate the systemic and skin toxicity of chronic intravenous administration of the h-R3 in a relevant species demonstrated by comparing the h-R3 binding affinity constants (Kd) in microsomal placental fractions from Homo sapiens and Cercopithecus aethiops monkeys using an EGF-Receptor radioligand competition assay. The Kd obtained for Nimotuzumab were 9.1 x 10(-8) M for monkeys and 4.5 x 10(-8) M for humans. Monkeys (n = 18) were distributed into 3 groups with 3 animals of each sex in each group. Group I received saline; group II received 2.85 mg/kg of h-R3; and group III received 28.57 mg/kg of the h-R3, which represent 1 and 10 times the human dose, and they were weekly intravenously treated during 26 weeks. During the study there were no deaths. Electroneurophysiological, sanguine chemistry and haematological results did not evidence alterations. Areas of haematomas, probably related with the administration procedure, were observed at the administration zones of all animals. The electrocardiography study showed at the end of the study a slight increase in the cardiac frequency of four treated animals without other signs. Unexpectedly, skin biopsies and a detailed clinical inspection of the animals did not detect the presence of cutaneous rash or any other skin toxicity sign reported for the majority of the anti-EGF-R monoclonal antibodies. It is concluded that doses up to 28.5 mg/kg of h-R3, intravenously administered during 26 weeks to Cercopithecus aethiops monkeys, do not produce considerable toxic effects.

Clinical evidences of GM3 (NeuGc) ganglioside expression in human breast cancer using the 14F7 monoclonal antibody labelled with (99m)Tc.

The relevance of certain gangliosides in tumour growth and metastatic dissemination has been well documented, reasons for considering these molecules as potential targets for cancer immunotherapy and diagnosis. GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues according to immunohistochemical studies, using either polyclonal or monoclonal antibodies. But both immunohistochemical and biochemical methods have strongly suggested its over-expression in human breast tumours. Nevertheless, the lack of a direct evidence of this antigenic display in human breast cancer has kept the subject controversial. For the first time, we described herein the "in vivo" detection of GM3(NeuGc) ganglioside in human breast primary tumours using a radioimmunoscintigraphic technique with 14F7, a highly specific anti-GM3(NeuGc) ganglioside monoclonal antibody, labelled with (99m)Tc. In an open, prospective Phase I/II clinical trial, including women diagnosed in stage II breast cancer, the 14F7 monoclonal antibody accumulation in tumours at doses of 0.3 (n=5), 1 (n=5) and 3 mg (n=4) was evaluated. Noteworthy, the immunoscintigraphic study showed antibody accumulation in 100% of patients' tumours for the 1 mg dose group. In turn, the radioimmunoconjugate injected at doses of 0.3 mg or 3 mg of the antibody, was uptaken by 60 and 33.3% of breast tumours, respectively. "In vivo" immune recognition of GM3(NeuGc) in breast tumours reinforces the value of this peculiar target for cancer immunotherapy.

Pharmacokinetic comparison of two recombinant human granulocyte colony-stimulating factor after subcutaneous administration in rabbits.

A pharmacokinetic comparison between two formulations (LeukoCIM, CIMAB SA versus Neupogen, Hoffman-La Roche, licensed by Amgen) of recombinant human granulocyte colony-stimulating factor (rhG-CSF) using non-compartmental analysis was performed in male F1 rabbits after a single subcutaneous 11.5 microg/kg dose to help decide whether to conduct further comparability tests. Unlike the absorption phase, a statistical difference was not detected between Neupogen and LeukoCIM for clearance (18.69+/-11.83 versus 28.42+/-12.11 mL/h/kg, P=0.22). In addition, using a multivariate statistical analysis by independent samples test, a significant difference was not found between the two formulations (P=0.88). Finally, the results obtained in this study confirmed the pharmacokinetic comparability between both formulations, supporting the claim for further assessments following the current protocol on biogeneric equivalence.

