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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 monobody SS , a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. Monobody SS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobody SS -based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobody SS -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. ER SS /28-PE25, with a K D 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, ER SS /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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Immunotoxins (ITs) are specialized therapeutic agents designed for targeted treatment, particularly in cancer therapy. They consist of a monoclonal antibody or antibody fragment linked to a potent cytotoxic agent, such as bacterial- or plant-derived toxins like diphtheria toxin, ricin, or pseudomonas exotoxin. The monoclonal antibody component specifically binds to antigens expressed on the surface of target cells, facilitating the internalization of the IT. Once inside the cell, the cytotoxic agent is released, disrupting essential cellular processes and leading to cell death. This targeted approach minimizes damage to healthy tissues while effectively eliminating diseased cells. The production of ITs involves two primary methods: recombinant fusion and chemical conjugation. In recombinant fusion, genetic engineering is used to create a fusion protein that combines the antibody and toxin, ensuring precise control over their ratio and functionality. In chemical conjugation, pre-existing antibodies are chemically linked to toxins, allowing for greater flexibility in combining different antibodies and cytotoxic agents. Each method has its advantages and challenges, influencing the specificity, production complexity, and therapeutic potential of the resulting ITs. As research advances, ITs continue to show promise not only in oncology but also in treating other diseases, including inflammatory conditions and atherosclerosis. The precise targeting and potent effects of ITs make them a valuable tool in the development of new therapeutic strategies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers., (© 2024 Federation of American Societies for Experimental Biology.)

Background: Immunotoxins are antibody-toxin conjugates that bind to surface antigens and exert effective cytotoxic activity after internalization into tumor cells. Immunotoxins exhibit effective cytotoxicity and have been approved by the FDA to treat multiple hematological malignancies, such as hairy cell leukemia and cutaneous T-cell lymphoma. However, most of the internalized immunotoxin is degraded in lysosomes, and only approximately 5% of free toxin escapes into the cytosol to exert cytotoxicity. Many studies have improved immunotoxins by engineering the toxin fragment to reduce immunogenicity or increase stability, but how the antibody fragment contributes to the activity of immunotoxins has not been well demonstrated., Methods: In the current study, we used 32A9 and 42A1, two anti-GPC3 antibodies with similar antigen-binding capabilities and internalization rates, to construct scFv-mPE24 immunotoxins and evaluated their in vitro and in vivo antitumor activities. Next, the antigen-binding capacity, trafficking, intracellular protein stability and release of free toxin of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 were compared to elucidate their different antitumor activities. Furthermore, we used a lysosome inhibitor to evaluate the degradation behavior of 32A9 scFv-mPE24 and 42A1 scFv-mPE24. Finally, the antigen-binding patterns of 32A9 and 42A1 were compared under neutral and acidic pH conditions., Results: Although 32A9 and 42A1 had similar antigen binding capacities and internalization rates, 32A9 scFv-mPE24 had superior antitumor activity compared to 42A1 scFv-mPE24. We found that 32A9 scFv-mPE24 exhibited faster degradation and drove efficient free toxin release compared to 42A1 scFv-mPE24. These phenomena were determined by the different degradation behaviors of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 in lysosomes. Moreover, 32A9 was sensitive to the low-pH environment, which made the 32A9 conjugate easily lose antigen binding and undergo degradation in lysosomes, and the free toxin was then efficiently produced to exert cytotoxicity, whereas 42A1 was resistant to the acidic environment, which kept the 42A1 conjugate relatively stable in lysosomes and delayed the release of free toxin., Conclusions: These results showed that a low pH-sensitive antibody-based immunotoxin degraded faster in lysosomes, caused effective free toxin release, and led to improved cytotoxicity compared to an immunotoxin based on a normal antibody. Our findings suggested that a low pH-sensitive antibody might have an advantage in the design of immunotoxins and other lysosomal degradation-dependent antibody conjugate drugs., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Cutaneous T-cell lymphoma (CTCL) encompasses two main subtypes: mycosis fungoides and Sezary syndrome. Global response rates for the systemic treatment of mycosis fungoides and Sezary syndrome are approximately 30%, and none of these treatments are thought to be curative. C-C chemokine receptor type 4 (CCR4) and CD25 are encouraging targets for the treatment of CTCL and are individually targeted by mogamulizumab and denileukin diftitox, respectively. We developed a novel CCR4-IL2 bispecific immunotoxin (CCR4-IL2 IT) targeting both CCR4 and CD25. CCR4-IL2 IT demonstrated superior efficacy against CCR4 + CD25 + CD30 + CTCL in an immunodeficient NSG mouse tumor model. Investigative New Drug-enabling studies of CCR4-IL2 IT are ongoing, including Good Manufacturing Practice production and toxicology studies. In this study, we compared the in vivo efficacy of CCR4-IL2 IT versus the US Food and Drug Administration-approved drug, brentuximab, using an immunodeficient mouse CTCL model. We demonstrated that CCR4-IL2 IT was significantly more effective in prolonging survival than brentuximab, and combination treatment of CCR4-IL2 IT and brentuximab was more effective than brentuximab or CCR4-IL2 IT alone in an immunodeficient NSG mouse CTCL model. Thus, CCR4-IL2 IT is a promising novel therapeutic drug candidate for CTCL treatment., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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 (V HH 7D12) as the targeting domain. First, we generated a nanoITX, combining V HH 7D12 and the fungal ribotoxin α-sarcin (αS) as the toxic moiety (V HH EGFRαS). Then, we incorporated a trimerization domain (TIE XVIII ) into the construct, obtaining a trimeric nanoITX (TriV HH EGFRαS). Finally, we designed and characterized a bispecific ITX, combining the V HH 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.

