RESUMEN

Breast cancer is one of the leading causes of cancer deaths worldwide. Thereafter, designing new treatments with higher specificity and efficacy is urgently required. In this regard, targeted immunotherapy using immunotoxins has shown great promise in treating cancer. To target a breast cancer cell, the authors used the antibody fragment against a receptor tyrosine kinase, EphA2, which is overexpressed in many cancers. This fragment was conjugated to a plant toxin, subunit A of ricin, in two different orientations from N to C-terminal (EphA2- C-Ricin and EphA2- N-Ricin). Then, these two immunotoxins were characterized using in vitro cell-based assays. Three different cell lines were treated, MDA-MB-231 (breast cancer) which has a high level of EphA2 expression, MCF-7 (breast cancer) which has a low level of EphA2 expression, and HEK293 (human embryonic kidney) which has a very low level of EphA2 expression. Moreover, binding ability, cytotoxicity, internalization, and apoptosis capacity of these two newly developed immunotoxins were investigated. The flow cytometry using Annexin V- Propidium iodide (PI) method indicated significant induction of apoptosis only in the MDA-MB-231 cells at different concentrations. It was also found that construct I, EphA2- C-Ricin immunotoxin, could bind, internalize, and induce apoptosis better than the EphA2- N-Ricin immunotoxin. In addition, the obtained data suggested that the N or C-terminal orientation conformation is of significant importance.

Asunto(s)

Apoptosis , Neoplasias de la Mama , Inmunotoxinas , Receptor EphA2 , Anticuerpos de Cadena Única , Humanos , Neoplasias de la Mama/inmunología , Receptor EphA2/inmunología , Receptor EphA2/metabolismo , Receptor EphA2/genética , Inmunotoxinas/farmacología , Inmunotoxinas/inmunología , Inmunotoxinas/genética , Femenino , Células HEK293 , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Células MCF-7 , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/farmacología , Ricina/inmunología , Línea Celular Tumoral

RESUMEN

Antibody-mediated depletion studies have demonstrated that CD8+ T cells are required for effective immune control of SIV. However, this approach is potentially confounded by several factors, including reactive CD4+ T cell proliferation, and provides no information on epitope specificity, a likely determinant of CD8+ T cell efficacy. We circumvented these limitations by selectively depleting CD8+ T cells specific for the Gag epitope CTPYDINQM (CM9) via the administration of immunotoxin-conjugated tetrameric complexes of CM9/Mamu-A*01. Immunotoxin administration effectively depleted circulating but not tissue-localized CM9-specific CD8+ T cells, akin to the bulk depletion pattern observed with antibodies directed against CD8. However, we found no evidence to indicate that circulating CM9-specific CD8+ T cells suppressed viral replication in Mamu-A*01+ rhesus macaques during acute or chronic progressive infection with a pathogenic strain of SIV. This observation extended to macaques with established infection during and after continuous antiretroviral therapy. In contrast, natural controller macaques experienced dramatic increases in plasma viremia after immunotoxin administration, highlighting the importance of CD8+ T cell-mediated immunity against CM9. Collectively, these data showed that CM9-specific CD8+ T cells were necessary but not sufficient for robust immune control of SIV in a nonhuman primate model and, more generally, validated an approach that could inform the design of next-generation vaccines against HIV-1.

Asunto(s)

Linfocitos T CD8-positivos , Inmunotoxinas , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Linfocitos T CD8-positivos/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Inmunotoxinas/inmunología , Inmunotoxinas/farmacología , Productos del Gen gag/inmunología , Replicación Viral/inmunología , Replicación Viral/efectos de los fármacos , Depleción Linfocítica/métodos

