Recombinant Immunotoxin Research Articles

Design of a recombinant immunotoxin against the human granulocyte-colony stimulating factor receptor.

Immunotoxin is a new strategy for protein therapy of cancer. This engineered protein contains two parts, the immune part which is an antibody or cytokine, directed against the cancer cell receptor, and the toxin part consisting of a plant or bacterial toxin leading to apoptosis by protein synthesis inhibition. The knowledge of cell-surface receptor overexpression in cancer cells can help scientists to construct new anti-cancer agents. The granulocyte colony stimulating factor (G-CSF) receptor is expressed on the cell surface of some blood cancers such as acute myeloid leukemia (AML). Therefore, this receptor can be used as an immunotoxin for treatment of some cancers. The aim of this work was to design and produce DT-GCSF immunotoxin using truncated DT fused to G-CSF. For fusion protein construction, DT389 and G-CSF fragments, were amplified by PCR using specific primers. A flexible linker SerGly4SerMet (SG4SM) was used to fuse the PCR products by SOEing PCR procedure to achieve an appropriate fusion protein, and the fused fragment was subcloned into pET21b. The new construction (pET-DT389GCSF) was transformed into E. coli strain BL21 (DE3) and the expression of the construction was confirmed by SDS-PAGE and Western blotting techniques. The data demonstrated the expression and purity rates of DT389GCSF about 25% and 90%, respectively. This chimeric protein construction can be used as a new anti-AML drug, but its in vitro and in vivo biological activity should be analyzed.

Reduction of non-specific toxicity of immunotoxin by intein mediated reconstitution on target cells

Recombinant immunotoxins are chimeric proteins composed of a targeting peptide that binds to a specific tumor antigen and a toxin protein killing target cells. Recombinant immunotoxin exhibits potent cancer inhibiting effects both in vivo and in vitro. However, the non-specific toxicity causes severe syndromes limiting their clinical application. To reduce toxicity caused by recombinant immunotoxins in general, we divided an immunotoxin into two nontoxic segments that may restore toxic bioactivity on tumor cell surface based on the intein mediated trans-splicing reaction. Both split and reconstituted immunotoxins were tested for their biological activities. We found that the reconstituted immunotoxin retained antigen specificity and affinity toward cancer cells overexpressing HER2/neu. After being internalized into HER2/neu positive cells, the reconstituted immunotoxin showed comparable cytotoxicity as the original immunotoxin, while the split immunotoxin fragments showed no toxic activity to cells with or without HER2/neu expression. This approach can potentially be used under clinical settings to reduce non-specific toxicity by administering patients with inactive immunotoxin fragments. Cytotoxic effect only occurs at tumor sites where the inactive fragments bind, trans-splice and become active toxin.

Molecular Remissions with Anti-CD22 Recombinant Immunotoxin Moxetumomab Pasudotox Are Associated with Improved Complete Remission Durations during Phase I and III Testing