Background: Human epidermal growth factor receptor 2 (HER2) positive breast cancer is an aggressive subtype, accounting for around 20% of all breast cancers. The development of HER2-targeted therapy has substantially improved patient outcomes. Nevertheless, the increasing rate of side effects and resistance to targeted drugs limit their efficacy in clinical practice. In this study, we designed and synthesized a new immunotoxin, 4D5Fv-PE25, which targets HER2-positive breast cancer, and evaluated its effectiveness in vitro and in vivo., Results: The 4D5Fv-PE25 was expressed in high-density Escherichia coli (E. coli.) using the fermentor method and refined via hydrophobicity, ion exchange, and filtration chromatography, achieving a 56.06% recovery rate. Additionally, the semi-manufactured product with 96% purity was prepared into freeze-dried powder by the lyophilized process. Flow cytometry was used to detect the expression of HER2 in SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 breast cancer cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used for cytotoxicity assay, and the half-maximal inhibitory concentration (IC 50 ) of 4D5Fv-PE25 lyophilized products to HER2-positive cell line SK-BR-3 was 12.53 ng/mL. The 4D5Fv-PE25 was injected into xenograft tumor mice via the tail vein on the 1st, 4th, and 8th day, it indicated that the growth of tumor volume was effectively inhibited for 24 days, although the 4D5Fv-PE25 was metabolized within 60 min by measuring the release of 3 H-Thymidine radiation., Conclusion: we succeeded in producing the 4D5Fv-PE25 freeze-dried powder using the prokaryotic expression method, and it could be employed as a potential drug for treating HER2-positive breast cancer., (© 2023. The Author(s).)

Angiogenesis, the formation of new vessels, is a critical step in the malignancy progression of solid tumors. Many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. Pharmaceutically, Vascular Endothelial Growth Factor (VEGF) can deliver coupled toxins to the tumor vessels through VEGF Receptors. In the current study, we designed, expressed, and assessed the in vitro and in vivo toxicities of a novel immunotoxin consisting of mouse VEGF and heminecrolysin toxin (mVEGF-HNc). The fusion protein was expressed in E. coli and purified via Ni +2 affinity chromatography. The biological activity of immunotoxin was evaluated on NIH/3T3 cells and TC1-tumorized mouse model. The mVEGF-NHc showed significant cytotoxicity on the cells as VEGFR-expressing cells. Moreover, the size of the tumor in the mVEGF-HNc-treated group started to reduce after six injections, while it continued to grow in the PBS-received mice. Efficacious targeting of solid tumor cells via mVEGF-HNc suggests its prospective therapeutic potential for cancer therapy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in a variety of cancers such as colon, stomach, pancreas, and prostate adenocarcinomas. Inhibition of EpCAM is considered as a potential target for cancer therapy. In current study, anti-EpCAM immunotoxin (α-EpCAM IT) was developed using genetic fusion of α-EpCAM single domain antibody (nanobody) (α-EpCAM Nb) to truncated form of diphtheria toxin. The expression of recombinant α-EpCAM IT was induced by Isopropyl β-d-1-thiogalactopyranoside (IPTG) and confirmed by SDS-PAGE and western blot. Recombinant α-EpCAM IT was purified from the inclusion bodies and refolded using urea gradient procedure. The cytotoxicity and apoptosis activity of α-EpCAM IT on EpCAM over-expressing (MCF7), low-expressing (HEK293), and no-expressing (HUVEC) cells were evaluated by 3-4,5-Dimethylthiazol-2-yl (MTT) assay and annexin V-FITC-PI assay as well. In addition, anti-tumor activity of α-EpCAM IT was evaluated on nude mice bearing MCF7 tumor cells. Results showed success expression and purification of α-EpCAM IT. The α-EpCAM IT showed time and dose-dependent anti-proliferative activity on MCF-7 cells. However, α-EpCAM IT did not show any anti-proliferative activity on HEK293 and HUVEC cells as well. In addition, the annexin V-FITC-PI assay results showed that α-EpCAM IT significantly increased apoptotic rate in MCF-7 cells with no effect on HEK293 and HUVEC as well. Moreover, α-EpCAM IT significantly reduced tumor size in vivo study. The achieved results indicate the potential of designing α-EpCAM IT as a novel therapeutic for cancer therapy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Propose: Human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of some kinds of cancer cells including breast cancer. In this study, we designed and produced a novel immunotoxin consisting anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified form of Pseudomonas exotoxin (PE35KDEL)., Methods: The three-dimensional (3D) structure of the fusion protein (anti-HER IT) was predicted by MODELLER 9.23 and its interaction with HER2 receptor was assessed using HADDOCK web server. Anti-HER2 IT, anti-HER2 scFv, and PE35KDEL proteins were expressed by Escherichia coli BL21 (DE3). After purification of the proteins using Ni 2+ affinity chromatography and refolding through dialysis, the cytotoxicity of proteins against breast cancer cell lines was examined by MTT assay., Results: In-silico studies showed that (EAAAK)2 linker can efficiently prevent the formation of salt bridges between two functional domains and the constructed fusion protein has a high affinity to HER2 receptor. The optimum condition of anti-HER2 IT expression was 25 °C and 1 mM IPTG. The protein was successfully purified and refolded by dialysis with a final yield of 45.7 mg per 1 L of bacterial culture. The cytotoxicity results showed that anti-HER2 IT was much more toxic on HER2-overexpressing cells, BT-474 (IC 50  ~ 95 nM) compared with HER2-negative cells, MDA-MB-23 (IC 50 ˃ 200 nM)., Conclusion: This novel immunotoxin has the potential to be applied as a therapeutic candidate for HER2-targeted cancer therapy. However further in vitro and in vivo evaluations are still required to confirm the efficacy and safety of this protein., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Senescent cells were recently shown to play a role in aging-related malfunctions and pathologies. This consensus has been facilitated by evidence from senolytic model mice capable of eliminating senescent cells in tissues using well-characterized senescent markers, such as p16 INK4a (hereafter p16). However, since the incomplete or artificial gene expression regulatory regions of manipulated marker genes affect their cognate expression, it currently remains unclear whether these models accurately reflect physiological senescence. We herein describe a novel approach to eliminate p16-expressing cells from mice at any given point in time, generating a new type of knock-in model, p16hCD2 mice and a toxin-conjugated anti-human CD2 antibody (hCD2-SAP) as an inducer. p16hCD2 mice possess an intact Cdkn2a locus that includes a p16 coding region and human CD2 (hCD2) expression unit. We confirmed cognate p16-associated hCD2 expression in mouse embryonic fibroblasts (MEFs) and in several tissues, such as the spleen, liver, and skin. We detected chronological increases in the hCD2-positive population in T lymphocytes that occurred in a p16-dependent manner, which reflected physiological aging. We then confirmed the high sensitivity of hCD2-SAP to hCD2 and validated its efficacy to remove p16-positive cells, particularly in T lymphocytes. The multiple administration of hCD2-SAP for a prolonged p16-positive cell deficiency partially restored aging-related phenotypes in T lymphocytes, such as the contraction of the CD4 + naïve population and expansion of senescence-associated T cells. Our novel approach of targeting p16-positive senescent cells will provide novel insights into the mechanisms underlying physiological aging in vivo., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and laser irradiation. The effectiveness of current PDT using red light for advanced cancers is limited, because red light can only reach depths within a few millimeters. To enhance the antitumor effect for lung cancers, we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin and a PS, mono-L-aspartyl chlorin e6 (NPe6)., Methods: We examined whether cetuximab encapsulated in endosomes was released into the cytosol by PS in PDT under light irradiation. A431 cells were treated with fluorescein isothiocyanate-labeled cetuximab, NPe6, and light irradiation and were observed with fluorescence microscopy. We analyzed the cytotoxicity of saporin-conjugated cetuximab (IT-cetuximab) in A431, A549, and MCF7 cells and the antitumor effect in model A549-bearing mice in vivo using the iTAP method., Results: Fluorescent microscopy analysis showed that the photodynamic effect of NPe6 (20 μM) and light irradiation (37.6 J/cm 2 ) caused the release of cetuximab from the endosome into the cytosol. In vitro analysis demonstrated that the iTAP method enhanced the cytotoxicity of IT-cetuximab by the photodynamic effect. In in vivo experiments, compared with IT-cetuximab alone or PDT alone, the iTAP method using a low dose of IT-cetuximab showed the greatest enhancement of the antitumor effect., Conclusions: Our study is the first report of the iTAP method using NPe6 for lung cancer cells. The iTAP method may become a new, minimally invasive treatment superior to current PDT methods., (© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Immunotoxins (ITs), which are toxin-fused tumor antigen-specific antibody chimeric proteins, have been developed to selectively kill targeted cancer cells. The epidermal growth factor receptor (EGFR) is an attractive target for the development of anti-EGFR ITs against solid tumors due to its overexpression on the cell surface of various solid tumors. However, the low basal level expression of EGFR in normal tissue cells can cause undesirable on-target/off-tumor toxicity and reduce the therapeutic window of anti-EGFR ITs. Here, based on an anti-EGFR monobody with cross-reactivity to both human and murine EGFR, we developed a strategy to tailor the anti-EGFR affinity of the monobody-based ITs carrying a 24-kDa fragment of Pseudomonas exotoxin A (PE24), termed ER-PE24, to distinguish tumors that overexpress EGFR from normal tissues. Five variants of ER-PE24 were generated with different EGFR affinities ( K D ≈ 0.24 nM to 104 nM), showing comparable binding activity for both human and murine EGFR. ER/0.2-PE24 with the highest affinity ( K D ≈ 0.24 nM) exhibited a narrow therapeutic window of 19 pM to 93 pM, whereas ER/21-PE24 with an intermediate affinity ( K D ≈ 21 nM) showed a much broader therapeutic window of 73 pM to 1.5 nM in in vitro cytotoxic assays using tumor model cell lines. In EGFR-overexpressing tumor xenograft mouse models, the maximum tolerated dose (MTD) of intravenous injection of ER/21-PE24 was found to be 0.4 mg/kg, which was fourfold higher than the MTD (0.1 mg/kg) of ER/0.2-PE24. Our study provides a strategy for the development of IT targeting tumor overexpressed antigens with basal expression in broad normal tissues by tailoring tumor antigen affinities.

