Recombinant Immunotoxin Research Articles

In silico and in vitro evaluation of a PE38 and Nb-based recombinant immunotoxin targeting the GRP78 receptor in cancer cells.

Cancer is a global health problem despite the most developed therapeutic modalities. The delivery of specific therapeutic agents to a target increases the effectiveness of cancer treatment by reducing side effects and post-treatment issues. Our aim in this study was to design a recombinant protein consisting of nanobody molecules and exotoxin that targets the surface GRP78 receptor on tumor cells. Bioinformatics methods make drug design and recombinant protein evaluation much easier before the laboratory steps. Two constructs were designed from a single-variable domain on heavy chain nanobody domains and PE toxin domains II, Ib, and III. The physicochemical properties, secondary structure, and solubility of the chimeric protein were analyzed using different software. Prostate cancer DU-145 and breast cancer MDA-MB-468 cell lines were used as GRP78-positive and negative controls, respectively. Accordingly, the cytotoxicity, binding affinity, cell internalization, and apoptosis were evaluated using MTT, enzyme-linked immunosorbent assay, and western blot. The results showed that in the DU-145 cell line, the cytotoxicity of two recombinant immunotoxins is dose and time-dependent. In MDA-MB-468 and HEK-293 cells, such an event does not occur. It is possible that two constructs designed for immunotoxins can attach to GRP78-positive cancer cells and then eradicate cancer cells by internalization and apoptosis. As our in vitro results were in line with in silico data confirming the Bioinformatics predictions, it can be concluded that the designed recombinant immunotoxins may exhibit therapeutic potential against GRP78-positive tumor cells.

Humanized recombinant immunotoxin targeting hCG demonstrates therapeutic potential for advanced stage difficult to treat cancers

We report the development of an immunotherapeutic molecule, a Humanized immunotoxin, for treating hCG-expressing advanced-stage cancers. PiPP, a high-affinity anti-hCG monoclonal antibody, is used in the immunotoxin for ‘homing’ hCG-positive cancer cells. The deimmunized (DI) form of α-Sarcin, a fungal-origin toxin that lacks functional T-cell epitopes, is used in the design to ensure minimal immunogenicity of the immunotoxin for repetitive use in humans. A single-chain variable fragment (scFv) of PiPP was constructed by linking the Humanized VH and VL regions of the antibody. The scFv part of the antibody was further linked to the toxin α-Sarcin (DI) at the gene level and expressed as a recombinant protein in E. coli. The immunotoxin was purified from the bacterial cell lysate and analysed for binding and cytotoxicity to hCG-secreting colorectal and pancreatic cancer cells. The results showed that the scFv(PiPP)-Sarcin immunotoxin was able to bind to colorectal and pancreatic cancer cells and killed approximately 85% of the cells. In vivo testing of the immunotoxin produced results similar to those of in vitro testing against colorectal adenocarcinoma-induced tumours. This immunotoxin could be a promising immunotherapeutic agent for treating colorectal, pancreatic and other terminal-stage hCG-expressing cancers.

A novel shark VNAR antibody-based immunotoxin targeting TROP-2 for cancer therapy

TROP-2, a tumor-associated antigen, has been implicated in the progression of various epithelial tumors. Due to its favorable expression profile, TROP-2 has emerged as a promising target for antibody–drug conjugates (ADCs) based anti-tumor therapies. Although ADCs have shown efficacy in cancer treatment, their application in solid tumors is hindered by their high molecular weight, poor tumor penetration, and release of cytotoxic molecules. Therefore, a recombinant immunotoxin was developed based on a shark-derived variable domain of immunoglobulin new antigen receptor (VNAR) antibody. VNARs are only one-tenth the size of IgG antibodies and possess remarkable tissue penetration capabilities and high stability. In this study, a shark VNAR phage display library was created, leading to the identification of shark VNAR-5G8 that targets TROP-2. VNAR-5G8 exhibited a high affinity and cellular internalization ability towards cells expressing high levels of TROP-2. Epitope analysis revealed that VNAR-5G8 recognizes a hidden epitope consisting of CRD and TY-1 on TROP-2. Subsequently, VNAR-5G8 was fused with a truncated form of Pseudomonas exotoxin (PE38) to create the recombinant immunotoxin (5G8-PE38), which exhibited significant anti-tumor activity in vitro and in vivo. Overall, this study highlights the promise of 5G8-PE38 as a valuable candidate for cancer therapy.

