Recombinant Immunotoxin Research Articles

Phase I study of mesothelin-targeted immunotoxin LMB-100 given as a long infusion with or without nab-paclitaxel.

3553 Background: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting a modified Pseudomonas exotoxin A payload. Previous Phase 1 clinical testing of a 30-minute LMB-100 “short” infusion format identified a serum half-life of ~1 hour. Pre-clinical data suggested that extending infusion time could improve anti-tumor efficacy by increasing tumor cell duration of exposure to LMB-100. The primary objective of this study was to determine the safety and tolerability of administering LMB-100 in a long infusion format over 24-48 hours alone or with nab-paclitaxel chemotherapy in patients with mesothelin-expressing solid tumors. Methods: Patients (n = 15) with pancreatic adenocarcinoma and other mesothelin-expressing solid tumors (n = 3; mesothelioma, colon, and ampullary cancers) treated on 3 dose levels received long infusion of LMB-100 (65 or 100 mcg/kg/day) for 24 hour on Days 1 and 4 (n = 6) or 48 hour on Day 1 (n = 9) with or without a loading dose (40 mcg/kg over 30 minutes) for up to 2 cycles. In the second arm, patients (n = 5) with pancreatic adenocarcinoma were treated with LMB-100 over 24 hours on Day 1 concurrently with nab-paclitaxel (125 mg/m2) for up to 3 cycles. Results: DLT of proteinuria (grade 3) in one patient and acute kidney injury (grade 1) in one patient were observed amongst patients receiving 100 mcg/kg/day over 48 hours and 24 hours, respectively. No objective responses were seen but all patients receiving nab-paclitaxel had > 50% decrease in CA 19-9. Patients at all single agent dose levels (8 of 10 evaluable) developed high titer anti-drug antibodies (ADAs) against LMB-100. Those with ADAs (8 of 8) had undetectable cycle 2 peak plasma LMB-100 concentration. Development of high titer ADAs occurred more frequently with long infusion than seen previously with “short” infusion LMB-100. Most long infusion patients (19 of 20) developed increased serum IL-6 within 24 hours of LMB-100 infusion. However, the systemic inflammatory response to LMB-100 (as measured by increased serum CRP) which occurs in most “short” infusion patients was not observed. Conclusions: Long infusion format LMB-100 is generally well tolerated but immunogenicity limits treatment to 1 effective cycle. No anti-tumor efficacy of the single agent was observed. Clinical trial information: NCT02810418 .

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

Background: Denileukin diftitox (trade name, Ontak) is the first recombinant immunotoxin (IM), in which the binding domain of diphtheria toxin has been replaced by the amino acid sequence of human interleukin-2 (DT389IL-2) using genetic engineering. Purity, stability, and structural property of the protein are critical factors for the scale-up production of this fusion protein. In this IM, location 519 has free cysteine residue that leads to cross S-S bound formation in the refolding process and, as a result, misfolding/aggregation of the protein may occur. Materials and Methods: To inhibit misfolding/aggregation, we substituted cysteine 519 by a serine residue with site-directed mutagenesis, and then the ability of the mutated protein for binding to the IL-2 receptor was predicted and determined by bioinformatics tools. For this purpose, the sequence of the denileukin diftitox was adopted from Drugbank, and the mentioned substitution applied. Two methods determined the folding of the fusion protein: de novo modeling method (by utilizing the I-TASSER database) and homology modeling method (by using some databases and tools, including Swiss-Model, PHYRE2, M4T, ModWeb, RaptorX, and EasyModeller). Finally, the ability of the proteins for binding to the IL-2 receptor was investigated by pyDock and Zdock docking servers, as well as Hex software. Results: The result showed that the mutated form (C519S) of this protein folds appropriately, and the ΔG of the models, measured by STRUM, showed no significant variation. Also, docking analysis has shown that the protein can efficiently bind to the IL-2 receptor without any substantial changes in the binding energy. Conclusion: The present study shows that the suggested mutation of this protein can be an acceptable replacement for denileukin diftitox with a similar affinity and a more proper refolding process.

