Toxicology, pharmacokinetics, and immunogenicity studies of CCR4-IL2 bispecific immunotoxin in rats and minipigs.

We have developed a diphtheria toxin-based recombinant human CCR4-IL2 bispecific immunotoxin (CCR4-IL2-IT) for targeted therapy of cutaneous T-cell lymphoma (CTCL). CCR4-IL2-IT demonstrated superior efficacy in an immunodeficient mouse CTCL model. Recently, we have compared the in vivo efficacy of CCR4-IL2-IT versus Brentuximab (FDA approved leading drug in CTCL market) in the same immunodeficient mouse CTCL model. The comparison demonstrated that CCR4-IL2-IT was significantly more effective than Brentuximab. In this study, we have performed non-GLP (Good Laboratory Practice) toxicology, pharmacokinetics, immunogenicity studies of CCR4-IL2-IT in both rats and minipigs. CCR4-IL2-IT demonstrated excellent safety profiles in both rats and minipigs. The maximum tolerated dose of CCR4-IL2-IT was determined as 0.4 mg/kg in both rats and minipigs. Complete blood count and chemistry analysis did not show significant difference for all measured parameters between the blood samples of pre-injection versus post-injection from the five-day toxicology studies of CCT4-IL2-IT in both rats and minipigs. Histology analysis did not show difference between the PBS treatment group versus CCR4-IL2-IT treatment group at 50 µg/kg in both rats and minipigs. The half-life of CCR4-IL2-IT was determined as about 45 min in rats and 30 min in minipigs. The antibodies against CCR4-IL2-IT were detected in about two weeks after CCR4-IL2-IT treatment. CCR4-IL2-IT did not induce cytokine release syndrome in a peripheral blood mononuclear cell derived humanized mouse model. The depletion of CCR4+ cell and CD25+ cell (two target cell populations of CCR4-IL2-IT) was observed in minipigs. The excellent safety profile promoted us to further develop CCR4-IL2-IT towards clinical trials.

Hosts and Heterologous Expression Strategies of Recombinant Toxins for Therapeutic Purposes.

The production of therapeutic recombinant toxins requires careful host cell selection. Bacteria, yeast, and mammalian cells are common choices, but no universal solution exists. Achieving the delicate balance in toxin production is crucial due to potential self-intoxication. Recombinant toxins from various sources find applications in antimicrobials, biotechnology, cancer drugs, and vaccines. "Toxin-based therapy" targets diseased cells using three strategies. Targeted cancer therapy, like antibody-toxin conjugates, fusion toxins, or "suicide gene therapy", can selectively eliminate cancer cells, leaving healthy cells unharmed. Notable toxins from various biological sources may be used as full-length toxins, as plant (saporin) or animal (melittin) toxins, or as isolated domains that are typical of bacterial toxins, including Pseudomonas Exotoxin A (PE) and diphtheria toxin (DT). This paper outlines toxin expression methods and system advantages and disadvantages, emphasizing host cell selection's critical role.

Diphtheria toxin-derived, anti-PD-1 immunotoxin, a potent and practical tool to selectively deplete PD-1+ cells.

