RESUMEN

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

Asunto(s)

Endocitosis , Exotoxinas , Inmunotoxinas , Humanos , Inmunotoxinas/farmacología , Inmunotoxinas/química , Animales , Endocitosis/efectos de los fármacos , Ratones , Línea Celular Tumoral , Exotoxinas/farmacología , Exotoxinas/química , Exotoxina A de Pseudomonas aeruginosa , ADP Ribosa Transferasas/farmacología , ADP Ribosa Transferasas/química , Ensayos Antitumor por Modelo de Xenoinjerto , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacología , Oxidación-Reducción/efectos de los fármacos , Femenino

RESUMEN

Most plant and bacterial toxins are highly immunogenic with non-specific toxic effects. Human ribonucleases are thought to provide a promising basis for reducing the toxic agent's immunogenic properties, which are candidates for cancer therapy. In the cell, the ribonuclease inhibitor (RI) protein binds to the ribonuclease enzyme and forms a tight complex. This study aimed to engineer and provide a gene construct encoding an improved version of Human Pancreatic RNase 1 (HP-RNase 1) to reduce connection to RI and modulate the immunogenic effects of immunotoxins. To further characterize the interaction complex of HP-RNase 1 and RI, we established various in silico and in vitro approaches. These methods allowed us to specifically monitor interactions within native and engineered HP-RNase 1/RI complexes. In silico research involved molecular dynamics (MD) simulations of native and mutant HP-RNase 1 in their free form and when bound to RI. For HP-RNase 1 engineering, we designed five mutations (K8A/N72A/N89A/R92D/E112/A) based on literature studies, as this combination proved effective for the intended investigation. Then, the cDNA encoding HP-RNase 1 was generated by RT-PCR from blood and cloned into the pSYN2 expression vector. Consequently, wild-type and the engineered HP-RNase 1 were over-expressed in E. coli TG1 and purified using an IMAC column directed against a poly-his tag. The protein products were detected by SDS-PAGE and Western blot analysis. HP-RNase 1 catalytic activity, in the presence of various concentrations of RI, demonstrated that the mutated version of the protein is able to escape the ribonuclease inhibitor and target the RNA substrate 2.5 folds more than that of the wild type. From these data, we tend to suggest the engineered recombinant HP-RNase 1 potentially as a new immunotherapeutic agent for application in human cancer therapy.

Asunto(s)

Proteínas Portadoras , Simulación de Dinámica Molecular , Ingeniería de Proteínas , Ribonucleasa Pancreática , Humanos , Ingeniería de Proteínas/métodos , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismo , Neoplasias/terapia , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Inmunoterapia/métodos , Escherichia coli/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Inmunotoxinas/química , Inmunotoxinas/genética , Inmunotoxinas/farmacología , Mutación

RESUMEN

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Asunto(s)

Antineoplásicos , Toxinas Bacterianas , Inmunotoxinas , Neoplasias , Anticuerpos de Dominio Único , Animales , Ratones , Humanos , Exotoxinas/genética , Exotoxinas/farmacología , Exotoxinas/química , Inmunotoxinas/genética , Inmunotoxinas/farmacología , Inmunotoxinas/química , Mesotelina , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/farmacología , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Dominio Catalítico , Línea Celular Tumoral , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Neoplasias/tratamiento farmacológico