Mutations In The Furin Cleavage Site To Improve Cytotoxicity In Anti‐Mesothelin Recombinant Immunotoxins Based On Pseudomonas Exotoxin A

A recombinant immunotoxin (RIT) is a fusion protein composed of an antibody and a toxin. The antibody binds to receptors on target cells and promotes internalization. Once internalized, the toxin kills the cell. RITs based on Pseudomonas exotoxin A (PE) have been created to target and kill cancer cells. In order to do this, internalized PE must reach the cytosol through a retrograde trafficking route, during which it encounters and is cleaved by the protease furin. Furin has been established as an integral part of PE‐based RIT intoxication pathways. Mutations in the PE furin cleavage site have been shown to influence cleavage efficiency and RIT cytotoxicity. In order to explore the role of furin in RIT intoxication further, we have constructed anti‐mesothelin/PE‐based RITs with mutations on either side of the furin cleavage site. Based on previous results, we initially hypothesized that mutations N‐terminal to the cleavage site would increase both cleavage efficiency and cytotoxicity in these RITs, while mutations C‐terminal to the cleavage site would increase cleavage efficiency without significant improvements to cytotoxicity. We expressed, purified, and tested the cytotoxicity of these constructs in tissue culture and found that mutations N‐terminal to the cleavage site decreased cytotoxicity relative to the wild‐type RIT, while mutations C‐terminal to the cleavage site had minimal effect. We next plan to explore cleavage efficiency of the mutant RITs.Support or Funding InformationTowson University Department of Biological Sciences

Developing and Evaluating Recombinant Immunotoxins against Latent Epstein Barr Virus Infected Cells

Epstein Barr virus (EBV), commonly associated with acute infectious mononucleosis, is an oncogenic virus implicated in thousands of cancer‐related deaths every year worldwide. Estimates suggest that 90–95% of the population worldwide are infected with EBV. Individuals with healthy immune system can manage acute infections with minimal symptoms, but EBV often persists in a latent state even after the acute infection has cleared. Studies have shown a definitive link between latent EBV infections and the development of certain cancers such as nasopharyngeal cancer and Burkitt’s lymphoma, yet there are currently no targeted treatments against latent EBV induced cancers. A potential treatment for both latent EBV infections and EBV‐induced malignancies is recombinant immunotoxins (RITs) targeted to the virus‐specific membrane protein, LMP1. RITs are fusion proteins comprised of an antibody and protein toxin designed to identify and kill specific populations of cells. RITs using the bacterial toxin Pseudomonas exotoxin A (PE) have been extensively studied. Several RITs are in clinical trials, and one (moxetumomab pasudotox) has received FDA approval for the treatment of Hairy Cell Leukemia. Using a previously developed anti‐LMP1 antibody, we have cloned, expressed, and purified two anti‐LMP1 single‐chain variable fragment antibody joined recombinantly to either PE24 or PE38. We have begun to develop a HEK293 cell line that expresses LMP1 in order to evaluate the cytotoxicity of our anti‐LMP1 RITs. Subsequent experiments will evaluate these RITs as a therapeutic against EBV‐infected cell lines.Support or Funding InformationThis project was support by the Towson University’s Graduate Student Association and the Fisher College of Science and Mathematics