Programmed death-1 (PD-1), an immune checkpoint receptor, is expressed on activated lymphocytes, macrophages, and some types of tumor cells. While PD-1+ cells have been implicated in outcomes of cancer immunity, autoimmunity, and chronic infections, the exact roles of these cells in various physiological and pathological processes remain elusive. Molecules that target and deplete PD-1+ cells would be instrumental in defining the roles unambiguously. Previously, an immunotoxin has been generated for the depletion of PD-1+ cells though its usage is impeded by its low production yield. Thus, a more practical molecular tool is desired to deplete PD-1+ cells and to examine functions of these cells. We designed and generated a novel anti-PD1 diphtheria immunotoxin, termed PD-1 DIT, targeting PD-1+ cells. PD-1 DIT is comprised of two single chain variable fragments (scFv) derived from an anti-PD-1 antibody, coupled with the catalytic and translocation domains of the diphtheria toxin. PD-1 DIT was produced using a yeast expression system that has been engineered to efficiently produce protein toxins. The yield of PD-1 DIT reached 1-2 mg/L culture, which is 10 times higher than the previously reported immunotoxin. Flow cytometry and confocal microscopy analyses confirmed that PD-1 DIT specifically binds to and enters PD-1+ cells. The binding avidities between PD-1 DIT and two PD-1+ cell lines are approximately 25 nM. Moreover, PD-1 DIT demonstrated potent cytotoxicity toward PD-1+ cells, with a half maximal effective concentration (EC50 ) value of 1 nM. In vivo experiments further showed that PD-1 DIT effectively depleted PD-1+ cells and enabled mice inoculated with PD-1+ tumor cells to survive throughout the study. Our findings using PD-1 DIT revealed the critical role of pancreatic PD-1+ T cells in the development of type-1 diabetes (T1D). Additionally, we observed that PD-1 DIT treatment ameliorated relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), a mouse model of relapsing-remitting multiple sclerosis (RR-MS). Lastly, we did not observe significant hepatotoxicity in mice treated with PD-1 DIT, which had been reported for other immunotoxins derived from the diphtheria toxin. With its remarkable selective and potent cytotoxicity toward PD-1+ cells, coupled with its high production yield, PD-1 DIT emerges as a powerful biotechnological tool for elucidating the physiological roles of PD-1+ cells. Furthermore, the potential of PD-1 DIT to be developed into a novel therapeutic agent becomes evident.

CCR4-IL2 bispecific immunotoxin is more effective than brentuximab for targeted therapy of cutaneous T-cell lymphoma in a mouse CTCL model.

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.

New insights on Pseudomonas aeruginosa exotoxin A-based immunotoxins in targeted cancer therapeutic delivery.

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

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

A Novel EGFR Targeted Immunotoxin Based on Cetuximab and Type 1 RIP Quinoin Overcomes the Cetuximab Resistance in Colorectal Cancer Cells.

Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab's therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 µg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.

Development of a new minimally invasive phototherapy for lung cancer using antibody-toxin conjugate.

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/cm2 ) 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.

Systemic immune changes accompany combination treatment with immunotoxin LMB-100 and nab-paclitaxel.

LMB-100 is a novel immune-conjugate (immunotoxin) that targets mesothelin. A phase 1/2 clinical trial was conducted (NCT02810418) with primary objectives assessing the safety and efficacy of LMB-100 ± nab-paclitaxel. Participant blood samples were analyzed for changes in serum cytokines and circulating immune cell subsets associated with response or toxicity. On Arm A, participants (n = 20) received standard 30-minute LMB-100 infusion with nab-paclitaxel. Although clinical efficacy was observed, the combination caused intolerable capillary leak syndrome (CLS), a major toxicity of unclear etiology that affects many immunotoxin drugs. Participants developing CLS experienced rapid elevations in IFNγ and IL-8 compared to those without significant CLS, along with midcycle increases in Ki-67- CD4 T cells that were CD38, HLA-DR, or TIM3 positive. Additionally, a strong increase in activated CD4 and CD8 T cells and a concurrent decrease in Tregs were seen in the single Arm A patient achieving a partial response. In Arm B, administration of single agent LMB-100 to participants (n = 20) as a long infusion given over 24-48 h was investigated based on pre-clinical data that this format could reduce CLS. An optimal dose and schedule of long infusion LMB-100 were identified, but no clinical efficacy was observed even in patients receiving LMB-100 in combination with nab-paclitaxel. Despite this, both Arm A and B participants experienced increases in specific subsets of proliferating CD4 and CD8 T cells following Cycle 1 treatment. In summary, LMB-100 treatment causes systemic immune activation. Inflammatory and immune changes that accompany drug associated CLS were characterized for the first time.

