The immunotoxin targeting PRLR increases tamoxifen sensitivity and enhances the efficacy of chemotherapy in breast cancer.

BACKGROUND: Though tamoxifen achieves success in treating estrogen receptor α (ERα)-positive breast cancer, the followed development of tamoxifen resistance is a common challenge in clinic. Signals downstream of prolactin receptor (PRLR) could synergize with ERα in breast cancer progression. However, the potential effect of targeting PRL-PRLR axis combined with tamoxifen has not been thoroughly investigated. METHODS: High-throughput RNA-seq data obtained from TCGA, Metabric and GEO datasets were analyzed to explore PRLR expression in breast cancer cell and the association of PRLR expression with tamoxifen treatment. Exogenous or PRL overexpression cell models were employed to investigate the role of activated PRLR pathway in mediating tamoxifen insensitivity. Immunotoxin targeting PRLR (N8-PE24) was constructed with splicing-intein technique, and the efficacy of N8-PE24 against breast cancer was evaluated using in vitro and in vivo methods, including analysis of cells growth or apoptosis, 3D spheroids culture, and animal xenografts. RESULTS: PRLR pathway activated by PRL could significantly decrease sensitivity of ERα-positive breast cancer cells to tamoxifen. Tamoxifen treatment upregulated transcription of PRLR and could induce significant accumulation of PRLR protein in breast cancer cells by alkalizing lysosomes. Meanwhile, tamoxifen-resistant MCF7 achieved by long-term tamoxifen pressure exhibited both upregulated transcription and protein level of PRLR. Immunotoxin N8-PE24 enhanced sensitivity of breast cancer cells to tamoxifen both in vitro and in vivo. In xenograft models, N8-PE24 significantly enhanced the efficacy of tamoxifen and paclitaxel when treating PRLR-positive triple-negative breast cancer. CONCLUSIONS: PRL-PRLR axis potentially associates with tamoxifen insensitivity in ERα-positive breast cancer cells. N8-PE24 could inhibit cell growth of the breast cancers and promote drug sensitivity of PRLR-positive breast cancer cells to tamoxifen and paclitaxel. Our study provides a new perspective for targeting PRLR to treat breast cancer.

Neural processing in the primary auditory cortex following cholinergic lesions of the basal forebrain in ferrets.

Cortical acetylcholine (ACh) release has been linked to various cognitive functions, including perceptual learning. We have previously shown that cortical cholinergic innervation is necessary for accurate sound localization in ferrets, as well as for their ability to adapt with training to altered spatial cues. To explore whether these behavioral deficits are associated with changes in the response properties of cortical neurons, we recorded neural activity in the primary auditory cortex (A1) of anesthetized ferrets in which cholinergic inputs had been reduced by making bilateral injections of the immunotoxin ME20.4-SAP in the nucleus basalis (NB) prior to training the animals. The pattern of spontaneous activity of A1 units recorded in the ferrets with cholinergic lesions (NB ACh-) was similar to that in controls, although the proportion of burst-type units was significantly lower. Depletion of ACh also resulted in more synchronous activity in A1. No changes in thresholds, frequency tuning or in the distribution of characteristic frequencies were found in these animals. When tested with normal acoustic inputs, the spatial sensitivity of A1 neurons in the NB ACh- ferrets and the distribution of their preferred interaural level differences also closely resembled those found in control animals, indicating that these properties had not been altered by sound localization training with one ear occluded. Simulating the animals' previous experience with a virtual earplug in one ear reduced the contralateral preference of A1 units in both groups, but caused azimuth sensitivity to change in slightly different ways, which may reflect the modest adaptation observed in the NB ACh- group. These results show that while ACh is required for behavioral adaptation to altered spatial cues, it is not required for maintenance of the spectral and spatial response properties of A1 neurons.

Gliotoxin elicits immunotoxicity in the early innate immune system of ducks.