An Anti‐Mesothelin Recombinant Immunotoxin Based on Deoxyribonuclease‐I

Recombinant immunotoxins (RITs) are fusion proteins consisting of a toxin attached to an antibody fragment. Unlike traditional chemotherapy, RITs used in cancer therapy can be specifically targeted to tumor cells while sparing healthy cells. One shortcoming of RITs is their immunogenicity, which is the immune response of patients to treatment with RITs. Patients with intact immune systems often produce antibodies to the toxin that neutralize its function. For this experiment, we attempted to prepare a RIT using deoxyribonuclease‐ I (DNase‐I) instead of a typical bacterial or plant‐derived toxin. DNase‐I is an endogenous human protein that catalyzes the degradation of DNA into its component nucleotides. Previous research shows that DNase‐I can be toxic to cells if delivered through targeted endocytosis. Furthermore, we predict that DNase‐I will be less immunogenic than other toxins, because it is a human protein. We successfully cloned an expression vector joining DNase‐I to an anti‐mesothelin antibody fragment and expressed it in E. coli. The resulting protein, however, was unstable and we were unable to purify it after refolding from inclusion bodies. We concluded that the design of the fusion protein needs improvement to enhance its stability. It is also possible that the refolding conditions need further optimization. Future directions include exploring both of these possibilities.Support or Funding InformationI would like to thank the Towson Bridges Program and CCBC for the opportunity. Thank you to Dr. Laing, Dr. Snyder, Dr. Berlyn and Trudymae for guidance throughout the program. Thank you to the NIH Bridges Grant 5 R25 GM058264 16 for funding. Special thanks to Dr. Weldon, Jillian Baker and all students that have helped throughout the project.

Population pharmacokinetics, efficacy, and safety of moxetumomab pasudotox in patients with relapsed or refractory hairy cell leukaemia.

To characterize the pharmacokinetics (PK) of moxetumomab pasudotox, an anti-CD22 recombinant immunotoxin, in adults with relapsed or refractory hairy cell leukaemia, we examined data from a phase 1 study (Study 1001; n = 49) and from the pivotal clinical study (Study 1053; n = 74). Data from both studies were pooled (n = 123) to develop a population PK model. Covariates included demographics, disease state, liver and kidney function, prior treatment, and antidrug antibodies (ADAs). Exposure-response and exposure-safety were analysed separately by study. A 1-compartment model with linear elimination from the central compartment and 2 clearance (CL) rates was developed. Moxetumomab pasudotox was cleared more rapidly after cycle 1, day 1 (CL1 = 24.7 L/h) than subsequently (CL2 = 3.76 L/h), with high interindividual variability (116 and 109%, respectively). In Study 1053, patients with ADA titres >10 240 showed ~4-fold increase in CL. Higher exposures (≥median) were related to higher response rates, capillary leak syndrome and increased creatinine (Study 1053 only), or grade ≥3 adverse events (Study 1001 only). Clinical benefits were still observed in patients with lower exposure or high ADA titres. Despite a high incidence of immunogenicity with increased clearance, moxetumomab pasudotox demonstrated efficacy in hairy cell leukaemia.

Breast cancer targeted/ therapeutic with double and triple fusion Immunotoxins

Target-specific transport of therapeutic agents holds promise to increase the efficacy of cancer treatment by decreasing injury to normal tissues and post treatment problems. HER2 is a tumor cell surface marker that is expressed in 25–30 % of breast cancer patients. The significant role of HER2 in cancer development and its biological feature makes it a highly appealing goal for the therapeutic treatment of cancer targeted therapy using HER2 monoclonal antibody. This approach is currently used as a special treatment against breast cancer in some research. In the present study, HER2 monoclonal antibody (mAb), (Herceptin) fused to PE38 by recombinant DNA technology and a new recombinant IT was developed. The scFv(Herceptin)-PE-STXA and scFv(Herceptin)-PE fusions cloned in pET28a and recombinant protein expression was carried out and then the proteins were purified. MCF-7 and SKBR-3 cells were used as HER2-negative and HER2-positive breast cancer cells, respectively. The cytotoxicity of its evaluated using MTT assay. The cell ELISA was used to determine the binding ability of immunotoxins (ITs) to the cell receptor and internalization and apoptosis were also assessed. The results revealed that cell cytotoxicity occurred in SKBR-3 cells in a dose-dependent manner but not in MCF-7 cells. It is possible that this ITs can attach to HER2-positive breast cancer cells and then, internalize and eradicate cancer cells by apoptosis. Here, we concluded that the recombinant ITs have therapeutic potential against HER2-positive breast cancer.

