Novel Oxygen-Dependent Degradable Immunotoxin Regulated by the Ubiquitin-Proteasome System Reduces Nonspecific Cytotoxicity.

The use of bacterial toxins as antitumor agents has received considerable attention. Immunotoxins based on antigen recognition of single-chain antibodies have been widely explored for cancer therapy. Despite their impressive killing effect on tumor cells, immunotoxins still display unspecific toxicity with undesired side effects. High levels of hypoxia-inducible factor 1α (HIF-1α) are well-known indicators of hypoxia in cancer cells. In this study, different linkers were employed to fuse the immunotoxin DAB389-4D5 scFv (DS) with the oxygen-dependent degradation domain (ODDD) of HIF-1α, a domain selectively facilitating the accumulation of HIF-1α under hypoxia, to construct the oxygen-dependent degradable immunotoxin DS-ODDD (DSO). The engineered fusion protein DSO-2 containing a linker (G4S)3 possesses the best killing effect on cancer cells under hypoxia and displayed considerably reduced nonspecific toxicity to normal cells under normoxic conditions. Flow cytometry, immunofluorescence, and immunoblot analyses demonstrated that DSO-2 was degraded via the ubiquitin-proteasome pathway regulated by the oxygen-sensitive mechanism. Western blot analysis indicated that the degradation of DSO-2 significantly decreased the activation of apoptosis-related molecules in normal cells. The engineered immunotoxin with oxygen-sensing properties developed herein is a potential therapeutic agent for cancer treatment.

Directing ricin-based immunotoxins with targeting affibodies and KDEL signal peptide to cancer cells effectively induces apoptosis and tumor suppression.

The plant toxin ricin, especially its cytotoxic A chain (RTA), can be genetically engineered with targeting ligands to develop specific anti-cancer recombinant immunotoxins (RITs). Here, we used affibody molecules targeting two cancer biomarkers, the receptors HER2 and EGFR, along with the KDEL signal peptide to construct two cancer-specific ricin-based RITs, HER2Afb-RTA-KDEL and EGFRAfb-RTA-KDEL. The affibodies successfully provided target-specificity and subsequent receptor-mediated endocytosis and the KDEL signal peptide routed the RITs through the retrograde transport pathway, effectively delivering RTA to the cytosol as well as avoiding the alternate recycling pathway that typical cancer cells frequently have. The in vivo efficacy of RITs was enhanced by introducing the albumin binding domain (AlBD) to construct AlBD/HER2Afb/RTA-KDEL. Systemic administration of AlBD-containing RITs to tumor-bearing mice significantly suppressed tumor growth without any noticeable side-effects. Collectively, combining target-selective affibody molecules, a cytotoxic RTA, and an intracellularly designating peptide, we successfully developed cancer-specific and efficacious ricin-based RITs. This approach can be applied to develop novel protein-based "magic bullets" to effectively suppress tumors that are resistant to conventional anti-cancer drugs.

Recombinant immunotoxins development for HER2-based targeted cancer therapies.

Understanding the molecular mechanisms of cancer biology introduces targeted therapy as a complementary method along with other conventional therapies. Recombinant immunotoxins are tumor specific antibodies that their recognizing fragment is utilized for delivering modified toxins into tumor cells. These molecules have been considered as a targeted strategy in the treatment of human cancers. HER2 tumor biomarker is a transmembrane tyrosine kinase receptor that can be used for targeted therapies in the forms of anti-HER2 monoclonal antibodies, antibody-drug conjugates and immunotoxins. There have been many studies on HER2-based immunotoxins in recent years, however, little progress has been made in the clinical field which demanded more improvements. Here, we summarized the HER2 signaling and it's targeting using immunotherapeutic agents in human cancers. Then, we specifically reviewed anti-HER2 immunotoxins, and their strengths and drawbacks to highlight their promising clinical impact.

Biology and Treatment of Hairy Cell Leukemia.