RESUMEN

Immunotoxins (ITXs) are chimeric molecules that combine the specificity of a targeting domain, usually derived from an antibody, and the cytotoxic potency of a toxin, leading to the selective death of tumor cells. However, several issues must be addressed and optimized in order to use ITXs as therapeutic tools, such as the selection of a suitable tumor-associated antigen (TAA), high tumor penetration and retention, low kidney elimination, or low immunogenicity of foreign proteins. To this end, we produced and characterized several ITX designs, using a nanobody against EGFR (VHH 7D12) as the targeting domain. First, we generated a nanoITX, combining VHH 7D12 and the fungal ribotoxin α-sarcin (αS) as the toxic moiety (VHHEGFRαS). Then, we incorporated a trimerization domain (TIEXVIII) into the construct, obtaining a trimeric nanoITX (TriVHHEGFRαS). Finally, we designed and characterized a bispecific ITX, combining the VHH 7D12 and the scFv against GPA33 as targeting domains, and a deimmunized (DI) variant of α-sarcin (BsITXαSDI). The results confirm the therapeutic potential of α-sarcin-based nanoITXs. The incorporation of nanobodies as target domains improves their therapeutic use due to their lower molecular size and binding features. The enhanced avidity and toxic load in the trimeric nanoITX and the combination of two different target domains in the bispecific nanoITX allow for increased antitumor effectiveness.

Asunto(s)

Neoplasias Colorrectales , Inmunotoxinas , Anticuerpos de Dominio Único , Humanos , Inmunotoxinas/química , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/uso terapéutico , Antígenos de Neoplasias , Neoplasias Colorrectales/tratamiento farmacológico , Receptores ErbB

RESUMEN

Pseudomonas aeruginosa exotoxin A-based immunotoxins (PE-ITs) are fusion proteins that harness targeting and toxin moieties. Structural optimizations in PE and targeting moieties were implemented to lower their immunogenicity and alleviate undesirable side effects. PE moiety was engineered to lack its cell-binding domain and T cell epitope regions, whereas single chain (scFv) and disulfide Fv portions (dsFv), nanobodies, and monobodies were utilized as targeting moieties. This review discusses applications of PE-ITs on different types of cancer, structural optimizations to reduce PE-ITs drawbacks, and recent modifications applied for efficient therapeutic delivery. Finally, we draw attention to the possibility of combining radiotherapy, radionuclides, and RGDs with PE-IT to improve overall response rates of IT-based treatments and reduce cancer cell resistance.

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Exotoxin A-immunotoxins are proteins that have been used in cancer treatments. The building components of these proteins are very poisonous to both cancer and normal cells. Also, unfavorable body reactions and side effects were seen with their usage. To allow the safe use of these proteins, changes were made in their building components. These changes made them damaging only to cancer cells while being safe to normal non-cancerous cells. This review will talk about the use of exotoxin A-Immunotoxins in different cancer treatments, and how they are created to limit the poisonous effect of their building components to only cancer cells.

Asunto(s)

Toxinas Bacterianas , Inmunotoxinas , Neoplasias , Humanos , Inmunotoxinas/uso terapéutico , Inmunotoxinas/química , Exotoxinas/uso terapéutico , Exotoxinas/química , Neoplasias/tratamiento farmacológico , Pseudomonas aeruginosa , Exotoxina A de Pseudomonas aeruginosa

RESUMEN

Breast cancer remains the most frequently diagnosed cancer and the principal cause of mortality by malignancy in women. HER2 positive subtype includes 15-20% of breast cancer cases. This receptor could be an appropriate mark for targeting breast cancer cells. Immunotherapy methods compared to current cancer treatment methods have the lowest side effects. DELTA-stichotoxin-Hmg2a is isolated from the sea anemone and kills cells through pore formation. In the current study, we designed and evaluated an immunotoxin composed of pertuzumab and DELTA-stichotoxin-Hmg2a-derived scFv by bioinformatics tools. The designed immunotoxin was constructed using the amino acid sequences. Then, secondary structure and physico-chemical features were studied, and the tertiary structure of the immunotoxin was built according to the homology modeling methods. The validation and allergenicity of the model were assessed. The immunotoxin and receptor were docked and molecular dynamics simulation indicated the construct stability. The analysis results indicated that the construct is a stable protein that could have a natural-like structure and would not be an allergen, so this immunotoxin could effectively target HER2 receptors. Therefore, our designed immunotoxin could be an appropriate immunotoxin against HER2-positive breast cancer and could be a challenging topic for future in vitro and in vivo studies.