BACKGROUNDHairy cell leukemia (HCL) is an indolent B-cell malignancy strongly expressing CD22, with high sensitivity to purine analog chemotherapy, but relapses frequently occur, associated with minimal residual disease (MRD). Targeted non-chemotherapeutic approaches have shown efficacy but leave MRD. Moxetumomab pasudotox is an anti-CD22 recombinant immunotoxin recently reported in relapsed/refractory HCL to achieve 57% and 51% complete remission (CR) rates in phase 1 and 3 testing, respectively. Eradication of MRD by bone marrow flow cytometry or immunohistochemistry was associated with improved CR duration. We have previously reported that Taqman real-time quantitative PCR (RQPCR), which amplifies patient- and HCL-specific B-cell immunoglobulin rearrangements (IGHV), can detect one HCL cell in 106 normal cells. In the present study we determined whether MRD eradication measured by RQPCR is associated with improved CR duration. METHODSA total of 28 patients achieving CR to moxetumomab pasudotox in phase 1 (n=18) and phase 3 (n=10) trials were studied at the National Institutes of Health by multicolor flow cytometry of the bone marrow aspirate (BMA) and blood. Patients received moxetumomab pasudotox at 40-50 mcg/Kg on days 1, 3 and 5 of 2-7 cycles (median 5) at 4-week intervals. Blood and bone marrow samples were obtained to determine CR. In addition to end of treatment, bone marrow samples for MRD were generally obtained every 6-12 months for 2.5 years, then every other year. Blood flow cytometry was obtained every 6 months for 2.5 years, then yearly. Follow-up (time since treatment initiation) was 26.9-121.8 (median 84) months. For this analysis, CR for each bone marrow time point was determined by standard criteria for all patients independent of specific data-reporting requirements and data cut-off time points. RQPCR testing for each patient required cloning the immunoglobulin heavy chain variable regions and designing sequence-specific primers and probes. RNA samples were made from PAXgene tubes of blood and sodium-heparin tubes of BMA. RESULTSWe found that BMA RQPCR was most sensitive, followed by BMA flow cytometry, blood RQPCR, and blood flow cytometry. Of the 28 patients studied, 12 (43%) achieved MRD-free CR by all 4 tests and 2 of 12 patients (17%) relapsed. Seven (25%) were MRD+ by BMA RQPCR but negative by the other 3 studies, and none relapsed. One patient RQPCR+ and flow-negative in both blood and BMA relapsed. Seven patients flow-negative but RQPCR+ in blood, flow+ in BMA, and either BMA RQPCR+ or RQPCR not done, all relapsed. One patient with BMA RQPCR not done and the other 3 studies positive relapsed. With respect to RQPCR of blood as a single test, 3 of 20 negative vs 8 of 8 positive patients relapsed (p<0.001). Median CR duration was 13.0 months in blood RQPCR+ patients, vs not reached in blood RQPCR-negative patients, 17 (85%) of whom remain in CR at 11.6-112.9 (median 53) months of follow-up. With respect to BMA status, 2 of 12 patients negative vs 9 of 16 patients positive by either flow or RQPCR relapsed (p=0.054), and the 12 BMA MRD-negative patients had longer CR duration than the other 16 patients (p=0.018). CONCLUSIONSRQPCR negativity of blood is strongly associated with prolonged CR duration after moxetumomab pasudotox and may be a useful measure of MRD when bone marrow studies cannot be done. Additional testing will be needed to determine if blood RQPCR can be used to determine the optimal number of cycles of immunotoxin to administer. BMA RQPCR, though more sensitive than blood RQPCR, was not as useful due to fewer patients being evaluable and more time needed for BMA-RQPCR+ patients to relapse. Additional testing and follow-up may determine if BMA RQPCR negativity will correlate significantly with CR duration. [Display omitted] DisclosuresYao:MedImmune: Employment. Marshall:MedImmume: Employment. Pastan:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox. Kreitman:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox.

ATIM-36. DOSE ESCALATION TRIAL OF D2C7 IMMUNOTOXIN (D2C7-IT) ADMINISTERED INTRATUMORALLY VIA CONVECTION-ENHANCED DELIVERY (CED) FOR RECURRENT MALIGNANT GLIOMA (MG)