OPINION STATEMENT: Despite its rarity, hairy cell leukemia (HCL) remains a fascinating disease and the physiopathology is becoming more and more understood. The accurate diagnosis of HCL relies on the recognition of hairy cells by morphology and flow cytometry (FCM) in the blood and/or bone marrow (BM). The BRAF V600E mutation, an HCL-defining mutation, represents a novel diagnostic parameter and a potential therapeutic target. The precise cellular origin of HCL is a late-activated postgerminal center memory B cell. BRAF mutations were detected in hematopoietic stem cells (HSCs) of patients with HCL, suggesting that this is an early HCL-defining event. Watch-and-wait strategy is necessary in approximately 10% of asymptomatic HCL patients, sometimes for several years. Purine analogs (PNAs) are the established first-line options for symptomatic HCL patients. In second-line treatment, chemoimmunotherapy combining PNA plus rituximab should be considered in high-risk HCL patients. The three options for relapsed/refractory HCL patients include recombinant immunoconjugates targeting CD22, BRAF inhibitors, and BCR inhibitors. The clinical interest to investigate blood minimal residual disease (MRD) was recently demonstrated, with a high risk of relapse in patients with positive testing for MRD and a low risk in patients with negative testing. However, efforts must be made to standardize MRD analyses in the near future. Patients with HCL are at risk of second malignancies. The increased risk could be related to the disease and/or the treatment, and the respective role of PNAs in the development of secondary malignancies remains a topic of debate.

Genetic manipulation of specific neural circuits by use of a viral vector system.

To understand the mechanisms underlying higher brain functions, we need to analyze the roles of specific neuronal pathways or cell types forming the complex neural networks. In the neuroscience field, the transgenic approach has provided a useful gene engineering tool for experimental studies of neural functions. The conventional transgenic technique requires the appropriate promoter regions that drive a neuronal type-specific gene expression, but the promoter sequences specifically functioning in each neuronal type are limited. Previously, we developed novel types of lentiviral vectors showing high efficiency of retrograde gene transfer in the central nervous system, termed highly efficient retrograde gene transfer (HiRet) vector and neuron-specific retrograde gene transfer (NeuRet) vector. The HiRet and NeuRet vectors enable genetical manipulation of specific neural pathways in diverse model animals in combination with conditional cell targeting, synaptic transmission silencing, and gene expression systems. These newly developed vectors provide powerful experimental strategies to investigate, more precisely, the machineries exerting various neural functions. In this review, we give an outline of the HiRet and NeuRet vectors and describe recent representative applications of these viral vectors for studies on neural circuits.

Construction and characterization of the recombinant immunotoxin RTA-4D5-KDEL targeting HER2/neu-positive cancer cells and locating the endoplasmic reticulum.

The specific targeting of immunotoxins enables their wide application in cancer therapy. The A-chain of the ricin protein (RTA) is an N-glycosidase that catalyzes the removal of adenine from the 28S rRNA, preventing protein translation and leading to cell death. Ricin is highly toxic but can only exert its toxic effects from within the cytoplasm. In this study, we linked the anti-HER2 single-chain variable fragment 4D5 scFv and the endoplasmic reticulum-targeting peptide KDEL to the C-terminal of the RTA to construct immunotoxin RTA-4D5-KDEL. In vitro experiments showed that the anticancer effect of RTA-4D5-KDEL towards ovarian cancer cells SKOV-3 increased 440-fold and 28-fold relative to RTA and RTA-4D5, respectively. RTA-4D5-KDEL had a strong inhibitory effect on HER2-overexpressing SKOV-3 cells and caused little damage to normal HEK-293 cells and H460 lung cancer cells. Immunofluorescence experiments showed that the immunotoxin RTA-4D5 could specifically bind to SKOV-3 cells, but not to normal cells HEK-293. The immunotoxin RTA-4D5-KDEL could rapidly localize the recombinant protein to the endoplasmic reticulum. These results suggest that the recombinant immunotoxin RTA-4D5-KDEL has a strong inhibitory effect on ovarian cancer cells that overexpress HER2 but little harm to the normal cells.