Immunotoxin SS1P is rapidly removed by proximal tubule cells of kidney, whose damage contributes to albumin loss in urine

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration of l-lysine. Our data suggests that l-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

A new scfv-based recombinant immunotoxin against EPHA2-overexpressing breast cancer cells; High in vitro anti-cancer potency

Immunotoxin therapy is one of the immunotherapy strategies providing a new, effective and high potency treatment against various cancers. Breast cancer is the most common cancer among women in many countries. The EPH receptors are a large part of tyrosine kinase receptors family and play an effective role in tumor development and angiogenesis. Among EPH receptors, EPHA2 is more commonly well-known and widely expressed in many cancers like breast cancer. In this study, we evaluated the specification of a designed immunotoxin formed by EPHA2-specific scfv linked with PE38KDEL on EPHA2-overexpressing breast cancer cell line. This new scfv-based recombinant immunotoxin was studied in terms of features such as binding potency, cytotoxicity effects, apoptosis induction ability, and internalization. The flow cytometry results showed that the immunotoxin can significantly (approximately 99%) bind to EPHA2-overexpressing breast cancer cell line (MDA-MB-231) in a low concentration (2.5 ng/ul) while cannot significantly bind to the normal cell line (HEK-293) or even EPHA2-very low expressing cell line (MCF-7). Using the MTT assay and Annexin V/Propidium iodide (PI) double staining method by flow cytometry, we observed significant killing and apoptosis induction of the MDA-MB-231 cells at different concentrations. Immunotoxin tracking by confocal microscopy at 2 h and 6 h revealed a massive presence of immunotoxin in the cytoplasm. Finally, given the in vitro results, it seems that this immunotoxin is competent enough to serve as a good candidate for in vivo studies to further explore the possibility of breast cancer treatment.

A population-based study of hairy cell leukemia over a period of 20 years

There are limited population-based studies of hairy cell leukemia (HCL), a rare chronic lymphoproliferative disorder of B-cells. We conducted a population-based study that included all patients diagnosed with HCL between 1996 and 2016 in Western Normandy. Recorded data focused on medical history, clinical presentation, biological results, treatment modalities in the first line and in relapsed/refractory patients and the occurrence of secondary malignancies. One hundred and twenty-three HCL patients were registered in the database. HCL represented 0.7% of all malignant hematological disorders and 3.0% of all leukemia. The overall age-standardized incidence ratio (SIR) was 0.39/100,000 inhabitants in men and 0.09/100,000 in women, and it remained stable over the 20-year period analyzed. One hundred and seven patients (88%) received first-line treatment, 33 patients (27%) received at least 2 lines of treatment and 14 patients (11%) received more than 2 lines. Cladribine used as first-line treatment induced a high hematological complete response (HCR) rate of 92%. The median overall survival (OS) was over 15 years, with 5-year and 10-year survival rates of 84% and 70.5%. No significant differences in OS were observed between men and women, between the calendar periods studied or between patients who received a single line treatment with IFN-α or PNA. The risk of relapse was higher with IFN-α treatment, requiring subsequent treatments in that patients. The time to next treatment (TTN) tends to be longer for PNAs compared to IFN-α even if difference is not significant. Secondary cancers were observed in 9/123 patients (7.3%) with solid tumors in 8 patients and hematological malignancy in one patient. Our data confirm in real life that single courses of cladribine administered to patients with HCL induce high response rates, the majority of which are HCR. Relapses seem less frequent than with IFN-α and the administration schedule is less restrictive for the patients. The emergence of chemo-immunotherapy and the development of effective new drugs such as recombinant immunotoxins and BRAF targeting will offer new possibilities in the management of HCL patients.