Expanding the Therapeutic Window of EGFR-Targeted PE24 Immunotoxin for EGFR-Overexpressing Cancers by Tailoring the EGFR Binding Affinity.

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 (KD ≈ 0.24 nM to 104 nM), showing comparable binding activity for both human and murine EGFR. ER/0.2-PE24 with the highest affinity (KD ≈ 0.24 nM) exhibited a narrow therapeutic window of 19 pM to 93 pM, whereas ER/21-PE24 with an intermediate affinity (KD ≈ 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.

Designing a humanized immunotoxin based on DELTA-stichotoxin-Hmg2a toxin: an in silico study.

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

Construction and evaluation of GPC3-targeted immunotoxins as a novel therapeutic modality for hepatocellular carcinoma.

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.

EGFRvâ ¢-targeted immunotoxin combined with temozolomide and bispecific antibody for the eradication of established glioblastoma.

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 IC50 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.

Plant-Derived Type I Ribosome Inactivating Protein-Based Targeted Toxins: A Review of the Clinical Experience.

Targeted toxins (TT) for cancer treatment are a class of hybrid biologic comprised of a targeting domain coupled chemically or genetically to a proteinaceous toxin payload. The targeting domain of the TT recognises and binds to a defined target molecule on the cancer cell surface, thereby delivering the toxin that is then required to internalise to an appropriate intracellular compartment in order to kill the target cancer cell. Toxins from several different sources have been investigated over the years, and the two TTs that have so far been licensed for clinical use in humans; both utilise bacterial toxins. Relatively few clinical studies have, however, been undertaken with TTs that utilise single-chain type I ribosome inactivating proteins (RIPs). This paper reviews the clinical experience that has so far been obtained for a range of TTs based on five different type I RIPs and concludes that the majority studied in early phase trials show significant clinical activity that justifies further clinical investigation. A range of practical issues relating to the further clinical development of TT's are also covered briefly together with some suggested solutions to outstanding problems.

A New Combination: Anti Glypican-3 scFv and Diphtheria Toxin with the Best Flexible Linker.

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

Improved Therapy of B-Cell Non-Hodgkin Lymphoma by Obinutuzumab-Dianthin Conjugates in Combination with the Endosomal Escape Enhancer SO1861.

Immunotoxins do not only bind to cancer-specific receptors to mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. The plant glycoside SO1861 was previously reported to enhance the endolysosomal escape of antibody-toxin conjugates in non-hematopoietic cells, thus increasing their cytotoxicity manifold. Here we tested this technology for the first time in a lymphoma in vivo model. First, the therapeutic CD20 antibody obinutuzumab was chemically conjugated to the ribosome-inactivating protein dianthin. The cytotoxicity of obinutuzumab-dianthin (ObiDi) was evaluated on human B-lymphocyte Burkitt's lymphoma Raji cells and compared to human T-cell leukemia off-target Jurkat cells. When tested in combination with SO1861, the cytotoxicity for target cells was 131-fold greater than for off-target cells. In vivo imaging in a xenograft model of B-cell lymphoma in mice revealed that ObiDi/SO1861 efficiently prevents tumor growth (51.4% response rate) compared to the monotherapy with ObiDi (25.9%) and non-conjugated obinutuzumab (20.7%). The reduction of tumor volume and overall survival was also improved. Taken together, our results substantially contribute to the development of a combination therapy with SO1861 as a platform technology to enhance the efficacy of therapeutic antibody-toxin conjugates in lymphoma and leukemia.

Development of anti DLL4 Nanobody fused to truncated form of Pseudomonas exotoxin: As a novel immunotoxin to inhibit of cell proliferation and neovascularization.

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.

Emerging trends in immunotoxin targeting cancer stem cells.

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.

Development of a TNF-α-mediated Trojan Horse for bacteria-based cancer therapy.

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.

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.