An efficient scheme for purification of a novel recombinant immunotoxin, rCCK8PE38, for anti-tumour experiments.

rCCK8PE38 is a novel immunotoxin that targets choleystokinin B receptor, which is over-expressed in some tumor tissues. Although we constructed a prokaryotic expression vector to express rCCK8PE38 in our laboratory, thorough purification was necessary to quantitatively assess its anti-tumor effect. In this study, we established a purification protocol to obtain rCCK8PE38 with high purity from E. coli. Three different types of chromatography, hydrophobic chromatography, ion exchange chromatography and size exclusion chromatography, were used in combination. The purification technological parameters of each chromatography type were optimized. The whole process of purification was arranged to minimize the purification steps and achieve purity and bioactivity. Finally, through this optimized scheme, we obtained a recombinant protein with a purity of >95%; then, the protein was stored at -80°C after lyophilization. The purified protein was used in a tumor inhibition experiment and was effective in killing tumor cells that over-expressed choleystokinin B receptor. The results of this study may provide some valuable information about protein purification and lay the foundation for further clinical experiments with rCCK8PE38.

Production and quality control assessment of a GLP-grade immunotoxin, D2C7-(scdsFv)-PE38KDEL, for a phase I/II clinical trial.

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel recombinant Pseudomonas exotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. Initial pre-clinical testing demonstrated the anti-tumor efficacy of D2C7-IT against orthotopic glioblastoma xenograft models expressing EGFRwt, EGFRvIII, or both EGFRwt and EGFRvIII. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter in Escherichia coli BLR (λ DE3). D2C7-IT was produced by a 10-L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300 mg of purified protein. The final vialed batch of D2C7-IT for clinical testing was at a concentration of 0.12 ± 0.1 mg/mL, the pH was at 7.4 ± 0.4, and endotoxin levels were below the detection limit of 10 EU/mL (1.26 EU/mL). The stability of the vialed D2C7-IT has been monitored over a period of 42 months through protein concentration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, size exclusion chromatography, cytotoxicity, sterility, and pH measurements. The vialed D2C7-IT is currently being tested in a phase I/II clinical trial by intratumoral convection-enhanced delivery for 72 h in patients with recurrent glioblastoma (NCT02303678, D2C7 for Adult Patients with Recurrent Malignant Glioma; clinicaltrials.gov ).

Advances in anticancer immunotoxin therapy.

Immunotoxins are a novel class of antibody-conjugated therapeutics currently in clinical development for a variety of malignancies. They consist of an antibody-based targeting domain fused to a bacterial toxin payload for cell killing. Immunotoxins kill cells by inhibiting protein synthesis, a unique mechanism of action that is toxic to both dividing and nondividing cells. Recent advances in the design and administration of immunotoxins are overcoming historical challenges in the field, leading to renewed interest in these therapeutics.

Development of a recombinant hCG-specific single chain immunotoxin cytotoxic to hCG expressing cancer cells.

A large number of cancers express human chorionic gonadotropin (hCG) or its subunits ectopically. Patients harboring such cancers have poor prognosis and adverse survival. PiPP is a monoclonal antibody of high affinity and specificity for hCGß/hCG. Work was carried out to develop a PiPP based recombinant immunotoxin for the immunotherapy of hCG expressing cancers. Recombinant PiPP antibody was constructed in scFv format in which gene encoding the VH and VL domains were joined through a linker. This scFv gene was fused to the gene expressing Pseudomonas exotoxin (PE38), and cloned in a Escherichia coli based expression vector under the control of strong bacteriophage T7 promoter. Immunotoxin conjugating scFv(PiPP) and PE38, was expressed in E. coli as recombinant protein. Recombinant PiPP immunotoxin was purified from the bacterial cell lysate and tested for binding and killing of hCGß expressing lymphoma, T-lymphoblastic leukemia and lung carcinoma cells in vitro. Immunotoxin showed nearly 90% killing on the cells. This is the first ever report on recombinant immunotoxin for binding and cytotoxicity to hCG expressing cancer cells, and thus can be a potential candidate for the immunotherapy of hCG expressing cells.