Gliotoxin (GT) belongs to the epipolythiodioxopiperazine (ETP) family, which is considered a crucial virulence determinant among the secondary metabolites produced by Aspergillus fumigatus. The metabolites are commonly found in food and feed, contributing to the invasion and immune escape of Aspergillus fumigatus, thereby posing a significant threat to the health of livestock, poultry, and humans. Heterophil extracellular traps (HETs), a novel form of innate immune defense, have been documented in the chicken's innate immune systems for capturing and eliminating invading microbes. However, the effects and mechanisms of GT on the production of duck HETs in vitro remain unknown. In this study, we first confirmed the presence of HETs in duck innate immune systems and further investigated the molecular mechanism underlying GT-induced HETs release. Our results demonstrate that GT can trigger typical release of HETs in duck. The structures of GT-induced HETs structures were characterized by DNA decoration, citrullinated histones 3, and elastase. Furthermore, NADPH oxidase, glycolysis, ERK1/2 and p38 signaling pathway were found to regulate GT-induced HETs. In summary, our findings reveal that gliotoxin activates HETs release in the early innate immune system of duck while providing new insights into the immunotoxicity of GT towards ducks.

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.

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

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

Bioanalysis of the Bicycle® toxin conjugate BT5528 and released monomethyl auristatin E via liquid chromatography-tandem mass spectrometry.

Background: The Bicycle® toxin conjugate BT5528 is a novel peptide therapeutic conjugated to the cytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantify BT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a protein precipitation procedure followed by LC-MS/MS detection. Quantitation of MMAE required a selective offline and online solid-phase extraction with detection via LC-MS/MS. Results: BT5528 was quantified over the assay range of 5-2500 ng/ml and free MMAE was quantified over the assay range of 0.05-50 ng/ml. Conclusion: Bioanalytical methods were used in the bioanalysis of intact BT5528 and released MMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.

MHC class III lymphocyte antigens 6 as endogenous immunotoxins: Unlocking immunotherapy in proficient mismatch repair colorectal cancer.

A majority of cancers, including colorectal cancer (CRC) with intact DNA mismatch repair, exhibit a paralyzed antitumor immune response and resistance to immune checkpoint inhibitors. Members of MHC class III lymphocyte antigen 6G (LY6G) encode glycosylphosphatidylinositol (GPI) proteins anchored to the membrane. Snake venom neurotoxins and LY6G proteins share a three-finger (3F) folding domain. LY6 proteins such as LY6G6D are gaining a reputation as excellent tumor-associated antigens that can potently inhibit anti-tumor immunity in cancers with proficient mismatch repair. Thus, we called MHC class III LY6G endogenous immunotoxins. Since the discovery of LY6G6D as a tumor-associated antigen, T-cell engagers (TcEs) have been developed to simultaneously bind LY6G6D on cancer cells and CD3 on T cells, improving the treatment of metastatic solid tumors that are resistant to ICIs. We present a current understanding of how alterations in MHC class III genes inhibit antitumor immunity, and how these understandings can be turned into effective treatments for patients who are refractory to standard immunotherapy. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics Cancer > Molecular and Cellular Physiology.

Engineering Human Pancreatic RNase 1 as an Immunotherapeutic Agent for Cancer Therapy Through Computational and Experimental Studies.

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

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

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

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.

Birinapant selectively enhances immunotoxin-mediated killing of cancer cells conditional on the IAP protein levels within target cells.

Immunotoxins (ITs) target cancer cells via antibody binding to surface antigens followed by internalization and toxin-mediated inhibition of protein synthesis. The fate of cells responding to IT treatment depends on the amount and stability of specific pro-apoptotic and pro-survival proteins. When treated with a pseudomonas exotoxin-based immunotoxin (HB21PE40), the triple-negative breast cancer (TNBC) cell line MDA-MB-468 displayed a notable resistance to toxin-mediated killing compared to the epidermoid carcinoma cell line, A431, despite succumbing to the same level of protein synthesis inhibition. In a combination screen of ~1912 clinically relevant and mechanistically annotated compounds, we identified several agents that greatly enhanced IT-mediated killing of MDA-MB-468 cells while exhibiting only a modest enhancement for A431 cells. Of interest, two Smac mimetics, birinapant and SM164, exhibited this kind of differential enhancement. To investigate the basis for this, we probed cells for the presence of inhibitor of apoptosis (IAP) proteins and monitored their stability after the addition of immunotoxin. We found that high levels of IAPs inhibited immunotoxin-mediated cell death. Further, TNFα levels were not relevant for the combination's efficacy. In tumor xenograft studies, combinations of immunotoxin and birinapant caused complete regressions in MDA-MB-468tumor-bearing mice but not in mice with A431 tumors. We propose that IAPs constitute a barrier to immunotoxin efficacy which can be overcome with combination treatments that include Smac mimetics.