Hepatocellular carcinoma (HCC) accounts for ∼90 % of all liver cancer cases, which was the third most common cause of cancer death worldwide in 2020. Glypican-3 (GPC3) is highly and specifically expressed in HCC, which makes it a promising therapeutic target. We discovered novel antibody sequences against GPC3 from a phage display library and ranked the candidates by their binding affinity and epitope bins. Candidates with single- to double-digit nanomolar affinity were selected and expressed in Fab format and linked to a deimmunized bacterial exotoxin moiety via an intein trans-splicing reaction. The resulting immunotoxins were evaluated for their in vitro binding specificity and affinity, cell surface binding on the HepG2 or Huh7, rate of internalization, and potency of cytotoxicity. The immunotoxin called GT5 exhibited strong antigen binding and cell surface binding, as well as high internalization efficiency. The molecule GT5 was further evaluated for cytotoxicity in HepG2 and Huh7 cell-based assay and assessed for its pharmacokinetics and antitumor activity in a murine tumor xenograft model. GT5 significantly inhibited tumor growth and showed stronger potency than the chemotherapeutic drug sorafenib. In conclusion, GT5, a novel GPC3 targeting immunotoxin, was shown to have a high affinity towards GPC3 and effectively inhibit hepatocellular tumor growth in vitro and in vivo, thus providing the basis for further development of GT5 immunotoxin as a novel therapeutic modality for the treatment of liver cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

LMB-100 is a recombinant immunotoxin composed of a Fab linked to a toxin. It kills cells expressing human mesothelin (hMSLN), which is highly expressed on the surface of mesothelioma and many other cancer cells. Clinically, we observed some patients had delayed responses to an anti-hMSLN immunotoxin treatment, suggesting the induction of anti-tumor immunity. We aimed to develop a mouse model to investigate whether immunotoxin alone can induce anti-tumor immunity and to study the mechanism of this immunity. An immunocompetent transgenic mouse was used to grow mouse mesothelioma AB1 cells expressing hMSLN in the peritoneal cavity. Mice were treated with LMB-100, and mice with complete responses (CRs) were rechallenged with tumor cells to determine whether anti-tumor immunity developed. Changes in gene expression profiles were evaluated by Nanostring, and changes in cytokines and chemokines were checked by protein arrays. The distribution of various immune cells was assessed by immunohistochemistry. Our results show that the mice with tumor reached CRs and developed anti-tumor immunity after LMB-100 treatment alone. The primary response requires CD8 + T cells, CD4 + T cells, and B cells. Transcriptional profiling shows that LMB-100 treatment reshapes the tumor immune microenvironment by upregulating chemotaxis signals. LMB-100 treatment upregulates genes associated with tertiary lymphoid structures (TLS) development and induces TLS formation in tumors. In sum, immunotoxin-mediated cell death induces anti-tumor immunity and the development of TLS, which provides insights into how immunotoxins cause tumor regressions.