D2C7-IT is a recombinant immunotoxin comprised of a dual-specific antibody fragment targeting EGFRwt and EGFRvIII and a genetically engineered form of the Pseudomonas exotoxin, PE38-KDEL. We report results of a phase 1 trial evaluating D2C7-IT delivered intratumorally by CED. Eligible patients are adults with recurrent supratentorial WHO grade III or IV MG; solitary tumor; ≥4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; and KPS>70%. Planned enrollment of two patients per dose level (DL). As of 5/29/2018, 43 patients have been treated (2 each per DL, but for DLs 3, 6, 9, and 16). Observed dose limiting toxicities include: grade 4 seizure (n=1) on DL3, grade 3 confusion and pyramidal tract syndrome (n=1) on DL13, and grade 4 cerebral edema and grade 3 dysphasia (n=2) on DL17. Grade 2 or higher adverse events possibly related to D2C7-IT include: seizure (grade 4, n=2, grade 3, n=2, grade 2, n=4), cerebral edema (grade 4, n=1), headache (grade 3, n=4; grade 2, n=18), hemiparesis (grade 3, n=4, grade 2, n=10), dysphasia (grade 3, n=2; grade 2, n=9), confusion (grade 3, n=1; grade 2, n=5), thromboembolic event (grade 3, n=2; grade 2, n=1), fatigue (grade 2, n=6), visual field cut (grade 2, n=3), paresthesia (grade 2, n=2), stroke (grade 2, n=2), and hemineglect (grade 2, n=2); one each of grade 3 elevated ALT, urinary tract infection, fall, generalized muscle weakness, encephalopathy, and somnolence; one each of grade 2 elevated AST, papilledema, gait disturbance, intracranial hemorrhage, and urinary incontinence. Fifteen patients are alive. Two patients have partial response and are alive ≥8.2 months and ≥34 months after infusion. CONCLUSION: DL17 is above the maximal tolerated dose. Encouraging efficacy results were observed. After review of efficacy and safety data, DL13 seems the optimal dose, additional patients will be treated on DL13.

Expression and Evaluation of HuscFv Antibody -PE40 Immunotoxin for Target Therapy of EGFR-Overexpressing Cancers.

BackgroundEpidermal growth factor receptor (EGFR) plays an important role in the progression and tumorigenesis of the various cancers. In this regards, anti-EGFR antibodies are valuable approved therapeutics for the EGFR over-expressing cancers. However, the occurrence of mutations in the EGFR and/or KRAS genes; a common phenomenon which is seen in many cancers, lead to the resistance to the EGFR-directed antibodies. EGFR based immunotoxins are capable of overcoming this limitation by directing the toxin moieties to the cancer cells resulting in cell death.ObjectivesIn the present study, a novel immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE-40) and anti-EGFR huscFv was developed and evaluated for the induction of cell death in EGFR positive A431tumoral cells.Materials and MethodsPE-40 fragment of the exotoxin A was amplified by using PCR and ligated to pET22b-huscFv. The reaction was confirmed by PCR and restriction digestion. The immunotoxin was expressed in E. coli BL21 (plysS) and then was purified by Ni-NTA affinity column. Subsequently, the toxicity of the purified immunotoxin was evaluated on EGFR over-expressing epidermoid carcinoma of skin, A431 cell line.ResultsPCR and restriction digestion experiments have verified the integrity of the immunotoxin construct. Purification by affinity column resulted in a highly purified recombinant immunotoxin. MTT assay revealed the growth inhibitory effect of the huscFv-PE40 immunotoxin on EGFR-over-expressing A431 cells with an IC50 value of 250 ng.mL-1.ConclusionIn conclusion, the results indicated that the immunotoxin developed in this study has a high toxicity on the EGFR-over-expressing tumor cells and could be considered as a promising candidate for the treatment of the EGFR positive cancers.

Protein Synthesis Inhibition Activity of Mesothelin Targeting Immunotoxin LMB-100 Decreases Concentrations of Oncogenic Signaling Molecules and Secreted Growth Factors.

LMB-100 is a mesothelin-targeted recombinant immunotoxin (iTox) that carries a modified Pseuodomonas exotoxin A (PE) payload. PE kills cells by inhibiting synthesis of new proteins. We found that treatment of pancreatic cancer cells with LMB-100 for 24–48 h did not change total protein level despite inducing protein synthesis inhibition (PSI). Further, increased levels of ubiquitinated proteins were detected, indicating that cells may have limited ability to compensate for PSI by reducing protein degradation. Together, these data suggest that PE depletes concentrations of a minority of cellular proteins. We used reverse phase protein array and Luminex assay to characterize this subset. LMB-100 decreased the abundance of 24 of 32 cancer-related proteins (including Bcl-x, Her2, Her3 and MUC16) without compensatory increases in other analytes. Further, cancer cells failed to maintain extracellular concentrations of cancer cell secreted growth factors (CCSGFs), including Vascular Endothelial Growth Factor (VEGF) following treatment with cytostatic LMB-100 doses both in culture and in mouse tumors. Decreased VEGF concentration did not change tumor vasculature density, however, LMB-100 caused tissue-specific changes in concentrations of secreted factors made by non-cancer cells. In summary, our data indicate that PSI caused by cytostatic LMB-100 doses preferentially depletes short-lived proteins such as oncogenic signaling molecules and CCSGFs.