RESUMEN

In order to overcome limitations of conventional cancer therapy methods, immunotoxins with the capability of target-specific action have been designed and evaluated pre-clinically, and some of them are in clinical studies. Targeting cancer cells via antibodies specific for tumour-associated surface proteins is a new biomedical approach that could provide the selectivity that is lacking in conventional cancer therapy methods such as radiotherapy and chemotherapy. A successful example of an approved immunotoxin is represented by immunoRNases. ImmunoRNases are fusion proteins in which the toxin has been replaced by a ribonuclease. Conjugation of RNase molecule to monoclonal antibody or antibody fragment was shown to enhance specific cell-killing by several orders of magnitude, both in vitro and in animal models. There are several RNases obtained from different mammalian cells that are expected to be less immunogenic and systemically toxic. In fact, RNases are pro-toxins which become toxic only upon their internalization in target cells mediated by the antibody moiety. The structure and large size of the antibody molecules assembled with the immunoRNases have always been a challenge in the application of immunoRNases as an antitoxin. To overcome this obstacle, we have offered a new strategy for the application of immunoRNases as a promising approach for upgrading immunoRNAses with maximum affinity and high stability in the cell, which can ultimately act as an effective large-scale cancer treatment. In this review, we introduce the optimized antibody-like molecules with small size, approximately 10 kD, which are presumed to significantly enhance RNase activity and be a suitable agent with the potential for anti-cancer functionality. In addition, we also discuss new molecular entities such as monobody, anticalin, nonobody and affilin as refined versions in the development of immunoRNases. These small molecules express their functionality with the suitable small size as well as with low immunogenicity in the cell, as a part of immunoRNases.

Asunto(s)

Antineoplásicos Inmunológicos , Antineoplásicos , Inmunotoxinas , Neoplasias , Proteínas Recombinantes de Fusión , Ribonucleasas , Animales , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/farmacocinética , Antineoplásicos Inmunológicos/farmacología , Humanos , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Inmunotoxinas/farmacocinética , Inmunotoxinas/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , Ribonucleasas/genética , Ribonucleasas/inmunología , Ribonucleasas/farmacocinética , Ribonucleasas/farmacología

RESUMEN

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.

Asunto(s)

Colorimetría/métodos , Citotoxinas/análisis , Elapidae/inmunología , Inmunoensayo/métodos , Inmunotoxinas/análisis , Venenos de Serpiente/análisis , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Bungarus/genética , Bungarus/fisiología , Citotoxinas/genética , Citotoxinas/inmunología , Venenos Elapídicos/análisis , Venenos Elapídicos/genética , Venenos Elapídicos/inmunología , Elapidae/fisiología , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Naja naja/inmunología , Naja naja/fisiología , Venenos de Serpiente/inmunología , Viperidae/inmunología , Viperidae/fisiología

RESUMEN

Immunotoxins, as a class of antitumor agents, consist of tumor-selective ligands linked to highly toxic protein molecules. This type of modified antibody has been designed for the therapy of cancers and a few viral infections. In this study, we designed immunotoxin consisting of mouse programmed cell death protein-1 (PD1), which genetically fused to diphtheria toxin (DT) subunit A (DT386). DNA construct was cloned, expressed in a bacterial system, purified, and confirmed by western blotting. The immunotoxin potency in the treatment of tumorous C57BL/6 mice was evaluated. Immunotoxin was injected intratumoral to mice, and through eight injections, 67% of the tumor volume of the test group started shrinking dramatically. On the contrary, the tumor size of the control group, treated with phosphate-buffered saline, continued its growth. The successful targeting of solid tumor cells by PD1-DT immunotoxin demonstrates the potential therapeutic utility of these conjugates.