Immunotoxins have represented a great potency in targeted therapeutics to encounter tumors. They consist of a protein toxin conjugated to a targeting moiety, which recognizes a specific antigen on surface of cancer cells and accordingly induces cell death by toxin segment. The targeting part could be a nanobody, which is a group of antibodies composed of an only functional single variable heavy chain (VHH).Therefore, this study was done to produce an immunotoxin (VGRNb-DT) by chemical conjugation of a truncated diphtheria toxin moiety to an anti-vascular endothelial growth factor receptor 2(VEGFR-2) nanobody, and to identify effectiveness of immunotoxin in recognizing the VEGFR-2- positive cancer cells and inhibiting cell growth and survival. Diphtheria toxin was expressed and purified by nickel affinity chromatography, and accordingly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed its expression. Function of heterobifunctional crosslinkers, Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and SATP (N-succinimidyl-S- acetylthiopropionate) for bioconjugation purposes was acknowledged by cation exchange high-performance liquid chromatography (HPLC). Cytotoxicity of immunotoxin was evaluated on the VEGFR-2 positive PC-3 cell line by MTT assay. Overexpression of VEGFR-2 in the PC-3 cell line allowed immunotoxin to recognize them by anti-VEGFR-2 nanobodies. The concentrations above 5 μg/ml represented a significant decrease in cell survival rate in PC-3 cells compared to HEK293 cells (VEGFR-2 negative cells) as controls.VGRNb-DT demonstrated a successful bioconjugation; furthermore, variable concentrations were correlated with cell death in prostate cancer PC-3 cells., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cancer, as a disease characterized by uncontrolled growth of cells, is recognized as one of the significant challenges in the field of health and medicine. There are various treatments for cancer like surgery, hormone therapy, chemotherapy, etc., but they have negative effects on the patient's lifestyle. Numerous side effects, and recently the emergence of drug resistance to these methods are weaknesses of these treatments. The utilization of bacteria as a treatment for cancer has attracted scientists' attention in the last decade. There are various methods of using bacteria to treat cancer, including the use of live, attenuated, or genetically engineered microbes, the use of bacterial toxins as an immunotoxin or conjugated to tumor antigens, bacteria-based cancer immunotherapy, bacterial vectors for gene-directed enzyme prodrug, and also the undeniable role of probiotics in treatment, are the cases that today are used for treatment. Bacterial therapy has shown a greater promise in cancer treatment due to its ability to lyse the tumor cells and deliver therapeutic products. However, the potential cytotoxicity of bacteria for healthy tissues, their inability to entirely lyse cancerous cells, and the possibility of mutations in their genomes are among the challenges of bacteriotherapy for cancer. Herein, we summarize the mechanism of bacteria, their potential benefits and harms, and the future of research in this field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