Asunto(s)

Neoplasias de la Mama , Proteína HMGB3 , Inmunotoxinas , Humanos , Femenino , Inmunotoxinas/química , Inmunotoxinas/metabolismo , Inmunotoxinas/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Inmunoterapia

RESUMEN

Along with all cancer treatments, including chemotherapy, radiotherapy, and surgery, targeting therapy is a new treatment manner. Immunotoxins are new recombinant structures that kill cancer cells by targeting specific antigens. Immunotoxins are composed of two parts: toxin moiety, which disrupts protein synthesis process, and antigen binding moiety that bind to antigens on the surface of cancer cells. Glypican 3 (GPC3) is an oncofetal antigen on the surface of Hepatocellular carcinoma (HCC) cells. In this study, truncated Diphtheria toxin (DT389) was fused to humanized scFv YP7 by one, two and three repeats of GGGGS linkers (DT389-(GGGGS)1-3YP7). In-silico and experimental investigation were performed to find out how many repeats of linker between toxin and scFv moieties are sufficient. Results of in-silico investigations revealed that the difference in the number of linkers does not have a significant effect on the main structures of the immunotoxin; however, the three-dimensional structure of two repeats of linker had a more appropriate structure compared to others with one and three linker replications. In addition, with enhancing the number of linkers, the probability of protein solubility has increased. Generally, the bioinformatics results of DT389-(GGGGS)2-YP7 structure showed that expression and folding is suitable; and YP7 scFv has appropriate orientation to bind GPC3. The experimental investigations indicated that the fusion protein was expressed as near to 50% soluble. Due to the high binding affinity of YP7 scFv and the proven potency of diphtheria in inhibiting protein synthesis, the proposed DT389-(GGGGS)2-YP7 immunotoxin is expected to function well in inhibiting HCC.

Asunto(s)

Carcinoma Hepatocelular , Inmunotoxinas , Neoplasias Hepáticas , Toxina Diftérica/química , Toxina Diftérica/genética , Glipicanos/uso terapéutico , Humanos , Inmunotoxinas/química , Inmunotoxinas/uso terapéutico

RESUMEN

Glypican-1 (GPC1) is a cell surface proteoglycan that is upregulated in multiple types of human cancers including pancreatic cancer. Here, we investigated whether GPC1 could be a target of antibody-toxin fusion proteins (i.e., immunotoxins) for treating pancreatic cancer. We constructed a panel of GPC1-targeted immunotoxins derived from a functional domain of Pseudomonas exotoxin A. An albumin-binding domain was also introduced into the anti-GPC1 immunotoxin to improve serum half-life. Small-molecule screening was performed to identify irinotecan that shows synergistic efficacy with the immunotoxin. We showed that GPC1 was internalized upon antibody binding. Anti-GPC1 immunotoxins alone inhibited tumor growth in a pancreatic cancer xenograft model. The immunotoxin treatment reduced active ß-catenin expression in tumor cells. Furthermore, immunotoxins containing an albumin-binding domain in combination with irinotecan caused pancreatic tumor regression. GPC1 expression was reduced by the immunotoxin treatment due to the degradation of the internalized GPC1 and its short cellular turnover rate. Our data indicate that the GPC1-targeted immunotoxin inhibits pancreatic tumor growth via degradation of internalized GPC1, downregulation of Wnt signaling, and inhibition of protein synthesis. The anti-GPC1 immunotoxin in combination with irinotecan thus provides a potential new treatment strategy for patients with pancreatic tumors.