Efectos antipsoriasico, antiinflamatorio y analgésico del propoleo rojo colectado en Cuba / Antipsoriatic, antinflammatory, and analgesic effects of red propolis collected in Cuba

Se evaluaron los efectos antipsoriásico, antiinflamatorio y analgésico de un extracto de propóleo rojo. Este extracto induce la formaciónde la capa granular en la prueba de la cola de ratón usada como modelo de psoriasis. El propóleo a la dosis de 50 mg/kg (vía oral) mostró actividad antiinflamatoria en el modelo de granuloma por algodón en ratas, así como en la prueba de permedabilidad capilar en el peritoneo de ratas en la dosis de 10 mg/kg. El extracto de propóleo (25 mg/kg, vía oral) presentó propiedades analgésicas en el modelo de estiramiento por ácido acético, mientras que la dosis de 40 mg/kg fue efectiva en la prueba del plato caliente en ratones. Estos resultados demuestran evidencias acerca de la utilidad potencial del propóleo rojo en trastornos inflamatorios y particularmente en el tratamiento de la psoriasis (AU)

Efectos antipsoriásico, antiinflamatorio y analgésico del propoleo rojo colectado en Cuba

Se evaluaron los efectos antipsoriásico, antiinflamatorio y analgésico de un extracto de propóleo rojo. Este extracto induce la formación de la capa granular en la prueba de la cola de ratón usada como modelo de psoriasis. El propóleo a la dosis de 50 mg/kg (vía oral) mostró actividad antiinflamatoria en el modelo de granuloma por algodón en ratas, así como en la prueba de permeabilidad capilar en el peritoneo de ratas en la dosis de 10 mg/kg. El extracto de propóleo (25 mg/kg, vía oral) presentó propiedades analgésicas en el modelo de estiramiento por ácido acético, mientras que la dosis de 40 mg/kg fue efectiva en la prueba del plato caliente en ratones. Estos resultados demuestran evidencias acerca de la utilidad potencial del propóleo rojo en trastornos inflamatorios y particularmente en el tratamiento de la psoriasis.

The antipsoriatic, antiinflammatory, and analgesic effects of a red propolis extract, were assessed. This extract induces the formation of a granular layer in the mouse tail test, used as a model for psoriasis. Propolis at a 50 mg/kg dose (oral) showed antiinflammatory activity in the cotton granuloma model in rats, as well as in the capillary permeability test in rats peritoneum at a 10 mg/kg dose. Propolis extract (25 mg/kg, oral) presented analgesic properties in the acetic acid stretching model, while the 40 mg/kg dose was effective in the hot plate test in mice. These results demonstrate evidence about the potential usefulness of red propolis in inflammatory disorders, and particularly, in the management of psoriasis.

Estudio toxicológico agudo del mazineb / Acute toxicologic study of mazineb

Se realizó el estudio toxicológico agudo del mazineb en ratas por diferentes vías de administración. La LD50 a las 24 horas en ratones (IP) fue de 235 mg/kg y 2 380 mg/kg (PO), en ratas fue de 2 900 mg/kg (PO). El mazineb por otras vías de administración (inhalación, piel) es poco tóxico, exceptuando la vía intraocular, donde induce ceguera irreversible. El mazineb no posee efecto inhibidor de la acetilcolinesterasa en los órganos estudiados después de su administración oral

Estudio toxicológico agudo del mazineb / Acute toxicologic study of azineb

Se realizó el estudio toxicológico agudo del mazineb en ratas por diferentes vías de administración. La LD50 a las 24 horas en ratones (IP) fue de 235 mg/kg y 2 380 mg/kg (PO), en ratas fue de 2 900 mg/kg (PO). El mazineb por otras vías de administración (inhalación, piel) es poco tóxico, exceptuando la vía intraocular, donde induce ceguera irreversible. El mazineb no posee efecto inhibidor de la acetilcolinesterasa en los órganos estudiados después de su administración oral

Rol de la acetilcolinesterasa en la broncoconstricción por keroseno / Role of acetylcholinesterase in kerosene induced bronchoconstriction

El efecto del keroseno sobre la acetilcolinesterasa (AChõ) traqueal, pulmonar y plasmática fue evaluado en experimentos in vivo e in vitro. El aerosol de keroseno administrado a cobayos durante 20 minutos a una concentración media de 32,5 mg/L produjo una hora después de la inhalación una significativa inhibición en la AChõ traqueal en un 28,8 por ciento de los valores controles. No se observó inhibición significativa en la AChõ pulmonar o en plasma. Enlos experimentos in vitro a una concentración del 1 por ciento después de una hora de incubación el keroseno inhibió significativamente la AChõ traqueal en un 44,9 por ciento de los valores controles, la AChõ en sangre total fue inhibida enun 25,8 por ciento, mientras que la AChõ pulmonar tampoco fue inhibida en los experimentos in vitro. Se discute los posibles mecanismos inhibitorios del keroseno en los tejidos estudiados (AU)