Asunto(s)

Toxina Diftérica/farmacología , Inmunotoxinas/farmacología , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Supervivencia Celular , Toxina Diftérica/genética , Toxina Diftérica/inmunología , Humanos , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Ratones , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/inmunología

RESUMEN

Recombinant immunotoxins are fusion proteins composed of a peptide toxin and a specific targeting domain through genetic recombination. They are engineered to recognize disease-specific target receptors and kill the cell upon internalization. Full-sized monoclonal antibodies, smaller antibody fragments and ligands, such as a cytokine or a growth factor, have been commonly used as the targeting domain, while bacterial Pseudomonas aeruginosa exotoxin (PE) is the usual toxin fusion partner, due to its natural cytotoxicity and other unique advantages. PE-based recombinant immunotoxins have shown remarkable efficacy in the treatment of tumors and autoimmune diseases. At the same time, efforts are underway to address major challenges, including immunogenicity, nonspecific cytotoxicity and poor penetration, which limit their clinical applications. Recent strategies for structural optimization of PE-based immunotoxins, combined with mutagenesis approaches, have reduced the immunogenicity and non-specific cytotoxicity, thus increasing both their safety and efficacy. This review highlights novel insights and design concepts that were used to advance immunotoxins for the treatment of hematological and solid tumors and also presents future development prospect of PE-based recombinant immunotoxins that are expected to play an important role in cancer therapy.

Asunto(s)

Exotoxinas/uso terapéutico , Inmunotoxinas/uso terapéutico , Neoplasias/tratamiento farmacológico , Pseudomonas aeruginosa/química , Animales , Ensayos Clínicos como Asunto , Exotoxinas/química , Exotoxinas/farmacología , Humanos , Inmunotoxinas/química , Inmunotoxinas/inmunología , Inmunotoxinas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico

RESUMEN

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer (BC) patients. Hence, immunotherapy is a proper treatment option for HER2-positive BC patients. Accumulating evidence has indicated that immunotoxin therapy is a novel approach to improve the potency of targeted therapy. Immunotoxins are antibodies or antibody fragments coupled with a toxin. We designed an immunotoxin. The physicochemical properties were evaluated using ProtParam servers and secondary structure was examined by PROSO II and GORV. Using I-TASSER, a 3D model was built and refined by GalaxyRefine. The model was validated using PROCHECK and RAMPAGE. To predict immunotoxin allergenicity and mRNA stability, AlgPred server and RNAfold were used. Furthermore, the immunotoxin and HER2 were docked by ZDOCK. The scFv+RTX-A could be a non-allergenic and stable chimeric protein, and the secondary structure of its components did not alter, and this protein had a proper 3D structure that might have stable mRNA structure which could bind to HER2. Given the fact that the designed immunotoxin was a non-allergenic and stable chimeric protein and that it could bind with high affinity to HER2 receptors, we proposed that this chimeric protein could be a useful candidate for HER-2 positive BC patients.

Asunto(s)

Neoplasias de la Mama/inmunología , Diseño de Fármacos , Inmunotoxinas/inmunología , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Femenino , Humanos , Inmunotoxinas/química , Modelos Moleculares , Conformación Proteica , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología

RESUMEN

Multiple myeloma (MM) is a B-cell malignancy that is incurable for a majority of patients. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed in multiple myeloma cells but not in other normal tissues except normal plasma B cells. Due to the restricted expression and being a cell surface membrane protein, BCMA is an ideal target for immunotherapy approaches in MM. Recombinant immunotoxins (RITs) are a novel class of protein therapeutics that are composed of the Fv or Fab portion of an antibody fused to a cytotoxic agent. RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in E. coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays using BCMA expressing cell lines and on cells isolated from MM patients. The in vivo efficacy of RITs was tested in a xenograft mouse model using BCMA expressing multiple myeloma cell lines. Anti-BCMA recombinant immunotoxins are very effective in killing myeloma cell lines and cells isolated from myeloma patients expressing BCMA. Two mouse models of myeloma showed that the anti-BCMA immunotoxins can produce a long-term complete response and warrant further preclinical development.