Class II human leucocyte antigen DRB1*11 in hairy cell leukaemia patients with and without haemolytic uraemic syndrome.

Frequencies of human leucocyte antigens (HLA) were determined in 287 classic hairy cell leukaemia (HCL) patients. With respect to both population (n = 287) and allele (2n = 574) frequency respectively, the most common HLA class I and II antigens expressed were HLA-A*02 (49·1% and 28·6%), HLA-B*07 (21·3% and 11·1%), HLA-C*07 (46·7 and 28·2%), HLA-DQB1*03 (62·7% and 37·3%), HLA-DRB1*11 (30·0% and 16·0%) and HLA-DRB4*01 (45·3% and 29·6%). In comparing 6-14 databases of control Caucasians to 267 Caucasian HCL patients, only HLA-DRB1*11 was consistently over-represented in HCL, 31·1% of patients vs. 17-19·9% of controls (P = 0·0055 to <0·0001) and 16·5% of alleles vs. 6·5-12·3% of control alleles (P = 0·022 to <0·0001). HLA-DRB1*11 is a known risk factor for acquired thrombotic microangiopathy. Anti-CD22 recombinant immunotoxin BL22 in HCL was associated with a 12% incidence of completely reversible grade 3-4 haemolytic uraemic syndrome (HUS), mainly during the second or third retreatment cycle. Of 49 HCL patients receiving ≥2 cycles of BL22, 7 (14%) had HUS and HLA-DRB1*11 was expressed in 71% of 7 with HUS compared with only 21% of 42 without (P = 0·015). These data suggest that DBR1*11 may be a marker for increased susceptibility to HCL and, among HCL patients, could be a risk factor for BL22-induced HUS.

Depletion of folate receptor ß-expressing macrophages alleviates bleomycin-induced experimental skin fibrosis.

OBJECTIVES: Folate receptor ß (FRß)-expressing macrophages have been identified as activated macrophages. Here, we investigated the infiltration of FRß-expressing macrophages in a murine model of bleomycin (BLM)-induced skin fibrosis and assessed the antifibrotic effects of depletion of FRß-expressing macrophages in this model using a recombinant immunotoxin to FRß. METHODS: A recombinant immunotoxin (anti-FRß-PE38) was prepared by conjugating the Fv portion of the anti-mouse FRß heavy chain with truncated Pseudomonas exotoxin A (VH-PE38) and the Fv portion of the anti-mouse FRß light chain. BLM-induced skin fibrosis mice were intravenously treated with either anti-FRß-PE38 or VH-PE38 as a control protein. Skin fibrosis was evaluated by the change of skin thickness and hydroxyproline content on Day 29. The TGFß1 mRNA levels in the treated skin were assessed by quantitative real-time RT-PCR on Day 9. RESULTS: Numbers of FRß-expressing macrophages increased in BLM-injected skin. Anti-FRß-PE38 treatment led to a dramatic reduction in the number of FRß-expressing macrophages. Additionally, skin thickness and hydroxyproline content, were markedly reduced. TGFß1 mRNA levels were also down-regulated after the treatment. TGFß1 expression was enriched in FRß-expressing macrophages compared with FRß-negative macrophages. CONCLUSION: These results indicated that anti-FRß-PE38 treatment efficiently depleted FRß-expressing macrophages and consequently alleviated BLM-induced skin fibrosis.

Comparison of Five Escherichia coli Strains to Achieve the Maximum Yield of a Recombinant Immunotoxin Consisting of an Antibody against VEGF Conjugated with MAP30 Toxin in a Benchtop Bioreactor.