Effect of rituximab on remissions without minimal residual disease and immunogenicity in patients with relapsed/refractory hairy cell leukemia receiving moxetumomab pasudotox.

e19015 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) was FDA-approved for relapsed/refractory hairy cell leukemia (HCL) but is now unavailable due to vial expiration until a company resumes development. The phase 3 complete remission (CR) rate was 41%, higher in patients with lower anti-drug antibodies (ADA). Minimal residual disease (MRD) eradication led to longer CR duration. HCL and the poorer prognosis variant HCLv strongly express CD22 and CD20. A clinical trial was done to determine if Rituximab could decrease immunogenicity by killing normal B-cells in HCL patients receiving Moxe and hasten MRD-free CR by reducing HCL tumor burden. Methods: To permit Rituximab enough time to reduce ADA and tumor burden, it was administered 3 days before cycle 1 day 1 at 375 mg/m2, and Moxe was given by 30-minute infusion days 1, 3 and 5. On subsequent 28-day cycles, patients received Moxe days 1, 3 and 5, and Rituximab prior to Moxe on day 1. Patients received up to 4 cycles past MRD-free CR, or up to 8 total cycles if MRD-free CR was achieved after cycle 4. The ADA assay determined percent neutralization by serum of the cytotoxicity of Moxe on CD22+ Raji cells. Results: After 13 patients received Moxe-Rituximab (MoxeR) without dose-limiting toxicity (DLT), meeting the phase 1 endpoint, 5 additional patients received Moxe with the biosimilar Ruxience (MoxeR). The first 3 patients received Moxe at 30 mcg/Kg/dose and subsequent patients 40 mcg/Kg/dose. All 18 were evaluable for toxicity and response. Although no DLT, one patient had transient grade 3 hemolytic uremic syndrome during cycle 3 without significant symptoms or need for therapy. Of the 18 patients, 15 (83%) responded, 14 (78%) achieved CR and 13 (72%) MRD-free CR by blood and bone marrow (BM) aspirate flow cytometry and BM biopsy immunohistochemistry. MRD-free CRs included one with HCLv. Even though Moxe vial expiration prevented enrollment of the 26 planned patients needed for a 1-sided p-value <0.025 compared to Moxe alone where 30 (47%) of 64 phase 1-3 patients achieved MRD-free CR, the 53% relative improvement with MoxeR achieved a 1-sided p-value of 0.05. Compared to 29 (48%) of 61 evaluable phase 1-3 patients who received Moxe alone with high ADA levels, 4 (29%) of 18 patients had high ADA levels to MoxeR (p=0.048). At 14-51 (median 35.3) months of follow-up, all but 2 of 13 MRD-free CRs are continuing, with relapse-free survival 14-45 (median 34.5) months. Conclusions: Despite enrolling slightly fewer patients than planned, MoxeR was safe and more effective than Moxe alone at achieving MRD-free CR, probably due to lower immunogenicity and faster reduction of HCL/HCLv tumor burden. Since non-Hodgkin’s lymphoma (NHL) cells from patients are sensitive to Moxe like HCL, MoxeR could be tested after NHL treatment to convert MRD+ to MRD-free CRs. Clinical trial information: NCT03805932 .

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.

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. 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. 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. 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.

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.

Tofacitinib to prevent anti-drug antibody formation against LMB-100 immunotoxin in patients with advanced mesothelin-expressing cancers.