Asunto(s)

Inmunotoxinas , Neoplasias Pancreáticas , Albúminas , Animales , Regulación hacia Abajo , Glipicanos/genética , Humanos , Inmunotoxinas/química , Inmunotoxinas/farmacología , Irinotecán , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Vía de Señalización Wnt , Neoplasias Pancreáticas

RESUMEN

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B12 ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of these nanobodies bound to holo-TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC-Nb4) using fluorescent light microscopy. The co-crystal structure of holo-TC:TCblR with another nanobody (TC-Nb34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR, rationalizing the decrease in the affinity of TC-B12 binding caused by the Δ88 mutation in CD320.

Asunto(s)

Anticuerpos Monoclonales/química , Inmunoconjugados/farmacología , Receptores de Superficie Celular/metabolismo , Saporinas/química , Anticuerpos de Dominio Único/química , Transcobalaminas/metabolismo , Vitamina B 12/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Camélidos del Nuevo Mundo , Ciclo Celular , Proliferación Celular , Células HEK293 , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunotoxinas/química , Inmunotoxinas/farmacología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Saporinas/inmunología , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/inmunología

RESUMEN

Rationale: Therapeutic antibody conjugates allow for the specific delivery of cytotoxic agents or immune cells to tumors, thus enhancing the antitumor activity of these agents and minimizing adverse systemic effects. Most current antibody conjugates are prepared by nonspecific modification of antibody cysteine or lysine residues, inevitably resulting in the generation of heterogeneous conjugates with limited therapeutic efficacies. Traditional strategies to prepare homogeneous antibody conjugates require antibody engineering or chemical/enzymatic treatments, processes that often affect antibody folding and stability, as well as yield and cost. Developing a simple and cost-effective way to precisely couple functional payloads to native antibodies is of great importance. Methods: We describe a simple proximity-induced antibody conjugation method (pClick) that enables the synthesis of homogeneous antibody conjugates from native antibodies without requiring additional antibody engineering or post-synthesis treatments. A proximity-activated crosslinker is introduced into a chemically synthesized affinity peptide modified with a bioorthogonal handle. Upon binding to a specific antibody site, the affinity peptide covalently attaches to the antibody via spontaneous crosslinking, yielding an antibody molecule ready for bioorthogonal conjugation with payloads. Results: We have prepared well-defined antibody-drug conjugates and bispecific small molecule-antibody conjugates using pClick technology. The resulting conjugates exhibit excellent in vitro cytotoxic activity against cancer cells and, in the case of bispecific conjugates, superb antitumor activity in mouse xenograft models. Conclusions: Our pClick technology enables efficient, simple, and site-specific conjugation of various moieties to the existing native antibodies. This technology does not require antibody engineering or additional UV/chemical/enzymatic treatments, therefore providing a general, convenient strategy for developing novel antibody conjugates.

Asunto(s)

Química Clic/métodos , Inmunoconjugados/química , Inmunotoxinas/química , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígenos , Antineoplásicos/farmacología , Línea Celular , Humanos , Inmunoconjugados/farmacología , Inmunotoxinas/farmacología , Masculino , Ratones , Ratones Endogámicos NOD , Neoplasias/tratamiento farmacológico

RESUMEN

Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs' intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.

Asunto(s)

Muerte Celular/efectos de los fármacos , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/toxicidad , Inmunotoxinas/química , Inmunotoxinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Anémonas de Mar/química , Animales

RESUMEN

Glioblastomas (GBMs) are refractory to current treatments and novel therapeutic approaches need to be explored. Pro­apoptotic tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) is tumor­specific and has been shown to induce apoptosis and subsequently kill GBM cells. However, approximately 50% of GBM cells are resistant to TRAIL and a combination of TRAIL with other therapeutics is necessary to induce mechanism­based cell death in TRAIL­resistant GBMs. The present study examined the ability of the tumor cell surface receptor, interleukin (IL)­13 receptor α2 (IL13Rα2)­ and epidermal growth factor receptor (EGFR)­targeted pseudomonas exotoxin (PE) to sensitize TRAIL­resistant GBM cells and assessed the dual effects of interleukin 13­PE (IL13­PE) or EGFR nanobody­PE (ENb­PE) and TRAIL for the treatment of a broad range of brain tumors with a distinct TRAIL therapeutic response. Receptor targeted toxins upregulated TRAIL death receptors (DR4 and DR5) and suppressed the expression of anti­apoptotic FLICE­inhibitory protein (FLIP) and X­linked inhibitor of apoptosis protein (XIAP). This also led to the induction of the cleavage of caspase­8 and caspase­9 and resulted in the sensitization of highly resistant established GBM and patient­derived GBM stem cell (GSC) lines to TRAIL­mediated apoptosis. These findings provide a mechanism­based strategy that may provide options for the cell­mediated delivery of bi­functional therapeutics to target a wide spectrum of TRAIL­resistant GBMs.