BACKGROUND: Cancer is among the leading causes of death worldwide, imposing high costs on the health systems of all societies. Extensive biological studies are required to discover appropriate therapies. Escherichia coli has long been regarded as one of the main biotechnological bio-factories to produce recombinant protein-based therapeutics. In the present study, five strains of E. coli were compared to achieve the maximum production of a previously designed recombinant immunotoxin-carrying MAP30 toxin against VEGF-overexpressed cancer cells in a benchtop bioreactor. METHODS: The recombinant immunotoxin coding gene sequence was extracted from the NCBI database. The host used to produce the recombinant immunotoxin were five E. coli strains of BL21 (DE3), DH5α, SHuffle®T7, XL1-Blue, and Rosetta-gamiTM (DE3). CaCl2 method was used for bacterial transformation. Bacterial growth measurements were performed using optical density measurements at 600 nm. The immunotoxin production was measured using SDS-PAGE analysis. The best-producing strain was cultivated in a 10-L benchtop stirred tank bioreactor. Recent patents on this field were also studied. RESULTS: The results demonstrated that the BL21 (DE3) strain had the highest expression of recombinant protein in comparison to other strains. Moreover, the cell growth of E. coli BL21 (DE3) and SHuffle®T7 strains before transformation in the LB medium, were significantly higher in comparison to other strains. Additionally, the transformation of Rosettagami was associated with decreased cell proliferation. The transformation of the XL1-Blue strain did not effect cell growth. Analysis of the growth kinetics demonstrated appropriate proliferation of the transformed BL21 (DE3) cells in the laboratory benchtop bioreactor. CONCLUSIONS: Based on the results of this study, the BL21 (DE3) strain could be used as a suitable host for the production of the recombinant immunotoxin against VEGF in stirred tank bioreactor, which can be employed for the treatment of tumors. Yet, its precise mechanism must be explored in extensive studies.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

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.

Antitumor Efficacy of EGFR-Targeted Recombinant Immunotoxin in Human Head and Neck Squamous Cell Carcinoma.

Over 90% of head and neck squamous cell carcinoma (HNSCC) overexpresses the epidermal growth factor receptor (EGFR). However, the EGFR-targeted monotherapy response rate only achieves 10-30% in HNSCC. Recombinant immunotoxin (RIT) often consists of an antibody targeting a tumor antigen and a toxin (e.g., diphtheria toxin [DT]) that kills cancer cells. We produced a humanized RIT, designated as hDT806, targeting overexpressed EGFR and investigated its effects in HNSCC. Distinct from the EGFR-targeted tyrosine kinase inhibitor erlotinib or antibody cetuximab, hDT806 effectively suppressed cell proliferation in the four HNSCC lines tested (JHU-011, -013, -022, and -029). In JHU-029 mouse xenograft models, hDT806 substantially reduced tumor growth. hDT806 decreased EGFR protein levels and disrupted the EGFR signaling downstream effectors, including MAPK/ERK1/2 and AKT, while increased proapoptotic proteins, such as p53, caspase-9, caspase-3, and the cleaved PAPR. The hDT806-induced apoptosis of HNSCC cells was corroborated by flow cytometric analysis. Furthermore, hDT806 resulted in a drastic inhibition in RNA polymerase II carboxy-terminal domain phosphorylation critical for transcription and a significant increase in the γH2A.X level, a DNA damage marker. Thus, the direct disruption of EGFR signaling, transcription inhibition, DNA damage, as well as apoptosis induced by hDT806 may contribute to its antitumor efficacy in HNSCC.

Generation of Single-Domain Antibody-Based Recombinant Immunotoxins.

The discovery of single-domain antibodies has opened new avenues for drug development. Single-domain antibodies, also known as nanobodies, can access buried epitopes that are inaccessible to conventional antibodies. These antigen-binding domains have a high level of solubility and stability, which makes them well suited for therapeutic development. This chapter will discuss the design, production, and testing of single-domain antibody-based recombinant immunotoxins. Recombinant immunotoxins are chimeric proteins that combine the specificity of an antibody with the ribosomal-inhibitory domain of a bacterial toxin. Immunotoxins using the Pseudomonas exotoxin domain have been well studied in clinical trials. Recently, an anti-CD22 immunotoxin was granted marketing approval for use in patients with relapsed or refractory hairy cell leukemia. This supports the idea that treatment with recombinant immunotoxins can be explored for cancers that have not responded to standard therapies.