LMB-100 is a mesothelin (MSLN)-targeting recombinant immunotoxin (iTox) carrying a Pseudomonas exotoxin A payload that has shown promise against solid tumors, however, efficacy is limited by the development of neutralizing anti-drug antibodies (ADAs). Tofacitinib is an oral Janus Kinase (JAK) inhibitor that prevented ADA formation against iTox in preclinical studies. A phase 1 trial testing LMB-100 and tofacitinib in patients with MSLN-expressing cancers (pancreatic adenocarcinoma, n=13; cholangiocarcinoma, n=1; appendiceal carcinoma, n=1; cystadenocarcinoma, n=1) was performed to assess safety and to determine if tofacitinib impacted ADA formation. Participants were treated for up to 3 cycles with LMB-100 as a 30-minute infusion on days 4, 6, and 8 at two dose levels (100 and 140 µg/kg) while oral tofacitinib was administered for the first 10 days of the cycle (10 mg BID). Peripheral blood was collected for analysis of ADA levels, serum cytokines and circulating immune subsets. The study was closed early due to occurrence of drug-induced pericarditis in 2 patients. Pericarditis with the combination was not reproducible in a transgenic murine model containing human MSLN. Two of 4 patients receiving all 3 cycles of treatment maintained effective LMB-100 levels, an unusual occurrence. Sustained increases in systemic IL-10 and TNF-α were seen, a phenomenon not observed in prior LMB-100 studies. A decrease in activated T cell subsets and an increase in circulating immunosuppressive myeloid populations occurred. No radiologic decreases in tumor volume were observed. Further testing of tofacitinib to prevent ADA formation is recommended in applicable non-malignant disease settings. https://www.clinicaltrials.gov/study/NCT04034238.

The potential use of recombinant immunotoxin and BCL-2 inhibitors for leukemic stem cell elimination

Leukemia is a type of hematological malignancy caused by the dysfunctional proliferation of leukocyte cells. According to the Global Burden of Disease 2019 report, there were over 643,579 new cases of leukemia cancer worldwide in 2020, killing 334,592 people. Chemotherapy, an important part of the treatment of leukemia, not only harm cancer cells but also harm healthy cells, which can lead to negative side effects. This article examines the possibility for cutting-edge therapy using targeted recombinant immunotoxins and BCL-2 inhibitors as targeted therapy against leukemic stem cells. Recombinant immunotoxin is a protein-based therapeutic agent composed of toxins combined with specific antibodies or other molecules such as growth factors, cytokines, or toxins. Diphtheria Toxin (DT) is the most commonly used toxin because it is easily expressed, making the toxin easier to take. The toxin kills cancer cells by inhibiting enzymatic cell protein synthesis. While immunotoxins enhance therapeutic outcomes by targeting leukemia stem cells, their efficacy can be compromised in certain patients due to apoptosis resistance mediated by proteins like BCL-2. The presence of anti-apoptotic protein BCL-2 enables cell survival even after exposure to immunotoxins. Consequently, combining recombinant immunotoxins and BCL-2 inhibitors holds promise for eradicating LSC and preventing relapse.

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.

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.

Recombinant anti-human epidermal growth factor receptor type 2 single-chain variable fragment-alpha-luffin fusion protein as a putative immunotoxin against human epidermal growth factor receptor type 2-positive breast cancer cells: an experimental research.

Breast cancer is one of the most frequent causes of cancer death in women. The application of immunotoxins to target overexpressed biomarkers on the surface of cancer cells and delivery of the toxin molecules into these cells has attracted too much attention during the last decade. This study was conducted to investigate the possible in-vitro cytotoxic and apoptotic activity of previously designed recombinant immunotoxin compromising anti-HER2 single-chain variable fragment (scFv) and alpha-luffin protein in human epidermal growth factor receptor type 2 (HER2)-positive and HER2-negative breast cancer cell lines. The previously designed recombinant immunotoxin and alpha-luffin protein were expressed in E. coli host cells and purified using Ni-affinity chromatography. The cytotoxicity of the proteins was tested through MTT and apoptosis studies on HER2-positive and HER2-negative breast cancer cell lines. Treatment of SKBR3 and MDA-MB-468 cells with the immunotoxin caused differential cytotoxicity and apoptotic events. Flow cytometry analysis indicated that the immunotoxin could arrest SKBR3 cells at the G0/G1 phase and induce apoptosis and cell death which were not observed in HER2-negative MDA-MB-468 cells. Annexin V/PI staining revealed late apoptotic events in SKBR3 cells treated with the immunotoxin which was different from the early apoptosis induced by the alpha-luffin protein alone. This immunotoxin could be a promising tool in developing new targeted therapeutic agents against HER2-positive cancer cells. Animal experiments are needed before making firmed conclusions.