Asunto(s)

Antígeno de Maduración de Linfocitos B/uso terapéutico , Proliferación Celular/efectos de los fármacos , Inmunotoxinas/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Animales , Antígeno de Maduración de Linfocitos B/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/uso terapéutico , Inmunotoxinas/inmunología , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Membrana Celular/metabolismo , Ensayos Analíticos de Alto Rendimiento , Inmunoconjugados/farmacología , Inmunotoxinas/farmacología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Transporte Biológico , Membrana Celular/inmunología , Citotoxicidad Inmunológica , Toxina Diftérica/inmunología , Toxina Diftérica/metabolismo , Toxina Diftérica/farmacología , Exotoxinas/inmunología , Exotoxinas/metabolismo , Exotoxinas/farmacología , Humanos , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Inmunotoxinas/metabolismo , Interleucina-2/inmunología , Interleucina-2/metabolismo , Interleucina-2/farmacología , Liposomas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología

RESUMEN

Cancer cells frequently upregulate surface receptors that promote growth and survival. These receptors constitute valid targets for intervention. One strategy involves the delivery of toxic payloads with the goal of killing those cancer cells with high receptor levels. Delivery can be accomplished by attaching a toxic payload to either a receptor-binding antibody or a receptor-binding ligand. Generally, the cell-binding domain of the toxin is replaced with a ligand or antibody that dictates a new binding specificity. The advantage of this "immunotoxin" approach lies in the potency of these chimeric molecules for killing cancer cells. However, receptor expression on normal tissue represents a significant obstacle to therapeutic intervention.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Inmunotoxinas/inmunología , Neoplasias/inmunología , Receptores de Superficie Celular/inmunología , Toxinas Biológicas/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Humanos , Inmunotoxinas/metabolismo , Inmunotoxinas/uso terapéutico , Ligandos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Unión Proteica , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Toxinas Biológicas/metabolismo

RESUMEN

c(RGDyK)-modified liposomes have been shown to be immunogenic and potentially trigger acute systemic anaphylaxis upon repeated intravenous injection in both BALB/c nude mice and ICR mice. However, questions concerning the potential influence of mouse strains, immunization routes, drug carrier properties, and changes in c(RGDyK) itself on the immunogenicity and resultant immunotoxicity (anaphylaxis) of cyclic RGD peptide-modified nanodrug delivery systems remain unanswered. Here, these potential impact factors were investigated, aiming to better understand the immunological properties of cyclic RGD peptide-based nanodrug delivery systems and seek for solutions for this immunogenicity-associated issue. It was revealed that anaphylaxis caused by intravenous c(RGDyK)-modified drug delivery systems might be avoided by altering the preimmunization route (i.e., subcutaneous injection), introducing positively charged lipids into the liposomes and by using micelles or red blood cell membrane (RBC)-based drug delivery systems as the carrier. Different murine models showed different incidences of anaphylaxis following intravenous c(RGDyK)-liposome stimulation: anaphylaxis was not observed in both SD rats and BALB/c mice and was less frequent in C57BL/6 mice than that in ICR mice. In addition, enlarging the peptide ring of c(RGDyK) by introducing amino sequence serine-glycine-serine reduced the incidence of anaphylaxis post the repeated intravenous c(RGDyKSGS)-liposome stimulation. However, immunogenicity of cyclic RGD-modified drug carriers could not be reversed, although some reduction in IgG antibody production was observed when ICR mice were intravenously stimulated with c(RGDyK)-modified micelles, RBC membrane-based drug delivery systems and c(RGDyKSGS)-liposomes instead of c(RGDyK)-liposomes. This study provides a valuable reference for future application of cyclic RGD peptide-modified drug delivery systems.