Targeting regulatory T cells by E7777 enhances CD8 T-cell-mediated anti-tumor activity and extends survival benefit of anti-PD-1 in solid tumor models.

Introduction: Regulatory T cell (Treg)-targeting cancer immunotherapy aims to transiently deplete Treg cells in the tumor microenvironment, without affecting effector T cells (Teff), thus both enhancing anti-tumor activity and avoiding autoimmunity. This study evaluated whether adding E7777 (a new formulation of denileukin diftitox [DD]) improved the efficacy of anti-PD-1 antibody therapy. DD is a recombinant protein containing the hydrophobic and catalytic portions of diphtheria toxin fused to full-length human IL-2. E7777 has the same amino acid sequence and brief circulatory half-life as DD, but with greater purity and potency. Methods: Subcutaneous syngeneic murine solid tumor models (colon cancer CT-26 and liver cancer H22) were used to evaluate safety, efficacy, and overall survival with E7777 and anti-PD-1 antibodies, each administered as monotherapy or in concurrent or sequential combination. In Experiment 1, treatments were compared to assess anti-tumor activity at various time points, with tumors excised and dissociated and tumor leukocytes characterized. In Experiment 2, tumor growth, response, and overall survival were characterized for 100 days following a 3-week treatment. Results: E7777 administered in combination with anti-PD-1 led to significantly increased anti-tumor activity and durable, extended overall survival compared to either treatment alone. In both tumor models, the Treg cell infiltration induced by anti-PD-1 treatment was counterbalanced by co-treatment with E7777, suggesting potential synergistic activity. Combination therapy showed the most favorable results. Treatment with E7777 was safe and well-tolerated. Discussion: Combined E7777 and anti-PD-1 therapy was well tolerated and more effective than monotherapy with either drug.

Recombinant immunotoxin induces tumor intrinsic STING signaling against head and neck squamous cell carcinoma.

The innate immune stimulator of interferon genes (STING) pathway is known to activate type I interferons (IFN-I) and participate in generating antitumor immunity. We previously produced hDT806, a recombinant diphtheria immunotoxin, and demonstrated its efficacy against head and neck squamous cell carcinoma (HNSCC). However, it's unknown whether the tumor-intrinsic STING plays a role in the anti-HNSCC effects of hDT806. In this study, we investigated the innate immune modulation of hDT806 on HNSCC. hDT806 significantly upregulated the level of STING and the ratio of p-TBK1/TBK1 in the HNSCC cells. Moreover, intratumoral hDT806 treatment increased the expression of STING-IFN-I signaling proteins including IFNA1, IFNB, CXCL10 and MX1, a marker of IFN-I receptor activity, in the HNSCC xenografts. Overexpression of STING mimicked the hDT806-induced upregulation of the STING-IFN-I signaling and induced apoptosis in the HNSCC cells. In the mouse xenograft models of HNSCC with STING overexpression, we observed a significant suppression of tumor growth and reduced tumor weight with increased apoptosis compared to their control xenograft counterparts without STING overexpression. Collectively, our data revealed that hDT806 may act as a stimulator of tumor-intrinsic STING-IFN-I signaling to inhibit tumor growth in HNSCC.

The D2D3 form of uPAR acts as an immunotoxin and may cause diabetes and kidney disease.