Moxetumomab Pasudotox: First Global Approval.

Moxetumomab pasudotox-tdfk (LUMOXITI™), an anti CD22 recombinant immunotoxin, has been developed by MedImmune and its parent company AstraZeneca for the treatment of hairy cell leukaemia. The product, discovered at the National Cancer Institute, is an optimised version of immunotoxin CAT-3888. Moxetumomab pasudotox is composed of the Fv fragment of an anti-CD22 monoclonal antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A, PE38. The Fv portion of moxetumomab pasudotox binds to CD22, a cell surface receptor expressed on a variety of malignant B-cells, thereby delivering the toxin moiety PE38 directly to tumour cells. Once internalised, PE38 catalyses the ADP ribosylation of the diphthamide residue in elongation factor-2 (EF-2), resulting in the rapid fall in levels of the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1), leading to apoptotic cell death. This article summarizes the milestones in the development of moxetumomab pasudotox leading to this first approval for the treatment of adults with relapsed or refractory hairy cell leukaemia who received at least two prior systemic therapies, including treatment with a purine nucleoside analogue. Development of moxetumomab pasudotox for non-Hodgkin’s lymphoma, chronic lymphocytic leukaemia and precursor cell lymphoblastic leukaemia/lymphoma was discontinued.

Anti-drug antibodies to LMB-100 are enhanced by mAbs targeting OX40 and CTLA4 but not by mAbs targeting PD1 or PDL-1

LMB-100 is a recombinant immunotoxin being developed for cancer treatment that is composed of a Fab that binds to mesothelin and a portion of Pseudomonas exotoxin A. LMB-100 is in clinical trials for the treatment of mesothelioma and pancreatic cancer. To determine if check point modulating antibodies enhance the formation of anti-drug antibodies (ADA) against LMB-100, we treated mice with LMB-100 and four different immune modulating monoclonal antibodies that have different mechanisms of action; anti-CTLA4, anti-OX40, anti-PD-1 and anti-PDL-1. We found that anti-PD-1 and anti PDL-1 do not increase the formation of ADA, but anti-CTLA-4 and anti-OX-40 do increase the onset of ADA. These results indicate that combining anti-CTLA-4 and anti-OX-40 with antibodies and other protein-based therapeutics may enhance ADA formation in humans.

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

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

Preclinical development of anti-BCMA immunotoxins targeting multiple myeloma.

Multiple myeloma (MM) is a B-cell malignancy that is incurable for the majority of patients. New treatments are urgently needed. Recombinant immunotoxins (RITs) are chimeric proteins that are composed of the Fv or Fab portion of an antibody fused to a bacterial toxin. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein and an ideal target for antibody-based treatments for MM. RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in Escherichia coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays. We also measured their binding to human and mouse serum albumins and to BCMA and measured their serum half-life in mice. Using Fvs from different anti-BCMA antibodies, we produced RITs that specifically kill BCMA-expressing MM cells in vitro. To increase the serum half-life in vivo, we generated RITs that are fused with albumin-binding domains (ABDs). All RITs with ABDs have some decreased activity compared to the parent RIT, which is not due to decreased binding to BCMA. Various new anti-BCMA immunotoxins were produced and evaluated. None of these were better than LMB-75 (anti-BCMA BM306-disulfide-stabilized Fv-LRggs) supporting the further preclinical development of LMB-75.