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

ABSTRACTAntibody–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.

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.

Analysis of systemic immune changes in patients with advanced cancer treated with immunotoxin LMB-100 and tofacitinib.

e15017 Background: LMB-100 recombinant immunotoxin delivers a modified Pseudomonas exotoxin A payload to mesothelin-expressing tumors. Almost all patients receiving LMB-100 develop anti-drug antibodies (ADAs) after 2 cycles/ 6 doses of the drug, therefore reducing peak serum drug concentrations to near the lower limit of detection. Tofacitinib is an oral JAK inhibitor that had been shown to prevent ADA formation in pre-clinical studies. Apart from assessing safety and tolerability, this trial sought to determine whether co-administration of tofacitinib with LMB-100 delays the formation of neutralizing ADAs so more patients could achieve the peak LMB-100 concentration of &gt; 600 ng/mL during treatment cycle 2. Correlative data examining circulating immune cell subsets and cytokines pre- and on-treatment are presented here. Methods: Patients with pancreatic adenocarcinoma (n = 13) and other mesothelin-expressing solid tumors (n = 3; cholangiocarcinoma, appendix, cystadenocarcinoma) were treated for up to 3 cycles (21 days) with LMB-100 given on days 4, 6, and 8 at 2 dose levels (100 and 140 mcg/kg), accompanied by oral tofacitinib for the first 10 days of the cycle (10 mg BID). Adverse events were assessed as per CTCAE 5.0. Analysis of the cytokine data was performed using a Wilcoxon signed-rank test. Results: DLTs of grade 3 cardiac toxicity (n = 1) and grade 4 hyponatremia (n = 1) were seen at the 140mcg/kg dose of LMB-100. One patient treated at 100 mcg/kg developed a grade 4 pericardial effusion, resulting in early closure of the study for safety. The two patients who experienced cardiac toxicity had lower baseline CD4+ cells and increased CD19+ cells post-LMB-100 treatment. Four out of 8 evaluable patients achieved LMB-100 concentration above the threshold during cycle 2, and 2 out of 3 did so during cycle 3. Analysis of peripheral blood cytokine levels showed sustained increases in TNF-α, IL-8, and IL-10 following LMB-100 administration through to the start of cycle 2. Percentage of Tregs increased through cycle 1 in patients who developed drug-neutralizing ADAs and was significantly higher than in patients with LMB-100 levels above threshold at the start of cycle 2. Conclusions: Combination of LMB-100 with tofacitinib is not safe. An insufficient number of patients was treated to conclusively assess the ability of tofacitinib to stop or delay ADA formation. Increases in circulating Tregs were observed in patients with early neutralizing ADA formation. Clinical trial information: NCT04034238 .

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.

In silico and experimental methods for designing a potent anticancer arazyme-herceptin fusion protein in HER2-positive breast cancer

Breast cancer is the most prevalent type of malignancies among women worldwide and is associated with serious physical and mental consequences. Current chemotherapies may lack successful outcomes; thus, the development of targeted recombinant immunotoxins is plausible. The predicted B cell and T cell epitopes of arazyme of the fusion protein are able to elicit immune response. The results of codon adaptation tool of herceptin-arazyme have improved from 0.4 to 1. The in silico immune simulation results showed significant response for immune cells. In conclusion, our findings show that the known multi-epitope fusion protein may activate humoral and cellular immune responses and maybe a possible candidate for breast cancer treatment. In this study, the selected monoclonal antibody constituting herceptin and the bacterial metalloprotease, arazyme, was used with different peptide linkers to design a novel fusion protein to predict different B cell and T cell epitopes by the means of the relevant databases. Modeler 10.1 and I-TASSER online server were used to predict and validate the 3D structure and then docked to HER2-receptor using HADDOCK2.4 web server. The molecular dynamics (MD) simulations of the arazyme-linker-herceptin-HER2 complex were performed by GROMACS 2019.6 software. The sequence of arazyme-herceptin was optimized for the expression in prokaryotic host using online servers and cloned into pET-28a plasmid. The recombinant pET28a was transferred into the Escherichia coli BL21DE3. Expression and binding affinity of arazyme-herceptin and arazyme to human breast cancer cell lines (SK-BR-3/HER2 + and MDA-MB-468/HER2 -) were validated by the SDS-PAGE and cell‑ELISA, respectively.