EGFRvⅢ is an established target for immunotherapy of glioblastoma (GBM). Current study aims to explore the efficacy of EGFRvⅢ-targeted immunotoxin combined with temozolomide (TMZ) or T cell-engaged bispecific antibody for the treatment of GBM. We generated three rabbit monoclonal antibodies (R1, R2, and R6) that specifically bound to EGFRvⅢ, but not EGFR, with high affinity. Immunotoxins were made by fusing the scFv of these antibodies with engineered Pseudomonas exotoxin PE24. The in vitro cytotoxicity and specificity of the immunotoxins was rigorously validated by EGFRvⅢ and EGFR-expressed cell lines. The in vivo efficacy of immunotoxin monotherapy and in combination with TMZ or EGFRvⅢ-targeted bispecific antibody was evaluated in orthotopic and subcutaneous xenograft mouse models. EGFRvⅢ immunotoxins potently killed U87, U251 and GL261 cells that were forcefully expressing EGFRvⅢ, with IC 50 values bellow 1.2 ng/ml. In a subcutaneous model, multiple intratumoral injections of immunotoxin at a dose of 2 mg/kg resulted in complete tumor regression in 3/5 of mice. In a C57BL/6 orthotopic glioblastoma model transplanted with GL261 cells that expressed a mouse version of EGFRvⅢ, two injections of 10 micrograms of immunotoxin in the lateral ventricles significantly improved the survival, with 2/5 mice being completely cured. Furthermore, in a subcutaneous xenograft model transplanted with EGFRvⅢ-expressed U87 cells, a single intratumoral injection of immuntoxin followed by i.v. injections of TMZ or EGFRvⅢ-targeted bispecific antibody achieved complete regression in mice. Taken together, EGFRvⅢ immunotoxin combined with TMZ or T cell-engaged bispecific antibody offers promise for curative treatment of GBM., Competing Interests: Conflict of interest statement The authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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) 1-3 YP7). 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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Targeted tumor therapy is an attractive approach for cancer treatment. Delta-like ligand 4 (DLL4) is overexpressed in tumor vasculature and plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. Immunotoxins due to their superior cell-killing ability and the relative simplicity of their preparation, have great potential in the clinical treatment of cancer. The aim of this study was to develop a novel immunotoxin against DLL4 as a cell cytotoxic agent and angiogenesis maturation inhibitor. In present study, an immunotoxin, named DLL4Nb-PE, in which a Nanobody as targeting moiety fused to the Pseudomonas exotoxin A (PE) was constructed, expressed and assessed by SDS-PAGE, western blotting, ELISA and flowcytometry. The functional assessment was carried out via MTT, apoptosis and chicken chorioallantoic membrane (CAM) assays. It was demonstrated DLL4Nb-PE specifically binds to DLL4 and recognizes DLL4-expressing MKN cells. The cytotoxicity assays showed that this molecule could induce apoptosis and kill DLL4 positive MKN cells. In addition, it inhibited neovascularization in the chicken chorioallantoic membrane. Our findings indicate designed anti-DLL4 immunotoxin has valuable potential for application to the treatment of tumors with high DLL4 expression., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cancer stem cells (CSCs) are self-renewing multipotent cells that play a vital role in the development of cancer drug resistance conditions. Various therapies like conventional, targeted, and radiotherapies have been broadly used in targeting and killing these CSCs. Among these, targeted therapy selectively targets CSCs and leads to overcoming disease recurrence conditions in cancer patients. Immunotoxins (ITs) are protein-based therapeutics with selective targeting capabilities. These chimeric molecules are composed of two functional moieties, i.e., a targeting moiety for cell surface binding and a toxin moiety that induces the programmed cell death upon internalization. Several ITs have been constructed recently, and their preclinical and clinical efficacies have been evaluated. In this review, we comprehensively discussed the recent preclinical and clinical advances as well as significant challenges in ITs targeting CSCs, which might reduce the burden of drug resistance conditions in cancer patients from bench to bedside., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Immunotoxins are regarded as a type of targeted therapy for killing cells by highly potent bacterial, fungal or plant toxins. Shiga like toxins (SLTs) are a group of bacterial AB5 protein toxins that inhibit host cell protein synthesis through the removal of a single adenine residue from the 28S rRNA and lead to apoptosis. Here, we described the design and usage of a Stx-based immunotoxin that can induce the selective cytotoxicity and apoptosis in Fn-14-positive cells related to the colon and lung cancer. In the present study, the Stx2a-PE15-P4A8 fusion protein was expressed efficiently in E. coli (DE3) system when driven from inclusion bodies by 8 M urea. The Stx2a-PE15-P4A8 fusion protein was expressed efficiently in E. coli (DE3) system and then purified. The purified fusion protein could specifically target Fn-14 receptor existed on colon and lung cancer cell lines and suppress these cells in a dose-dependent manner. In addition, the protein was able to nearly 50 % of apoptotic cell death and maintains about 54 % of its stability after 24 h of incubation in mouse serum at 37 °C. Compared to PE38-P4A8 construct in our previous study, these results showed that the Stx2a-PE15-P4A8 construct can be an efficient therapeutic candidate for cancer immunotherapy., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tumor necrosis factor α (TNF-α) is upregulated in a chronic inflammatory environment, including tumors, and has been recognized as a pro-tumor factor in many cancers. Applying the traditional TNF-α antibodies that neutralize TNF-α activity, however, only exerts modest anti-tumor efficacy in clinical studies. Here, we develop an innovative approach to target TNF-α that is distinct from the neutralization mechanism. We employed phage display and yeast display to select non-neutralizing antibodies that can piggyback on TNF-α and co-internalize into cells through receptor ligation. When conjugating with toxins, the antibody exhibited cytotoxicity to cancer cells in a TNF-α-dependent manner. We further implemented the immunotoxin to an E. coli vehicle specially engineered for a high secretion level. In a syngeneic murine melanoma model, the bacteria stimulated TNF-α expression that synergized with the secreted immunotoxin and greatly inhibited tumor growth. The treatment also dramatically remodeled the tumor microenvironment in favor of several anti-tumor immune cells, including N1 neutrophils, M1 macrophages, and activated CD4 + and CD8 + lymphocytes. We anticipate that our new piggyback strategy is generalizable to targeting other soluble ligands and/or conjugates with different drugs for managing a diverse set of diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background/aim: Cytotoxic payload conjugation to antibodies efficiently suppresses tumors and contributes to the improvement of cancer survival. In our previous study, c-Kit targeting antibody-drug conjugate (2G4-DM1) with DM1, a microtubule inhibitor, efficiently suppressed tumor growth. However, slow-growing c-Kit-positive tumors, such as GIST-48, did not efficiently respond to DM1. In this study, we aimed to treat tumors using 2G4 immunotoxin with Pseudomonas exotoxin A (PE) as a payload., Materials and Methods: Modified FcBP-PE24 containing p-benzoyl-L-phenylalanine, unnatural amino acid, was expressed in E. coli and purified. Then, photoconjugation of 2G4 antibody and FcBP-PE24 at 365 nm was carried out and 2G4 immunotoxin was purified using anion exchange chromatography. In vitro cytotoxicity of 2G4 immunotoxins was assessed in HMC-1.2, GIST-48, and MDA-MB-453 cells. Then, in vivo efficacy analysis was performed using C.B-17 SCID mice., Results: 2G4 immunotoxin efficiently induced cytotoxicity in 2G4-DM1-resistant HMC-1.2 and GIST-48 cells by inhibiting protein synthesis but not in c-Kit-negative MDA-MB-453 cells. The results showed ~200-fold or more increase in cytotoxicity against c-Kit-positive cells compared to IC50 of 2G4-DM1. In addition, 2G4 immunotoxin suppressed tumor growth in the in vivo xenograft mouse model., Conclusion: 2G4 immunotoxins could be an alternative therapeutic strategy for microtubule inhibitor- resistant cancer cells., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The human transferrin receptor (TFR) is overexpressed in most breast cancers, including preneoplastic ductal carcinoma in situ (DCIS). HB21(Fv)-PE40 is a single-chain immunotoxin (IT) engineered by fusing the variable region of a monoclonal antibody (HB21) against a TFR with a 40 kDa fragment of Pseudomonas exotoxin (PE). In humans, the administration of other TFR-targeted immunotoxins intrathecally led to inflammation and vascular leakage. We proposed that for treatment of DCIS, intraductal (i.duc) injection of HB21(Fv)-PE40 could avoid systemic toxicity while retaining its potent antitumor effects on visible and occult tumors in the entire ductal tree. Pharmacokinetic studies in mice showed that, in contrast to intravenous injection, IT was undetectable by enzyme-linked immunosorbent assay in blood following i.duc injection of up to 3.0 μg HB21(Fv)-PE40. We demonstrated the antitumor efficacy of HB21(Fv)-PE40 in two mammary-in-duct (MIND) models, MCF7 and SUM225, grown in NOD/SCID/gamma mice. Tumors were undetectable by In Vivo Imaging System (IVIS) imaging in intraductally treated mice within 1 wk of initiation of the regimen (IT once weekly/3 wk, 1.5 μg/teat). MCF7 tumor-bearing mice remained tumor free for up to 60 d of observation with i.duc IT, whereas the HB21 antibody alone or intraperitoneal IT treatment had minimal/no antitumor effects. These and similar findings in the SUM225 MIND model were substantiated by analysis of mammary gland whole mounts, histology, and immunohistochemistry for the proteins Ki67, CD31, CD71 (TFR), and Ku80. This study provides a strong preclinical foundation for conducting feasibility and safety trials in patients with stage 0 breast cancer.