Moxetumomab pasudotox in relapsed/refractory hairy cell leukemia

This is a pivotal, multicenter, open-label study of moxetumomab pasudotox, a recombinant CD22-targeting immunotoxin, in hairy cell leukemia (HCL), a rare B cell malignancy with high CD22 expression. The study enrolled patients with relapsed/refractory HCL who had ≥2 prior systemic therapies, including ≥1 purine nucleoside analog. Patients received moxetumomab pasudotox 40 µg/kg intravenously on days 1, 3, and 5 every 28 days for ≤6 cycles. Blinded independent central review determined disease response and minimal residual disease (MRD) status. Among 80 patients (79% males; median age, 60.0 years), durable complete response (CR) rate was 30%, CR rate was 41%, and objective response rate (CR and partial response) was 75%; 64 patients (80%) achieved hematologic remission. Among complete responders, 27 (85%) achieved MRD negativity by immunohistochemistry. The most frequent adverse events (AEs) were peripheral edema (39%), nausea (35%), fatigue (34%), and headache (33%). Treatment-related serious AEs of hemolytic uremic syndrome (7.5%) and capillary leak syndrome (5%) were reversible and generally manageable with supportive care and treatment discontinuation (6 patients; 7.5%). Moxetumomab pasudotox treatment achieved a high rate of independently assessed durable response and MRD eradication in heavily pretreated patients with HCL, with acceptable tolerability.

Abstract 1928: Identification of interleukin 13 receptor alpha 2 (IL-13Ra2) overexpression in LSL-KrasG12D/+, LSL-Try53R172H/+, and PDx-1-Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma as a target for receptor directed anticancer therapy

Abstract Pancreatic ductal adenocarcinoma (PDA) is a uniformly lethal disease because it escapes early diagnosis and at an advanced stage there are no effective therapies available. Previously, we have demonstrated that IL-13R α2 is overexpressed in 71% of human PDA specimens. We have targeted IL-13Rα2 in an orthotopic mouse model of human PDA and shown effectiveness of the receptor targeting agent, IL-13-PE, a recombinant immunotoxin comprised of IL-13 and Pseudomonas exotoxin. To determine the effectiveness of IL-13-PE and develop receptor targeting approaches, we used a LSL-KrasG12D/+, LSL-Try53R172H/+ and PDx-1-Cre (KPC) genetically engineered mouse model that spontaneously develops PDA in C57BL/6J immunocompetent mice and simulates human PDA disease. These spontaneous PDA tumors showed significant overexpression of IL-13R α2 mRNA compared to normal mouse pancreas and surrounding non-cancerous pancreatic tissues as determined by RT-qPCR. By in situ hybridization (ISH) and immunohistochemistry (IHC) analysis, we confirmed these results at mRNA and protein levels of tumor and non-tumor tissue specimens by hybridizing the tumor sections with biotinylated anti-sense riboprobe and immunostaining with anti-murine IL-13R α2 antibody. We established two tumor cell lines from KPC mice PDA, which maintained abundant expression of IL-13R α2 mRNA and protein levels. The overexpressed IL-13R α2 on PDA tumor cells is found biologically active, as it binds to IL-13-PE in a concentration dependent manner killing tumors cells at IC50 of ~25ng/ml (concentration of IL-13-PE that killed 50% of tumor cells). In a clonogenic assay, PDA tumor cells formed colonies, which were again sensitive to IL-13-PE in a concentration dependent manner with IC50 of ~28ng/ml. Based on these data, we have initiated assessment of anti-cancer activity of IL-13-PE against spontaneously developed PDA tumors developed in mice between the ages of 14-16 weeks. These mice were randomized to either receive excipient control i.p. or 50 µg/Kg IL-13-PE every alternate day for two weeks and then evaluated for general health and tumor growth by ultrasound technique. Five week follow up data reveal that mice treated with excipient alone developed larger tumors compared to treated mice and the general health of IL-13-PE treated mice was better compared to excipient-treated mice. These animals are being followed for their overall survival. Our studies indicate that IL-13R α2 is overexpressed in genetically engineered mouse model of PDA suggesting that IL-13R α 2 is a useful drug target not only in orthotopic mouse model but also in spontaneously developed PDA in mice, which may mimic its biological function more closely to human PDA. Citation Format: Akiko Suzuki, Pamela Leland, Bharat H. Joshi, Raj K. Puri. Identification of interleukin 13 receptor alpha 2 (IL-13Ra2) overexpression in LSL-KrasG12D/+, LSL-Try53R172H/+, and PDx-1-Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma as a target for receptor directed anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1928.