Abstract 6344: Recombinant Immunotoxin exhibited targeted killing of Trop2 overexpressing tumour cells lines

Abstract Introduction: The high prevalence of cervical cancer remains a global challenge particularly in developing countries where its therapy is principally by chemoradiation marked by severe toxicities. Antibody-based therapy targeting specific overexpressed surface antigen has proven as a promising strategy. Tumor-associated calcium signal transducer 2 (Trop2), also known as epithelial glycoprotein-1 antigen, is a membrane protein found in epithelial tissues involved in cellular signalling, migration, proliferation, and differentiation in normal cells. Its overexpression is validated in several tumor types including basal, squamous and adenocarcinomas of the cervix, breast, colon, and gastrointestinal tracts. Due to its differential overexpression, Trop2 is a viable therapeutic marker whereby a recombinant immunotoxin composed of a single chain variable antibody fragment (scFv) armed with Pseudomonas exotoxin A (Anti-Trop2-ETA) may be delivered for targeted killing of such tumors. Methods: Plasmids encoding Anti-Trop2-ETA were designed in silico by fusion of anti-Trop2 single chain variable fragment (scFv) antibody to the N-terminus of Pseudomonas exotoxin A and then cloned into an pMT backbone. Expression was performed in BL21 E. Coli cells under osmotic stress and then Anti-Trop2-ETA was purified by IMAC via an N-terminus encoded polyhistidine tag. Purified fractions were structurally characterized by SDS PAGE gel and immunoblotting to confirm the presence of the Anti-Trop2-ETA protein to be used for treatment of tumor cell lines. Trop2 expression cervical cancer and breast tumor cell lines were confirmed, and cells were treated with Anti-Trop2-ETA. Results: Dose dependent killing by Anti-Trop2-ETA was observed in the Trop2 expressing tumor cell lines. IC50 values were significantly lower for Trop2 expressing cells as compared to control cells lacking Trop2 expression. Conclusion: Here, we demonstrated antigen specific killing of Trop2 overexpressing tumor cells in vitro. Targeted deployment of recombinant immunotoxin to specifically engage and kill Trop2 positive cells is a potential approach for cancer therapeutics. As such, the ability of the rIT to specifically target and kill Trop2 expressing tumors is an important initial finding towards the possibility of antigen-dependent elimination of Trop2 expressing cancer cells. Ongoing evaluations are aimed at antigen dependent biological activity as well as pharmacokinetics of the rIT in tumor bearing in vivo models. Citation Format: Dennis Makafui Dogbey, Sizalobuhle Masuku, Stefan Barth. Recombinant Immunotoxin exhibited targeted killing of Trop2 overexpressing tumour cells lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6344.

Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria.

Bacterial cancer therapy is a promising next-generation modality to treat cancer that often uses tumor-colonizing bacteria to deliver cytotoxic anticancer proteins. However, the expression of cytotoxic anticancer proteins in bacteria that accumulate in the nontumoral reticuloendothelial system (RES), mainly the liver and spleen, is considered detrimental. This study examined the fate of the Escherichia coli strain MG1655 and an attenuated strain of Salmonella enterica serovar Gallinarum (S. Gallinarum) with defective ppGpp synthesis after intravenous injection into tumor-bearing mice (~108 colony forming units/animal). Approximately 10% of the injected bacteria were detected initially in the RES, whereas approximately 0.01% were in tumor tissues. The bacteria in the tumor tissue proliferated vigorously to up to 109 colony forming units/g tissue, whereas those in the RES died off. RNA analysis revealed that tumor-associated E. coli activated rrnB operon genes encoding the rRNA building block of ribosome needed most during the exponential stage of growth, whereas those in the RES expressed substantially decreased levels of this gene and were cleared soon presumably by innate immune systems. Based on this finding, we engineered ΔppGpp S. Gallinarum to express constitutively a recombinant immunotoxin comprising TGFα and the Pseudomonas exotoxin A (PE38) using a constitutive exponential phase promoter, the ribosomal RNA promoter rrnB P1. The construct exerted anticancer effects on mice grafted with mouse colon (CT26) or breast (4T1) tumor cells without any notable adverse effects, suggesting that constitutive expression of cytotoxic anticancer protein from rrnB P1 occurred only in tumor tissue.