Asunto(s)

Formación de Anticuerpos/inmunología , Inmunotoxinas/inmunología , Nanopartículas/química , Péptidos Cíclicos/inmunología , Preparaciones Farmacéuticas/administración & dosificación , Animales , Línea Celular Tumoral , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Eritrocitos/inmunología , Inmunoglobulina G/inmunología , Liposomas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Desnudos , Micelas , Ratas , Ratas Sprague-Dawley

RESUMEN

Immunotoxins are a class of targeted cancer therapeutics in which a toxin such as Pseudomonas exotoxin A (PE) is linked to an antibody or cytokine to direct the toxin to a target on cancer cells. While a variety of PE-based immunotoxins have been developed and a few have demonstrated promising clinical and preclinical results, cancer cells frequently have or develop resistance to these immunotoxins. This review presents our current understanding of the mechanism of action of PE-based immunotoxins and discusses cellular mechanisms of resistance that interfere with various steps of the pathway. These steps include binding of the immunotoxin to the target antigen, internalization, intracellular processing and trafficking to reach the cytosol, inhibition of protein synthesis through ADP-ribosylation of elongation factor 2 (EF2), and induction of apoptosis. Combination therapies that increase immunotoxin action and overcome specific mechanisms of resistance are also reviewed.

Asunto(s)

ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas/inmunología , Resistencia a Antineoplásicos , Exotoxinas/inmunología , Inmunotoxinas/farmacología , Neoplasias/tratamiento farmacológico , Factores de Virulencia/inmunología , ADP Ribosa Transferasas/farmacología , Toxinas Bacterianas/farmacología , Supervivencia Celular/efectos de los fármacos , Ensayos Clínicos como Asunto , Citosol/metabolismo , Exotoxinas/farmacología , Humanos , Inmunotoxinas/inmunología , Neoplasias/inmunología , Factor 2 de Elongación Peptídica/metabolismo , Transporte de Proteínas , Factores de Virulencia/farmacología , Exotoxina A de Pseudomonas aeruginosa

RESUMEN

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. Pseudomonas exotoxin A (PE) is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.

Asunto(s)

Exotoxinas/inmunología , Inmunotoxinas/inmunología , Pseudomonas/inmunología , ADP Ribosa Transferasas/inmunología , Animales , Formación de Anticuerpos/inmunología , Toxinas Bacterianas/inmunología , Ensayos Clínicos como Asunto , Epítopos de Linfocito B/inmunología , Exotoxinas/química , Exotoxinas/farmacocinética , Humanos , Inmunoensayo , Inmunomodulación/efectos de los fármacos , Inmunotoxinas/química , Inmunotoxinas/farmacocinética , Mesotelina , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes/inmunología , Relación Estructura-Actividad , Resultado del Tratamiento , Factores de Virulencia/inmunología , Exotoxina A de Pseudomonas aeruginosa

RESUMEN

Hepatocellular carcinoma (HCC) accounts for most liver cancers and represents one of the deadliest cancers in the world. Despite the global demand for liver cancer treatments, there remain few options available. The U.S. Food and Drug Administration (FDA) recently approved Lumoxiti, a CD22-targeting immunotoxin, as a treatment for patients with hairy cell leukemia. This approval helps to demonstrate the potential role that immunotoxins can play in the cancer therapeutics pipeline. However, concerns have been raised about the use of immunotoxins, including their high immunogenicity and short half-life, in particular for treating solid tumors such as liver cancer. This review provides an overview of recent efforts to develop a glypican-3 (GPC3) targeting immunotoxin for treating HCC, including strategies to deimmunize immunotoxins by removing B- or T-cell epitopes on the bacterial toxin and to improve the serum half-life of immunotoxins by incorporating an albumin binding domain.

Asunto(s)

Toxinas Bacterianas/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Glipicanos/antagonistas & inhibidores , Inmunotoxinas/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Animales , Toxinas Bacterianas/inmunología , Humanos , Inmunotoxinas/inmunología