Improving the In Vivo Efficacy of an Anti-Tac (CD25) Immunotoxin by Pseudomonas Exotoxin A Domain II Engineering

Tac (CD25) is expressed on multiple hematologic malignancies and is a target for cancer therapies. LMB-2 is an extremely active anti-Tac recombinant immunotoxin composed of an Fv that binds to Tac and a 38-kDa fragment of Pseudomonas exotoxin A (PE38). Although LMB-2 has shown high cytotoxicity toward Tac-expressing cancer cells in clinical trials, its efficacy was hampered by the formation of anti-drug antibodies against the immunogenic bacterial toxin and by dose-limiting off-target toxicity. To reduce toxin immunogenicity and nonspecific toxicity, we introduced six point mutations into domain III that were previously shown to reduce T-cell immunogenicity and deleted domain II from the toxin, leaving only the 11aa furin cleavage site, which is required for cytotoxic activity. Although this strategy has been successfully implemented for mesothelin and CD22-targeting immunotoxins, we found that removal of domain II significantly lowered the cytotoxic activity of anti-Tac immunotoxins. To restore cytotoxic activity in the absence of PE domain II, we implemented a combined rational design and screening approach to isolate highly active domain II-deleted toxin variants. The domain II-deleted variant with the highest activity contained an engineered disulfide-bridged furin cleavage site designed to mimic its native conformation within domain II. We found that this approach restored 5-fold of the cytotoxic activity and dramatically improved the MTD. Both of these improvements led to significantly increased antitumor efficacy in vivo We conclude that the next-generation anti-Tac immunotoxin is an improved candidate for targeting Tac-expressing malignancies. Mol Cancer Ther; 17(7); 1486-93. ©2018 AACR.

Pharmacokinetics (PK), pharmacodynamics (PD) and immunogenicity of moxetumomab pasudotox (Moxe), an immunotoxin targeting CD22, in adult patients (Pts) with relapsed or refractory hairy cell leukemia (HCL).

7061Background: Moxe is a recombinant immunotoxin that specifically binds to CD22 on the B cell surface and internalizes leading to cell death. The PK, PD, and immunogenicity of moxe were evaluated...

Moxetumomab pasudotox in heavily pretreated patients with relapsed/refractory hairy cell leukemia: Results of a pivotal international study.

7004Background: A pivotal multicenter, single-arm study evaluated moxetumomab pasudotox, a first-in-class recombinant immunotoxin, in patients (pts) with relapsed/refractory hairy cell leukemia (HC...

Expression of VGRNb-PE immunotoxin in transplastomic lettuce (Lactuca sativa L.).

This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They consist of an antibody linked to a protein toxin and are designed to specifically kill the tumor cells. In our previous study, VGRNb-PE immunotoxin protein containing anti-VEGFR2 nanobody fused to the truncated form of Pseudomonas exotoxin A has been established. Here, we expressed this immunotoxin in lettuce chloroplasts. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, multigene engineering in a single transformation event and maternal inheritance of the transgenes. Site specific integration of transgene into chloroplast genomes, and homoplasmy were confirmed. Immunotoxin levels reached up to 1.1% of total soluble protein or 33.7µg per 100mg of leaf tissue (fresh weight). We demonstrated that transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. These results indicate that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins.

Antiviral Immunotoxin Against Bovine herpesvirus-1: Targeted Inhibition of Viral Replication and Apoptosis of Infected Cell.