CD45RA is a specific marker for leukemia stem cell (LSC) sub-populations in acute myeloid leukemia (AML). Ranpirnase (Rap), an amphibian RNase, has been extensively investigated in preclinical and clinical studies for its antitumor activity. Rap could be administered repeatedly to patients without inducing an immune response. Reversible renal toxicity has been reported to be dose-limiting. In this study, we generated a novel immunotoxin targeting LSCs: Hm3A4-Rap, which was composed of Rap and Hm3A4, a human-mouse chimeric antibody against CD45RA. This immunotoxin was generated recombinantly by fusing Rap to Hm3A4 at the Fc terminus and then produced by stably transfecting CHO cells. The immunotoxin was purified using Ni-NTA and then evaluated using RT-PCR, SDS-PAGE, antibody titer assays, competitive inhibition assays, and internalization assays. In addition, the purity, molecular integrity, and affinity to the CD45RA antigen were determined. In vitro studies demonstrated that Hm3A4-Rap could efficiently kill target cells. In vivo studies demonstrated that Hm3A4-Rap had potent anti-leukemia activity, with dosed mice showing a significant increase in survival time compared to control mice (P < 0.01). In summary, our immunotoxin had excellent biological activity suggesting its potential therapeutic value for treating AML patients. Additional preclinical and clinical studies are needed to develop this immunotoxin as a treatment option for patients with leukemia.

Background: Increased CCKBR expression density or frequency has been reported in many neoplasms., Objective: We aimed to investigate whether CCKBR drives the growth of gastric cancer (GC) and its potential as a therapeutic target of immunotoxins., Methods: A lentiviral interference system was used to generate CCKBR-knockdown gastric cancer cells. Cell Counting Kit-8 and clonogenic assays were used to evaluate cell proliferation. Woundhealing and cell invasion assays were performed to evaluate cell mobility. Cell cycle was analyzed by flow cytometry. Tumor growth in vivo was investigated using a heterologous tumor transplantation model in nude mice. In addition, we generated the immunotoxin FQ17P and evaluated the combining capacity and tumor cytotoxicity of FQ17P in vitro., Results: Stable downregulation of CCKBR expression resulted in reduced proliferation, migration and invasion of BGC-823 and SGC-7901 cells. The impact of CCKBR on gastric cancer cells was further verified through CCKBR overexpression studies. Downregulation of CCKBR expression also inhibited the growth of gastric tumors in vivo. Furthermore, FQ17P killed CCKBR-overexpressing GC cells by specifically binding to CCKBR on the tumor cell surface., Conclusion: The CCKBR protein drives the growth, migration, and invasion of gastric cancer cells, and it might be a promising target for immunotoxin therapy based on its aberrant expression, functional binding interactions with gastrin, and subsequent internalization., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Therefore, fighting against such cancer is reasonable. Chemotherapy drugs are sometimes inefficient and often accompanied by undesirable side effects for patients. On the other hand, the emergence of chemoresistant HCC emphasizes the need for a new high-efficiency treatment strategy. Immunotoxins are armed and rigorous targeting agents that can purposefully kill cancer cells. Unlike traditional chemotherapeutics, immunotoxins because of targeted toxicity, insignificant cross-resistance, easy production, and other favorable properties can be ideal candidates against HCC. In this review, the characteristics of proper HCC-specific biomarkers for immunotoxin targeting were dissected. After that, the first to last immunotoxins developed for the treatment of liver cancer were discussed. So, by reviewing the strengths and weaknesses of these immunotoxins, we attempted to provide keynotes for designing an optimal immunotoxin against HCC.

Immunotoxins (ITs) are protein-based drugs that compose of targeting and cytotoxic moieties. After binding the IT to the specific cell-surface antigen, the IT internalises into the target cell and kills it. Targeting and cytotoxic moieties usually include monoclonal antibodies and protein toxins with bacterial or plant origin, respectively. ITs have been successful in haematologic malignancies treatment. However, ITs penetrate poorly into solid tumours because of their large size. Use of camelid antibody fragments known as nanobodies (Nbs) as a targeting moiety may overcome this problem. Nbs are the smallest fragment of antibodies with excellent tumour tissue penetration. The ability to recognise cryptic (immuno-evasive) target antigens, low immunogenicity, and high-affinity are other fundamental characteristics of Nbs that make them suitable candidates in targeted therapy. Here, we reviewed and discussed the structure and function of ITs, Nbs, and nanobody-based ITs. To gain sound insight into the issue at hand, we focussed on nanobody-based ITs.

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-CUS 245C ) based on Cetuximab and recombinant Cucurmosin (CUS 245C )., Methods: E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS 245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS 245C . The chemical conjugation method was used for the preparation of C-CUS 245C . Then dialysis and IMAC were used to purify C-CUS 245C . Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS 245C . At last the anti-colorectal cancer activity of C-CUS 245C was investigated in vitro and in vivo., Results: CUS 245C with high purity could be obtained from the prokaryotic system. C-CUS 245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS 245C on EGFR-positive cells for 120 h with IC 50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS 245C was remarkably more potent than that of Cetuximab, CUS 245C , and C + CUS 245C (P < 0.001). Whereas the cytotoxicity of C-CUS 245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS 245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS 245C for the targeted therapy of colorectal neoplasms., Conclusions: C-CUS 245C 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.