Asunto(s)

Toxinas Bacterianas/farmacología , Exotoxinas/farmacología , Glioblastoma , Subunidad alfa2 del Receptor de Interleucina-13 , Interleucina-13/farmacología , Proteínas de Neoplasias , Pseudomonas/química , Anticuerpos de Dominio Único/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF , Toxinas Bacterianas/química , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Exotoxinas/química , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Inmunotoxinas/química , Inmunotoxinas/farmacología , Interleucina-13/química , Subunidad alfa2 del Receptor de Interleucina-13/genética , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Anticuerpos de Dominio Único/química , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo

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

OBJECTIVES: Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C). METHODS: E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo. RESULTS: CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms. CONCLUSIONS: C-CUS245C exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC.

Asunto(s)

Cetuximab/uso terapéutico , Neoplasias Colorrectales/terapia , Inmunotoxinas/uso terapéutico , Terapia Molecular Dirigida/métodos , Proteínas de Plantas/uso terapéutico , Animales , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cetuximab/farmacología , Cromatografía de Afinidad/métodos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Escherichia coli/metabolismo , Humanos , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Inmunotoxinas/química , Inmunotoxinas/aislamiento & purificación , Inmunotoxinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutagénesis Insercional/métodos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de Xenoinjerto

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

Saporin, a type I ribosome-inactivating protein from soapwort plant, is a potent protein synthesis inhibitor. Catalytically, saporin is a characteristic N-glycosidase, and it depurinates a specific adenine residue from a universally conserved loop of the major ribosomal RNA (rRNA) of eukaryotic cells. It is well-known that saporin induces apoptosis through different pathways, including ribotoxic stress response, cell signal transduction, genomic DNA fragmentation and RNA abasic lyase (RAlyase) activity, and NAD+ depletion by poly-(ADP)-ribose polymerase hyperactivation. Saporin's high enzymatic activity, high stability, and resistance to conjugation procedures make it a well-suited tool for immunotherapy approaches.In the present study, we focus on saporin-based targeted toxins that may be efficacious therapeutic agents for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our discussed points suggest that saporin may be a strategic molecule for therapeutic knockout treatments and a powerful candidate for novel drugs in the struggle against coronavirus 2019 (COVID-19).

Asunto(s)

Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Saporinas/química , Saporinas/farmacología , Antivirales/química , Apoptosis/efectos de los fármacos , Humanos , Inmunotoxinas/química , Inmunotoxinas/farmacología , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Cholesterol seems to play a central role in the augmentation of saporin-based immunotoxin (IT) cytotoxicity by triterpenoid saponins. Endolysosomal escape has been proposed as one mechanism for the saponin-mediated enhancement of targeted toxins. We investigated the effects of lipid depletion followed by repletion on Saponinum album (SA)-induced endolysosomal escape of Alexa Fluor labelled saporin and the saporin-based immunotoxin OKT10-SAP, directed against CD38, in Daudi lymphoma cells. Lipid deprived cells showed reduced SA-induced endolysosomal escape at two concentrations of SA, as determined by a flow cytometric method. The repletion of membrane cholesterol by low density lipoprotein (LDL) restored SA-induced endolysosomal escape at a concentration of 5 µg/mL SA but not at 1 µg/mL SA. When LDL was used to restore the cholesterol levels in lipid deprived cells, the SA augmentation of OKT10-SAP cytotoxicity was partially restored at 1 µg/mL SA and fully restored at 5 µg/mL SA. These results suggest that different mechanisms of action might be involved for the two different concentrations of SA and that endosomal escape may not be the main mechanism for the augmentation of saporin IT cytotoxicity by SA at the sub-lytic concentration of 1 µg/mL SA.