RESUMEN

BACKGROUND: Immunotoxin is a hybrid protein consisting of a toxin moiety that is linked to a targeting moiety for the purpose of specific elimination of target cells. Toxins used in traditional immunotoxins are practically difficult to be produced in large amount, have poor tissue penetration and a complex internalization process. We hypothesized that the smaller HALT-1, a cytolysin derived from Hydra magnipapillata, can be used as the toxin moiety in construction of a recombinant immunotoxin. RESULTS: In this study, pro-inflammatory macrophage was selected as the target cell due to its major roles in numerous inflammatory and autoimmune disorders. We aimed to construct macrophage-targeted recombinant immunotoxins by combining HALT-1 with anti-CD64-scFv in two orientations, and to assess whether their cytotoxic activity and binding capability could be preserved upon molecular fusion. The recombinant immunotoxins, HALT-1-scFv and scFv-HALT-1, were successfully constructed and expressed in Escherichia coli (E. coli). Our data showed that HALT-1 still exhibited significant cytotoxicity against CD64+ and CD64- cell lines upon fusion with anti-CD64 scFv, although it had half cytotoxic activity as compared to HALT-1 alone. As positioning HALT-1 at N- or C-terminus did not affect its potency, the two constructs demonstrated comparable cytotoxic activities with IC50 lower in CD64+ cell line than in CD64- cell line. In contrast, the location of targeting moieties anti-CD64 scFv at C-terminal end was crucial in maintaining the scFv binding capability. CONCLUSIONS: HALT-1 could be fused with anti-CD64-scFv via a fsexible polypeptide linker. Upon the successful production of this recombinant HALT-1 scFv fusion protein, HALT-1 was proven effective for killing two human cell lines. Hence, this preliminary study strongly suggested that HALT-1 holds potential as the toxin moiety in therapeutic cell targeting.

Asunto(s)

Hydra/efectos de los fármacos , Hydra/inmunología , Inmunotoxinas/inmunología , Animales , Línea Celular , Cnidarios , Escherichia coli/metabolismo , Humanos , Receptores de IgG , Anticuerpos de Cadena Única , Toxinas Biológicas

RESUMEN

Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Inmunotoxinas/inmunología , Mucina-1/química , Mucina-1/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Dominios Proteicos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Human epidermal growth factor receptor 2 (HER2) is an attractive target for cancer therapy, although a large fraction of tumors that express HER2 may still resist first-line therapies. Immunotoxins with antibodies that are armed with extremely potent cytotoxic toxin molecules may provide an important solution to this problem. In this work, we constructed three new anti-HER2 immunotoxins by using single-domain antibody (sdAb) molecules as the targeting moiety and the improved toxin PE24X7 as the effector with the aim of simplifying the preparation and reducing the off-target toxicity of the immunotoxins. Due to the beneficial outcomes of sdAb molecules, the synthesized immunotoxins were efficiently expressed in soluble form, avoiding the refolding process required by the common immunotoxin design and having high solubility and stability. Cell toxicity experiments showed that they have high cytotoxicity against various HER2-positive tumor cells and good selectivity (more than 1000-fold) towards HER2-positive rather than HER2-negative cells. Importantly, in vivo treatment experiments showed that one of the new immunotoxins could efficiently halt tumor growth at doses lower than 0.75 mg/kg, and it had a maximum tolerated dose (MTD) higher than 8.0 mg/kg, showing a substantially improved MTD and a broadened therapeutic window than the previously reported anti-HER2 immunotoxins. Given that PE toxin-based immunotoxins have been approved for clinical cancer therapy, the unique characteristics of the immunotoxins presented here make them promising for use in the development of anti-HER2 cancer therapeutics.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Antineoplásicos/inmunología , Inmunotoxinas/inmunología , Receptor ErbB-2/inmunología , Anticuerpos de Dominio Único/inmunología , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos

RESUMEN

Management of high-grade gliomas remains a complex challenge. Standard of care consists of microsurgical resection, chemotherapy and radiation, but despite these aggressive multimodality therapies the overall prognosis remains poor. A major focus of ongoing translational research studies is to develop novel therapeutic strategies that can maximize tumor cell eradication while minimizing collateral side effects. Particularly, biological intratumoral therapies have been the focus of new translational research efforts due to their inherent potential to be both dynamically adaptive and target specific. This two-part review will provide an overview of biological intratumoral therapies and summarize key advances and remaining challenges in intratumoral biological therapies for high-grade glioma. Part I focuses on discussion of the concepts of intratumoral delivery and immunotoxin therapies.