Abstract 2348: Investigating the role of furin in the intoxication pathway of recombinant immunotoxins, based on Pseudomonas exotoxin A

Abstract 2348: Investigating the role of furin in the intoxication pathway of recombinant immunotoxins, based on Pseudomonas exotoxin A

Abstract PR005: Intraductal administration of a recombinant transferrin receptor-directed immunotoxin clears ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer

Abstract Background: For DCIS, a preneoplastic lesion with a 15-50% chance of recurrence or progression, there is a reappraisal of the necessity of aggressive treatment with its attendant toxicities. HB21(Fv)-PE40, a conjugate of an Fv fragment of the human transferrin-receptor (TFR) mAb and a 40 kDa fragment of bacterial Pseudomonas exotoxin (PE), has potent tumor cell killing activity. HB21(Fv)-PE40 binds to abundantly expressed TFR on cancer cells; the molecule is internalized, processed, and trafficked to the cytosol. Inside the cell, PE-toxin catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor 2 (EF2), inactivates EF2, and leads to an arrest in protein translation and cell death by apoptosis. However, due to its abundant expression in the liver, the recombinant immunotoxin (RIT) is potentially toxic. We hypothesized that RIT administration via the mammary ductal opening at the teat will destroy preneoplasias, both visible and occult, in the entire ductal tree, while systemic effects will be minimal. The intraductal approach could provide a direct, safe, and highly effective treatment for DCIS. Methods: We tested this concept in two models of DCIS using an ER/PR-, HER2+ cell line derived from a DCIS, SUM225-Luc, and in the ER/PR+, HER2- breast cancer cell line, MCF-7-Luc. In both cell lines, the IC50 of the toxin was in the nM range. DCIS developed within one week in the MIND models following i.duc injection of tumor cells in the 4th pair of mammary glands of female NSG mice. On day 7, the tumor-bearing mice received vehicle, TFR mAb alone, or the HB21(Fv)-PE40 conjugate by the i.duc or i.v route three times at weekly intervals and were observed for 1-6 months. Within two weeks of the treatment, IVIS imaging signals were undetectable in the mammary gland treated with RIT i.duc in both the models (P&amp;lt;0.0001). Mammary gland whole mount analysis, histopathology, and immunohistochemistry for Ki67, Ku80, TFR and CD31 showed that i.duc toxin rendered more than 90% of the mice tumor-free. Pharmacokinetics studies were conducted in mice with the RIT administered i.v. or i.duc. RIT was undetectable by TFR-ELISA in the blood of i.duc-treated mice. Conclusions. Overall, we have shown that HB21(Fv)-PE40 has remarkable antitumor activity by i.duc administration in two MIND models of DCIS. RIT was undetectable in blood after i.duc injection by ELISA, suggesting that the toxin remains inside the ductal system during its 30 min half-life. This preclinical study provides a solid foundation for a clinical study. Dose escalation studies of HB21(Fv)-PE40 by the i.duc route could be performed in patients with DCIS and early breast cancer who will undergo mastectomy as part of their treatment regimen. This study design will allow a detailed examination of the local (breast) and systemic effects of i.duc HB21(Fv)-PE40. It will introduce a potent agent for DCIS treatment and cancer interception in women at high risk of developing breast cancer. The contribution of the immune system remains to be explored. Citation Format: Saraswati Sukumar. Intraductal administration of a recombinant transferrin receptor-directed immunotoxin clears ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR005.