Bovine herpesvirus 1 (BoHV-1) is a highly contagious viral pathogen which causes infectious bovine rhinotracheitis in cattle worldwide. Currently, there is no antiviral prophylactic treatment available capable of mitigating the disease impact and facilitating recovery from latent infection. In this study, we have engineered a novel recombinant anti-BoHV-1 immunotoxin construct termed “BoScFv-PE38” that consists of a single-chain monoclonal antibody fragment (scFv) fused with an active domain of Pseudomonas exotoxin A as a toxic effector (PE38). The recombinant BoScFv-PE38 immunotoxin expressed in a prokaryotic expression system has specific binding affinity for BoHV-1 glycoprotein D (gD) with a dissociation constant (Kd) of 12.81 nM and for BoHV-1 virus particles with a Kd value of 97.63 nM. We demonstrate that the recombinant BoScFv-PE38 is internalized into MDBK cell compartments that inhibit BoHV-1 replication with a half-maximal inhibitory concentration (IC50) of 4.95 ± 0.33 nM and a selective index (SI) of 456 ± 31. Furthermore, the BoScFv-PE38 exerted a cytotoxic effect through the induction of ATP and ammonia, leading to apoptosis of BoHV-1-infected cells and the inhibition of BoHV-1 replication in MDBK cells. Collectively, we show that BoScFv-PE38 can potentially be employed as a therapeutic agent for the treatment of BoHV-1 infection.

The Role of the Furin‐Cleavable Linker and KDEL Sequence in Cytotoxicity of Recombinant ImmunoDNase

The lack of specificity of standard chemotherapies to identify and kill cancer cells has challenged researchers to engineer targeted therapies to replace conventional treatments. One strategy that is currently being optimized to treat cancer is the use of recombinant immunotoxins (RITs). RITs are chimeric proteins that are engineered to contain an antibody conjugated to a toxin. The antibody allows for specific targeting of cancer cells while the toxin domain is internalized by the targeted cell and subsequently induces cell death. These therapies suffer from several problems, including non‐specific toxicities, low anti‐tumor activity, and immunogenicity. A bacterial moiety introduced into a patient with an intact immune system can elicit an immune response that consequently eliminates the possibility of retreatment. Deimmunized, humanized, and fully human immunoconjugates have been engineered, but often lack efficacy. We are using specific sequences known to assist cellular intoxication by Pseudomonas exotoxin A (PE) to enhance the activity of human‐derived immunotoxins. We are developing a recombinant immunotoxin based on human DNase I, using a furin‐cleavable linker and KDEL ER retention sequence adapted from the PE intoxication strategy. We expect this construct to have low immunogenicity, high specificity for dividing cells, and enhanced toxicity relative to the unmodified DNase I construct.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recombinant immunotoxins with albumin-binding domains have long half-lives and high antitumor activity

Recombinant immunotoxins (RITs) are chimeric proteins consisting of a Fv that binds to a cancer cell and a portion of a protein toxin. One of these, Moxetumomab pasudotox, was shown to be effective in treating patients with some leukemias, where the cells are readily accessible to the RIT. However, their short half-life limits their efficacy in solid tumors, because penetration into the tumors is slow. Albumin and agents bound to albumin have a long half-life in the circulation. To increase the time tumor cells are exposed to RITs, we have produced and evaluated variants that contain either an albumin-binding domain (ABD) from Streptococcus or single-domain antibodies from Llama. We have inserted these ABDs into RITs targeting mesothelin, between the Fv and the furin cleavage site. We find that these proteins can be produced in large amounts, are very cytotoxic to mesothelin-expressing cancer cell lines, and have a high affinity for human or mouse serum albumin. In mice, the RIT containing an ABD from Streptococcus has a longer half-life and higher antitumor activity than the other two. Its half-life in the circulation of mice ranges from 113 to 194 min compared with 13 min for an RIT with no ABD. Cell uptake studies show the RIT enters the target cell bound to serum albumin. We conclude that RITs with improved half-lives and antitumor activity should be evaluated for the treatment of cancer in humans.

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

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