Novel EGFR­bispecific recombinant immunotoxin based on cucurmosin shows potent anti­tumor efficiency in vitro.

Epidermal growth factor receptor (EGFR) is overexpressed in various tumors and is associated with cancer initiation, progression, and poor prognosis. Despite the achievements made by tyrosine kinase inhibitors and monoclonal antibodies in certain cases, many patients have not benefited from such treatment due to resistance. Immunotoxins (ITs) are antibody­cytotoxin chimeric molecules with specific cell killing ability, which have achieved different degrees of success in the treatment of a wide range of cancers in clinical trials. The aim of the current study was to examine a novel targeting EGFR recombinant immunotoxin Bs/cucurmosin (CUS) generated by fusing CUS to the EGFR­specific nanobody 7D12­9G8. Bs/CUS was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. Furthermore, it retained binding capacity and specificity with EGFR and was superior to rE/CUS, a monospecific IT we reported previously. In vitro results showed that Bs/CUS could be internalized into the cytoplasm and selectively kill cells in the picomolar range. Flow cytometry showed that Bs/CUS killed the cells mediated by the apoptosis pathway. Taken together, results of the current study indicated that Bs/CUS is a promising candidate that should be further evaluated as a cancer therapeutic for the treatment of EGFR­positive tumors.

Anti-cancer Immunotoxins, Challenges, and Approaches.

The development of recombinant immunotoxins (RITs) as a novel therapeutic strategy has made a revolution in the treatment of cancer. RITs result from the fusion of antibodies to toxin proteins for targeting and eliminating cancerous cells by inhibiting protein synthesis. Despite indisputable outcomes of RITs regarding inhibition of multiple cancer types, high immunogenicity has been known as the main obstacle in the clinical use of RITs. Various strategies have been proposed to overcome these limitations, including immunosuppressive therapy, humanization of the antibody fragment moiety, generation of immunotoxins originated from endogenous human cytotoxic enzymes, and modification of the toxin moiety to escape the immune system. This paper is devoted to review recent advances in the design of immunotoxins with lower immunogenicity.

DT389-YP7, a Recombinant Immunotoxin against Glypican-3 That Inhibits Hepatocellular Cancer Cells: An In Vitro Study.

Hepatocellular carcinoma (HCC) is one of the high-metastatic types of cancer, and metastasis occurs in one-third of patients with HCC. To maintain the effectiveness of drug compounds on cancer cells and minimize their side effects on normal cells, it is important to use new approaches for overcoming malignancies. Immunotoxins (ITs), an example of such a new approach, are protein-structured compounds consisting of toxic and binding moieties which can specifically bind to cancer cells and efficiently induce cell death. Here, we design and scrutinize a novel immunotoxin against an oncofetal marker on HCC cells. We applied a truncated diphtheria toxin (DT389) without binding domain as a toxin moiety to be fused with a humanized YP7 scFv against a high-expressed Glypican-3 (GPC3) antigen on the surface of HCC cells. Cytotoxic effects of this IT were investigated on HepG2 (GPC3+) and SkBr3 (GPC3-) cell lines as positive- and negative-expressed GPC3 antigens. The dissociation constant (Kd) was calculated 11.39 nM and 18.02 nM for IT and YP7 scfv, respectively, whereas only IT showed toxic effects on the HepG2 cell line, and decreased cell viability (IC50 = 848.2 ng/mL). Changing morphology (up to 85%), cell cycle arrest at G2 phase (up to 13%), increasing intracellular reactive oxygen species (ROSs) (up to 50%), inducing apoptosis (up to 38% for apoptosis and 23% for necrosis), and an almost complete inhibition of cell movement were other effects of immunotoxin treatment on HepG2 cells, not on SkBr3 cell line. These promising results reveal that this new recombinant immunotoxin can be considered as an option as an HCC inhibitor. However, more extensive studies are needed to accomplish this concept.

Development of Glypican-3 Targeting Immunotoxins for the Treatment of Liver Cancer: An Update.

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.