Site-Specific PEGylation of Recombinant Immunotoxin DAB389IL-2: Structural and Functional Assessment

BackgroundDAB389IL-2 is considered a fusion immunotoxin and it is used for the CTCL therapy. DAB389IL-2 includes of two distinct portions; the catalytic domain of diphtheria toxin and IL-2. DAB389IL-2 duo to the presence of a free cysteine residue (Cys 513 in IL-2 part) is prone to unwanted intramolecular and intermolecular disulfide bonds formation and aggregation problems. Aggregation is considered as the most common physical instability. PEGylation is an effective approach to increase the stability and half-life of therapeutic proteins.Materials and methodsIn this study, the PEGylation of recombinant DAB389IL-2 was performed by mPEG-vinylsulfone, through partial denaturation condition at 4 0C for 24 h. To confirm the PEGylation reaction, SDS-PAGE and Dynamic Light Scattering (DLS) was used. The structure of DAB389IL-2 and PEGylated immunotoxin DAB389IL-2 was analyzed using the circular dichroism (CD) and fluorescence methods. Also, K562 cells line were treated with various concentrations of DAB389IL-2 and conjugated form. In the following, the nuclease activity of DAB389IL-2 and PEGylated form was determined.ResultsThe SDS-PAGE result confirmed the site-specific PEGylation of DAB389IL-2. Spectroscopy results exhibited that the PEGylation doesn’t affect the protein native structure. Also, cytotoxicity assay and nuclease activity test confirmed that PEGylated protein induces death in K562 cells line and DNA degradation respectively. ConclusionIt is concluded that the PEGylated immunotoxin DAB389IL-2 has a proper structure and function; thus, PEGylated immunotoxin requires more survey due its unique properties.

Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization.

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Abstract PR07: Phase I/II study of mesothelin-targeted immunotoxin LMB-100 with nab-paclitaxel for patients with advanced pancreatic adenocarcinoma

Abstract Purpose: LMB-100 recombinant immunotoxin (iTox) consists of a mesothelin-binding Fab for targeting and a modified Pseudomonas exotoxin A payload. Preclinical studies showed that combining taxanes with iTox results in synergistic antitumor activity. The primary objectives of this phase I/II study were to determine the maximum tolerated dose (MTD) of LMB-100 when given with nanoalbumin bound (nab)-paclitaxel to participants with previously treated advanced pancreatic adenocarcinoma and to assess the objective response rate. Patients and Methods: Patients (n = 20) received fixed-dose nab-paclitaxel (125 mg/m2 on Days 1 and 8) with LMB-100 (65 or 100 mcg/kg on Days 1, 3, and 5) in 21-day cycles. Treatment was limited to 2-3 cycles by protocol. Safety and tolerability, pharmacokinetics, immunogenicity, and relationship of activity to tumor mesothelin expression were assessed. Results: Dose-limiting toxicity (DLT) of Grade 3 myalgia was observed in 2 of 5 patients treated with 100 mcg/kg. DLT of Grade 3 edema from capillary leak syndrome (CLS) was observed in 1 of 6 patients at MTD of 65 mcg/kg. Severe toxicities of Grade 4 left ventricular dysfunction and hypotension from CLS were observed in 1 patient in the phase II expansion, resulting in study closure. LMB-100 anti-drug antibody formation limited LMB-100 exposure in 5 of 13 patients during cycle 2. Seven of 17 patients with evaluable CA19-9 experienced &amp;gt;50% decrease of this tumor marker, and one patient developed a partial response. Response was associated with higher tumor mesothelin expression and Cycle 2 LMB-100 exposure. Conclusions: The combination caused serious toxicity. Despite some observed clinical activity, further testing of this combination will not be pursued. This abstract is also being presented as Poster B01. Citation Format: Christine C. Alewine, Mehwish Ahmad, Cody J. Peer, Zishuo Hu, Min-jung Lee, Akiro Yuno, Jessica Kindrick, Anish Thomas, Seth Steinberg, Jane B. Trepel, William D. Figg, Raffit Hassan, Ira Pastan. Phase I/II study of mesothelin-targeted immunotoxin LMB-100 with nab-paclitaxel for patients with advanced pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR07.