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa , consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli , and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli . Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC 50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM ( n = 9) and 19 ± 1.4 pM ( n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

Pancreatic cancer, as one of the most aggressive and lethal malignancies in the world, is lack of effective treatment. Constructing immunotoxin molecules to target the mesothelin (MSLN) receptor is a potential therapeutic strategy for pancreatic cancer and other related malignant tumors, with some molecules being tested in clinical trials. However, currently, there are still some limitations in its applications, such as the difficulty of the preparation of drug molecules, the limited effectiveness of drugs, and the inadequacy of drug safety and immunogenicity. In this study, we constructed a novel type of anti-MSLN immunotoxin, A1-PE24X7, in which a single domain antibody (sdAb) molecule was used as the target ligand and an improved PE24X7 toxin with reduced off-target toxicity and immunogenicity was used as the effector. Unlike conventional immunotoxins, the designed A1-PE24X7 could be easily expressed in the E. coli system in the form of a soluble protein with a good yield (15--20 mg/L), avoiding the complex process of denaturation and refolding of inclusion bodies, and it can be conveniently stored in PBS solution for more than 7 days at 4 °C, showing high storage stability. Cell-based experiments showed that A1-PE24X7 entered MSLN-expressing tumor cells in a receptor-mediated manner and killed these cells with an EC 50 in the low nanomolar range (0.13 nM against NCI-N87 cells and 0.79 nM against AsPC-1 cells) and it showed ideal selectivity for the MSLN receptor (>100 nM against receptor negative PC3 cells). In animal-based experiments, A1-PE24X7 had tumor enrichment ability in relation to MSLN-positive tumors and showed strong tumor killing and inhibition in mouse models of pancreatic cancer and gastric cancer. Five injections of 3.0 mg/kg A1-PE24X7 significantly reduced the tumor volume of gastric NCI-N87 cancer and also significantly inhibited the growth of pancreatic AsPC-1 cancer. In addition, the maximum tolerable dosage (MSD) of A1-PE24X7 to mice was higher than 15 mg/kg, showing that A1-PE24X7 has a relatively broad therapeutic window. These preclinical results indicate that this strategy has good potential for application to the treatment of pancreatic cancer and other tumors with high MSLN expression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

Neuropilin-1 (NRP-1) regulates a range of physiological and pathological processes, including angiogenesis. Targeting of NRP1 is considered a significant approach in cancer therapy. In the present study, a novel antiNRP1 immunotoxin (αNRP1 IT) was developed by genetic fusion of a single domain (VHH) anti-NRP-1 antibody fragment to a truncated diphtheria toxin. The αNRP1 IT was expressed into bacterial cells as an inclusion body (IB). Expression of αNRP1 IT was confirmed by SDS-PAGE and western blotting. Recombinant αNRP1 IT was purified using nickel affinity chromatography. Toxicity and antiangiogenesis effect of αNRP1 IT was investigated both in vitro and in vivo . Results showed that αNRP1 IT significantly reduced the viability of human umbilical vein endothelial cell line (HUVEC) ( p  < .05). The αNRP1 IT significantly inhibited tube formation of HUVEC cells ( p  < .001). Furthermore, αNRP1 IT inhibited angiogenesis in Chick Chorioallantoic Membrane (CAM) Assay. These data suggest the potential of αNRP1 IT as a novel therapeutic in targeted cancer therapy.

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.

Background: LMB-100 is an antibody-toxin conjugate with an antimesothelin Fab linked to a 24-kilodalton portion of Pseudomonas exotoxin A with mutations that decrease immunogenicity. The objective of the current first-in-human phase 1 study was to determine the maximum tolerated dose (MTD) and safety in patients with advanced solid tumors expressing mesothelin., Methods: Cohorts of 1 to 7 patients received intravenous LMB-100 at 7 dose levels from 40 µg/kg to 250 µg/kg intravenously on days 1, 3, and 5 of a 21-day cycle., Results: Of the 25 patients accrued, 17 had mesothelioma, 3 each had ovarian or pancreatic cancer, and 2 patients had gastric cancer. Dose-limiting toxicities occurred in 2 of 4 patients treated at a dose of 250 µg/kg (capillary leak syndrome) and in 3 of 7 patients treated at a dose of 170 µg/kg (creatinine increase). The MTD of LMB-100 was 140 µg/kg. Of the 10 patients with mesothelioma who were treated at doses of 170 µg/kg or 140 µg/kg, 8 had stable disease and 2 developed progressive disease. Peak LMB-100 plasma concentrations were dose-dependent during cycle 1. The development of antidrug antibodies decreased LMB-100 blood levels in 8 of 21 patients (38%) who received cycle 2 and 9 of 11 patients (81.8%) who received cycle 3., Conclusions: The MTD for single-agent LMB-100 was found to be 140 µg/kg given on a schedule of every other day for 3 doses every 3 weeks. Although less immunogenic than the first-generation antimesothelin immunotoxin SS1P, the majority of patients developed antidrug antibodies after 2 cycles, indicating that LMB-100 has limited antitumor efficacy as a single agent. Phase 2 studies of LMB-100 plus pembrolizumab currently are ongoing for patients with mesothelioma and lung cancer., Lay Summary: Mesothelin, a cell surface antigen, is an attractive target for cancer therapy given its limited expression in normal human tissues and high expression in many human cancers. LMB-100 is a recombinant antimesothelin immunotoxin consisting of a humanized antimesothelin antibody fragment fused to a truncated Pseudomonas exotoxin A. In the current study, the authors determined the safety, maximum tolerated dose, and pharmacokinetics of LMB-100, as well as the generation of antidrug antibodies. Ongoing phase 2 clinical trials are evaluating the combination of LMB-100 plus pembrolizumab in patients with treatment-refractory mesothelioma and non-small cell lung cancer., (© 2020 American Cancer Society.)

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.

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.