Asunto(s)

Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Inmunotoxinas/uso terapéutico , Antineoplásicos/inmunología , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Sistemas de Liberación de Medicamentos , Glioma/patología , Humanos , Inmunotoxinas/inmunología , Infusiones Intralesiones , Inyecciones Intralesiones , Clasificación del Tumor , Pronóstico , Ensayos Clínicos Controlados Aleatorios como Asunto

RESUMEN

BACKGROUND: Mesothelin (MSLN) is a cell surface glycoprotein expressed at a high level on many malignancies, including pancreatic adenocarcinoma, serous ovarian cancer, and epithelioid mesothelioma. MSLN-targeted recombinant immunotoxins (RITs) consist of an anti-MSLN Fv fused to the catalytic domain of Pseudomonas exotoxin A. Recent data has also shown that MSLN is expressed at clinically relevant levels on the surface of colorectal cancer (CRC). In this study, CRC cell lines were tested for MSLN expression and susceptibility to MSLN-targeted RITs. MATERIALS AND METHODS: CRC cell lines were tested for membranous MSLN expression via flow cytometry. Cell lines expressing MSLN were tested by WST-8 cell viability assay for sensitivity to various RITs and chemotherapeutic agents. CRC cell line SW-48 was tested in a mouse model for response to RIT as a single agent or in combination with actinomycin D and oxaliplatin. RESULTS: CRC cell lines were susceptible to anti-MSLN RITs at half maximal inhibitory concentration levels comparable with those previously described in pancreatic cancer cell lines. In a nude mouse model, MSLN-targeted RIT treatment of SW48 CRC tumors resulted in a significant decrease in tumor volume. Although combination therapy with standard of care chemotherapeutic oxaliplatin did not improve tumor regressions, combination therapy with actinomycin D resulted in > 90% tumor volume reduction with 50% complete regressions. CONCLUSIONS: These data support the development of anti-MSLN RITs as well as other MSLN-targeted therapies for CRC.

Asunto(s)

ADP Ribosa Transferasas/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Exotoxinas/administración & dosificación , Proteínas Ligadas a GPI/antagonistas & inhibidores , Inmunotoxinas/administración & dosificación , Factores de Virulencia/administración & dosificación , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/inmunología , Animales , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Antineoplásicos Inmunológicos/inmunología , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Línea Celular Tumoral , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Exotoxinas/genética , Exotoxinas/inmunología , Femenino , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Humanos , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Mesotelina , Ratones , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Exotoxina A de Pseudomonas aeruginosa

RESUMEN

Proteus mirabilis is a major cause of complicated urinary tract infections (UTIs). P. mirabilis' urease activity hydrolyzes urea and raises urine pH levels, which can catalyze bladder and kidney stone formation. This urolithiasis leads to harder-to-treat infections, possible urinary blockage, and subsequent septicemia. Development of a mucosal vaccine against P. mirabilis urinary tract infections is critical to protect against this potentially deadly infection process. Here, we describe the methodology necessary to produce a vaccine candidate conjugated to cholera toxin, administer the vaccine via the intranasal route, and test efficacy in a murine transurethral P. mirabilis infection model.

Asunto(s)

Anticuerpos Antibacterianos/metabolismo , Inmunotoxinas/administración & dosificación , Infecciones por Proteus/prevención & control , Proteus mirabilis/inmunología , Infecciones Urinarias/prevención & control , Administración Intranasal , Animales , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Toxina del Cólera/administración & dosificación , Toxina del Cólera/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunotoxinas/inmunología , Ratones , Ratones Endogámicos CBA , Vacunación