Soluble urokinase plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. In addition to suPAR, proteolysis of membrane-bound uPAR results in circulating D1 and D2D3 proteins. We showed that when exposed to a high-fat diet, transgenic mice expressing D2D3 protein developed progressive kidney disease marked by microalbuminuria, elevated serum creatinine, and glomerular hypertrophy. D2D3 transgenic mice also exhibited insulin-dependent diabetes mellitus evidenced by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic ß cell mass, and high fasting blood glucose. Injection of anti-uPAR antibody restored ß cell mass and function in D2D3 transgenic mice. At the cellular level, the D2D3 protein impaired ß cell proliferation and inhibited the bioenergetics of ß cells, leading to dysregulated cytoskeletal dynamics and subsequent impairment in the maturation and trafficking of insulin granules. D2D3 protein was predominantly detected in the sera of patients with nephropathy and insulin-dependent diabetes mellitus. These sera inhibited glucose-stimulated insulin release from human islets in a D2D3-dependent manner. Our study showed that D2D3 injures the kidney and pancreas and suggests that targeting this protein could provide a therapy for kidney diseases and insulin-dependent diabetes mellitus.

Improving the cytotoxicity of immunotoxins by reducing the affinity of the antibody in acidic pH.

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.

Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia.

Antibody-drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg-1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

Toxin type and domain sequence affect accumulation of recombinant immunotoxins.Transient expression in plant cell packs and intact plants correlates well.IC₅₀ values of toxicity correlate with the cell surface receptor concentration.

Nanobody-Based EGFR-Targeting Immunotoxins for Colorectal Cancer Treatment.

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

A novel EGFR-specific recombinant ricin-panitumumab (scFv) immunotoxin against breast and colorectal cancer cell lines; in silico and in vitro analyses.

The Epidermal Growth Factor Receptor (EGFR) has been of high importance as it is over expressed in a wide diversity of epithelial cancers, promoting cell proliferation and survival pathways. Recombinant immunotoxins (ITs) have emerged as a promising targeted therapy for cancer treatment. In this study, we aimed to investigate the antitumor activity of a novel recombinant immunotoxin designed against EGFR. Using an in silico approach, we confirmed the stability of the RTA-scFv fusion protein. The immunotoxin was successfully cloned and expressed in the pET32a vector, and the purified protein was analyzed by electrophoresis and western blotting. In vitro evaluations were conducted to assess the biological activities of the recombinant proteins (RTA-scFv, RTA, scFv). The novel immunotoxin demonstrated significant anti-proliferative and pro-apoptotic effects against cancer cell lines. The MTT cytotoxicity assay revealed a decrease in cell viability in the treated cancer cell lines. Additionally, Annexin V/Propidium iodide staining followed by flow cytometry analysis showed a significant induction of apoptosis in the cancer cell lines, with half maximal inhibitory concentration (IC50) values of 81.71 nM for MDA-MB-468 and 145.2 nM for HCT116 cells (P < 0.05). Furthermore, the EGFR-specific immunotoxin exhibited non-allergenic properties. The recombinant protein demonstrated high affinity binding to EGFR. Overall, this study presents a promising strategy for the development of recombinant immunotoxins as potential candidates for the treatment of EGFR-expressing cancers.

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.

Crotamine-based recombinant immunotoxin targeting HER2 for enhanced cancer cell specificity and cytotoxicity.

Crotamine, one of the major toxins present in the venom of the South American rattlesnake Crotalus durissus terrificus, exhibits potent cytotoxic properties and has been suggested for cancer therapy applications. However, its selectivity for cancer cells needs to be improved. This study designed and produced a novel recombinant immunotoxin, HER2(scFv)-CRT, composed of crotamine and single-chain Fv (scFv) derived from trastuzumab targeting human epidermal growth factor receptor 2 (HER2). The recombinant immunotoxin was expressed in Escherichia coli and purified using various chromatographic techniques. The cytotoxicity of HER2(scFv)-CRT was assessed in three breast cancer cell lines, demonstrating enhanced specificity and toxicity in HER2-expressing cells. These findings suggest that the crotamine-based recombinant immunotoxin has the potential to expand the repertoire of recombinant immunotoxin applications in cancer therapy.