Immunotoxins are Ab-cytotoxin chimeric molecules with mighty cytotoxicity. Programmed cell death 1-ligand 1 (PD-L1), is a transmembrane protein expressed mainly in inflammatory tumor tissues and plays a pivotal role in immune escape and tumor progression. Although PD-L1 immune checkpoint therapy has been successful in some cases, many patients have not benefited enough due to primary/secondary resistance. In order to optimize the therapeutic efficacy of anti-PD-L1 mAb, we used durvalumab as the payload and CUS 245C , a type I ribosome-inactivating protein isolated from Cucurbita moschata, as the toxin moiety, to construct PD-L1-specific immunotoxin (named D-CUS 245C ) through the engineered cysteine residue. In vitro, D-CUS 245C selectively killed PD-L1 + tumor cells. In vivo studies also showed that D-CUS 245C had obvious antitumor effect on PD-L1 + human xenograft tumors in nude mice. In conclusion, in the combination of the toxin with mAb, this study developed a new immunotoxin targeting PD-L1, emphasizing a novel and promising treatment strategy and providing a valuable way to optimize cancer immunotherapy., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris . In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn + cell lines was higher than that of granulysin alone.

Background: Treatment of hepatocellular carcinoma (HCC) using antibody-based targeted therapies, such as antibody conjugates and chimeric antigen receptor T (CAR-T) cell therapy, shows potent antitumor efficacy. Glypican-3 (GPC3) is an emerging HCC therapeutic target; therefore, antibodies against GPC3 would be useful tools for developing immunotherapies for HCC., Methods: We isolated a novel human monoclonal antibody, 32A9, by phage display technology. We determined specificity, affinity, epitope and anti-tumor activity of 32A9, and developed 32A9-based immunotherapy technologies for evaluating the potency of HCC treatment in vitro or in vivo., Results: 32A9 recognized human GPC3 with potent affinity and specificity. The epitope of 32A9 was located in the region of the GPC3 protein core close to the modification sites of the HS chain and outside of the Wnt-binding site of GPC3. The 32A9 antibody significantly inhibited HCC xenograft tumor growth in vivo. We then pursued two 32A9-based immunotherapeutic strategies by constructing an immunotoxin and CAR-T cells. The 32A9 immunotoxin exhibited specific cytotoxicity to GPC3-positive cancer cells, while 32A9 CAR-T cells efficiently eliminated GPC3-positive HCC cells in vitro and caused HCC xenograft tumor regressions in vivo., Conclusions: Our study provides a rationale for 32A9 as a promising GPC3-specific antibody candidate for HCC immunotherapy.

A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug resistant (MDR) gene deficient mice so that only MDR deficient cells expressing the target molecule would be depleted while target molecule expressing, but MDR sufficient, cells are spared. This allows targeted depletion at a higher degree of specificity than has been previously achieved. We have applied this technique to study trogocytosis, the intercellular transfer of cell surface molecules, but this principle could also be adapted using technology already available for use in other fields of study., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background/aim: Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of cancer worldwide. Our study focused on the axon guidance receptor roundabout guidance receptor 1 (ROBO1) as a target for monoclonal antibody therapy of HNSCC. We previously showed that saporin-conjugated anti-ROBO1 (B5209B) immunotoxin (IT-ROBO1) enhanced cytotoxic effects on HNSCC cells in combination with the photosensitizer aluminum phthalocyanine disulphonate (AlPcS2a) and illumination. We examined the effects of this combination therapy in a mouse xenograft model., Materials and Methods: IT-ROBO1 was intraperitoneally administered to HSQ-89 (derived from Japanese maxillary sinus squamous carcinoma, RCB0789; RIKEN, Tsukuba, Japan) xenografted mice. After 3 days, AlPcS2a was injected subcutaneously around the tumor and the area was illuminated at 650 nm for 30 min. The growth of the tumor was evaluated and the effects on the tumor were examined., Results: Pronounced anti-tumor effects were elicited by the administration of IT-ROBO1 and AlPcS2a with light illumination on tumor size and pathological characteristics., Conclusion: The results showed that photosensitizer treatment with illumination robustly enhanced the antitumor effect of the IT-ROBO1 immunotoxin., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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., Competing Interests: The authors declare no conflicts of interest.

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., Competing Interests: The authors declare no conflict of interest.

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 IC 50 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.

The majority of clinically diagnosed cutaneous T-cell lymphomas (CTCL) highly express the cell-surface markers CC chemokine receptor 4 (CCR4) and/or CD25. Recently, we have developed diphtheria toxin-based recombinant Ontak®-like human IL2 fusion toxin (IL2 fusion toxin) and anti-human CCR4 immunotoxin (CCR4 IT). In this study, we first compared the efficacy of the CCR4 IT vs IL2 fusion toxin for targeting human CD25 + CCR4 + CTCL. We demonstrated that CCR4 IT was more effective than IL2 fusion toxin. We further constructed an IL2-CCR4 bispecific IT. The bispecific IT was significantly more effective than either IL2 fusion toxin or CCR4 IT alone. The bispecific IT is a promising novel targeted therapeutic drug candidate for the treatment of refractory and recurrent human CD25 + and/or CCR4 + CTCL., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

The present study was performed to design an immunotoxin consisting of engineered RNase A and scFv of Cetuximab. To accomplish this study goal, at first to evade RNase A from its inhibitors in the cytoplasm, six amino acids of RNase A were substituted, then the physicochemical features of engineered RNase A were assessed. To investigate the interaction between the engineered RNase A and the ribonuclease inhibitor, protein-protein docking was performed. After engineering the RNase A, it was theoretically conjugated with scFv of Cetuximab using a cleavable linker to produce scFv-engineered RNase A. Then, wild-RNase A (14 kD), engineered RNase A (14 kD) and scFv-engineered RNase A (42 kDa) were expressed in the BL21 (DE3) strain of Escherichia coli and purified by Ni-NTA columns. To confirm the expressed proteins, western blot analysis was performed. The functioning of wild-RNase A and engineered RNase A were investigated by RNA fragmentation assay. Finally, to evaluate the cytotoxicity of scFv-engineered RNase A, a dose-response cytotoxicity assay was performed on Her1-positive and Her1-negative cell lines. The results showed that engineered RNase A could maintain its structure and disulfide bonds and evade its inhibitor. Expression and purification were successfully conducted and both enzymes could degrade yeast RNA. The result of cytotoxicity showed that the engineered immunotoxin could induce cell death to Her1-positive cell lines with an IC 50 of 50 nM. It appears that scFv-engineered RNase A can be a promising molecule for use., (© 2019 Wiley Periodicals, Inc.)