Abstract CN07-03: Recombinant immunotoxins for hematologic malignancies

Abstract Recombinant immunotoxins are composed of fragments of monoclonal antibodies (Mabs) and protein toxins, enabling the toxin to bind to a target cell recognized by the antibody, and the toxin to kill the cell after internalization. Recombinant immunotoxins are similar to but distinct from growth-factor fusions toxins like denileukin diftitox and Tagraxofusp, FDA-approved in 1999 and 2018, respectively. Unlike chemical conjugates, Recombinant immunotoxins have a peptide that links the cell-binding to the toxin domains. After proteolytic cleavage, the toxin separates from the binding domain, undergoes intracellular trafficking and enters the cytosol, resulting in apoptotic cell death. Recombinant immunotoxins containing Pseudomonas exotoxin A contain an Fv or Fab fragment of a Mab replacing the native cell-binding toxin domain. The first recombinant immunotoxin we made contained an anti-CD25 single-chain Fv fused to a 38 kDa fragment of Pseudomonas exotoxin called PE38. Anti-CD25 recombinant immunotoxin LMB-2 was active in several hematologic malignancies, notably hairy cell leukemia (HCL) and adult T-cell leukemia (ATL). LMB-2 achieved a high complete remission (CR) rate in ATL when combined with chemotherapy to reduce immunogenicity and progression between cycles. Improved targeting of HCL was achieved with anti-CD22 recombinant immunotoxin Moxetumomab Pasudotox (Moxe) that was stabilized with a disulfide bond in the Fv and has a high affinity for CD22. Moxe achieved investigator-assessed CR rates of 51-64% in relapsed/refractory HCL in Phase 1 and 3 trials, leading to FDA-approval in 2018. Minimal residual disease (MRD) was negative in most CRs. On the phase I trial, which had adequate follow-up, MRD eradication by the most sensitive standard assay, flow cytometry of the bone marrow aspirate (BMA), was associated with longer CR duration. Extra or ‘consolidation’ cycles past documentation of CR was also associated with longer CR duration. To detect MRD with higher sensitivity, patient specific immunoglobulin heavy chain (IgH) rearrangements were cloned and real-time quantitative PCR (RQ-PCR) performed. Using patient-specific primers and probe, 1 HCL cell could be detected in 106 normal cells. We found that phase 1-3 patients achieving MRD-free CR by blood RQ-PCR had significantly prolonged CR duration (p&amp;lt;0.0001) compared to those remaining blood RQ-PCR+. Both BMA flow cytometry and RQ-PCR were associated with prolonged CR duration if negative, but blood flow cytometry was not helpful since only one patient with CR remained flow-positive. Molecular remissions assessed by blood analysis may be useful to determine the number of cycles of Moxe to administer to achieve long-term CR or possibly cure. To achieve molecular remissions with fewer cycles, Moxe is being tested with rituximab to facilitate tumor reduction and decrease anti-drug antibody formation. Citation Format: Robert Kreitman, Daniel Gorelik, Maryalice Stetler-Stevenson, Constance M Yuan, Hao-Wei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan. Recombinant immunotoxins for hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-03. doi:10.1158/1535-7163.TARG-19-CN07-03

Abstract B099: Characterization of monoclonal antibodies generated to the 287-302 amino acid loop of the human epidermal growth factor receptor