Asunto(s)

Colesterol/química , Endosomas/efectos de los fármacos , Inmunotoxinas/metabolismo , Lisosomas/efectos de los fármacos , Saponinas/farmacología , Saporinas/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colesterol/metabolismo , LDL-Colesterol/farmacología , Relación Dosis-Respuesta a Droga , Endosomas/química , Endosomas/metabolismo , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Inmunotoxinas/química , Linfocitos/química , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Lisosomas/química , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Saporinas/química , Ácidos Sulfónicos/química , Triterpenos/farmacología

RESUMEN

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

Asunto(s)

ADP Ribosa Transferasas/farmacología , Antineoplásicos/farmacología , Toxinas Bacterianas/farmacología , Exotoxinas/farmacología , Inmunotoxinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Anticuerpos de Dominio Único/farmacología , Factores de Virulencia/farmacología , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/genética , Antineoplásicos/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Exotoxinas/química , Exotoxinas/genética , Femenino , Humanos , Inmunotoxinas/química , Inmunotoxinas/genética , Modelos Moleculares , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Factores de Virulencia/química , Factores de Virulencia/genética , Exotoxina A de Pseudomonas aeruginosa

RESUMEN

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.

Asunto(s)

Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/química , Citoplasma/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Inteínas , Neoplasias/tratamiento farmacológico , Animales , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Línea Celular Tumoral , Citoplasma/genética , Toxina Diftérica/administración & dosificación , Toxina Diftérica/química , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Femenino , Xenoinjertos , Humanos , Inmunotoxinas/administración & dosificación , Inmunotoxinas/química , Inmunotoxinas/genética , Inmunotoxinas/metabolismo , Ratones , Ratones Desnudos , Neoplasias/genética , Neoplasias/metabolismo , Dominios Proteicos , Transporte de Proteínas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo

RESUMEN

Mesothelin (MSLN) is a cell surface glycoprotein normally expressed only on serosal surfaces, and not found in the parenchyma of vital organs. Many solid tumors also express MSLN, including mesothelioma and pancreatic adenocarcinoma. Due to this favorable expression profile, MSLN represents a viable target for directed anti-neoplastic therapies, such as recombinant immunotoxins (iToxs). Pre-clinical testing of MSLN-targeted iTox's has yielded a strong body of evidence for activity against a number of solid tumors. This has led to multiple clinical trials, testing the safety and efficacy of the clinical leads SS1P and LMB-100. While promising clinical results have been observed, neutralizing anti-drug antibody (ADA) formation presents a major challenge to overcome in the therapeutic development process. Additionally, on-target, off-tumor toxicity from serositis and non-specific capillary leak syndrome (CLS) also limits the dose, and therefore, impact anti-tumor activity. This review summarizes existing pre-clinical and clinical data on MSLN-targeted iTox's. In addition, we address the potential future directions of research to enhance the activity of these anti-tumor agents.

Asunto(s)

Proteínas Ligadas a GPI/metabolismo , Inmunotoxinas/uso terapéutico , Mesotelioma/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/metabolismo , Ensayos Clínicos como Asunto , Proteínas Ligadas a GPI/química , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Inmunotoxinas/química , Inmunotoxinas/farmacología , Mesotelina , Mesotelioma/metabolismo , Terapia Molecular Dirigida , Neoplasias Pancreáticas/metabolismo