Abstract Dysregulation of the epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of malignancies including glioblastoma and various epithelial cancers. Oncogenesis occurs from overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287-302 loop from the extracellular domain is exposed completely on EGFRvIII (deletion of exons 2-7), partially exposed on some cancers but cryptic on normal cells expressing WT levels of EGFR. We report on the generation of antibodies to this loop. A peptide corresponding to amino acids 286-303 was synthesized and then coupled chemically to Keyhole Limpet Hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V-regions were cloned and sequenced. Receptor binding was characterized using ELISA and flow cytometry. Monoclonal antibodies (n=7) reactive for the 287-302 loop were characterized further. Each one reacted with EGFRvIII but not EGFR WT. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be potently cytotoxic for MDA-468, A431 and F98-EGFRvIII-expressing cells. We conclude that immunization with a peptide corresponding to a cryptic EGFR epitope can produce cancer-binding antibodies. By way of example, the 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be developed as a therapeutic agent for clinical use. Citation Format: Eric Chun Hei Ho, Antonella Antignani, Robert Sarnovsky, David J FitzGerald. Characterization of monoclonal antibodies generated to the 287-302 amino acid loop of the human epidermal growth factor receptor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B099. doi:10.1158/1535-7163.TARG-19-B099

Abstract A081: Phase I study of mesothelin-targeted immunotoxin LMB-100 given as a long infusion

Abstract Purpose: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting and a modified Pseudomonas exotoxin A payload. Previous Phase 1 clinical testing of a 30-minute LMB-100 infusion format identified dose-limiting toxicity (DLT) of capillary leak syndrome (CLS) and a serum half-life of ~60 minutes. Pre-clinical data suggested that extending infusion time could improve anti-tumor efficacy by increasing tumor cell duration of exposure to LMB-100. The primary objective of this study was to determine the safety and tolerability of administering LMB-100 over a longer infusion time. We hypothesized that reduced CMAX would result in decreased severity of off-target toxicities like CLS without decreasing anti-tumor activity. Patients and Methods: Patients (n = 15) with pancreatic adenocarcinoma and other mesothelin-expressing solid tumors (n = 3; mesothelioma, colon adenocarcinoma, ampullary carcinoma) treated on 3 dose levels received long infusion (24-48 hours) LMB-100 (65 or 100 mcg/kg/day) with or without a loading dose (40 mcg/kg over 30 minutes) for up to 2 cycles. Safety and tolerability, pharmacokinetics, immunogenicity and anti-tumor activity were assessed. Results: DLT of proteinuria (grade 3) was observed in 1 of 6 patients receiving 100 mcg/kg/day over 48 hours with pre-load. Three of 6 patients at this dose level developed significant CLS (&amp;gt;5 kg weight gain from fluid edema) that was not dose-limiting. No CLS was observed in the 6 patients who received 100 mcg/kg over 24 hours on days 1 and 4 with pre-load. Increased immunogenicity was observed with the long infusion compared to standard administration format: 8 of 9 patients receiving 2nd cycle treatment had undetectable plasma LMB-100 concentration during this cycle that was associated with the development of high titer anti-LMB-100 antibodies. Progressive disease was observed in 11 of 12 evaluable patients, and none of 8 evaluable patients with pancreatic adenocarcinoma had a clinically significant decline in CA 19-9 tumor marker. Conclusions: Long infusion format of delivery for LMB-100 can reduce CLS but increases immunogenicity. No anti-tumor activity was observed. Citation Format: Christine Alewine, Mehwish Iqra Ahmad, Cody J Peer, Seth Steinberg, William D Figg, Raffit Hassan, Ira Pastan. Phase I study of mesothelin-targeted immunotoxin LMB-100 given as a long infusion [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A081. doi:10.1158/1535-7163.TARG-19-A081

Abstract CN07-02: Immunotoxins targeting GPC3 for liver cancer

Abstract Glypican-3 (GPC3) is a cell surface glypican that serves as a co-receptor for Wnt. Our laboratory has generated two high affinity antibodies targeting GPC3, HN3 and YP7. The HN3 human nanobody recognizes the N-lobe of GPC3, whereas the YP7 antibody recognizes the C-lobe of GPC3. Mutating residue F41 on the N-lobe of GPC3 inhibits binding of Wnt and the HN3 nanobody and inhibits activation of β-catenin. Both antibodies are fused to a fragment of Pseudomonas exotoxin A (PE38) to create recombinant immunotoxins. Interestingly, the HN3-derived immunotoxin (HN3-PE38) has superior antitumor activity as compared with the YP7-derived immunotoxin (YP7-PE38). Intravenous administration of HN3-PE38 causes regression of hepatocellular carcinoma (HCC) tumor xenografts in mice. Our study establishes GPC3 as a promising target for immunotoxin-based liver cancer therapy and demonstrates immunotoxin-induced tumor regression via dual mechanisms: inactivation of Wnt signaling via the HN3 nanobody and inhibition of protein synthesis via the PE bacteria toxin. However, immunogenicity and a short serum half-life may limit the ability of immunotoxins to transition to the clinic. To address these issues, we have recently engineered HN3-based immunotoxins to contain various deimmunized PE toxins. These new immunotoxins include HN3-T20, which is modified to remove domain II of the PE toxin and six T-cell epitopes. All of our immunotoxins display high affinity to human GPC3, with HN3-T20 having a KD value of 7.4 nM. A real-time cell growth inhibition assay demonstrates that a single dose of HN3-T20 at 1.6 nM is capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20’s serum retention, we further engineer the HN3-T20 by adding a streptococcal albumin binding domain (ABD) and a llama single-domain antibody fragment (ALB1) specific for serum albumin. For the detection of immunotoxin in mouse serum, we develop a highly sensitive ELISA and find that HN3-ABD-T20 has a 45-fold higher serum half-life than HN3-T20 (326 min vs 7.3 minutes); consequently, HN3-ABD-T20 shows the best serum retention and remains detectible even after a 24-hour period in mice. Furthermore, addition of an albumin binding domain results in HN3-ABD-T20 mediated tumor regression in mice at a low dose (1 mg/kg). These data show that the albumin binding deimmunized immunotoxins are high potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer. Citation Format: Mitchell Ho, Bryan D Fleming, Tim F Greten, Ira Pastan. Immunotoxins targeting GPC3 for liver cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-02. doi:10.1158/1535-7163.TARG-19-CN07-02

ATIM-24. DOSE FINDING AND DOSE EXPANSION TRIAL OF D2C7 IMMUNOTOXIN (D2C7-IT) ADMINISTERED INTRATUMORALLY VIA CONVECTION-ENHANCED DELIVERY (CED) FOR RECURRENT MALIGNANT GLIOMA (MG)

Abstract BACKGROUND 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, with dose expansion at the selected phase 2 dose, evaluating D2C7-IT delivered intratumorally by CED. METHODS 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 &gt;70%. Two patients per dose level (DL) were to be enrolled in the dose escalation portion (dose range: 40ng/mL to 23,354ng/mL), followed by dose expansion at the selected phase 2 dose (DL13). RESULTS As of 6/07/2019, 51 patients have been treated; 10 patients on the phase 2 dose. 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 (n=1) and grade 3 dysphasia (n=1) on DL17. Grade 3 or higher adverse events possibly related to D2C7-IT include: seizure (grade 4, n=2, grade 3, n=3), cerebral edema (grade 4, n=1), hydrocephalus (grade 3, n=5), headache (grade 3, n=4), hemiparesis (grade 3, n=4), dysphasia (grade 3, n=4), lymphopenia (grade 3, n=3), thromboembolic event (grade 3, n=3); and one each of grade 3 elevated ALT, urinary tract infection, fall, wound complication, generalized muscle weakness, confusion, encephalopathy, and somnolence. Fourteen patients are alive. Three patients have partial radiographic response and remain alive without additional therapy more than 46, 27 and 21 months after D2C7-IT infusion. CONCLUSION Dose level 13 was selected as the optimal phase 2 dose and patient accrual is ongoing on the dose expansion arm. Encouraging efficacy results have been observed. A trial of D2C7-IT with checkpoint inhibitor is planned to start in the near future.

A novel approach to assess domain specificity of anti-drug antibodies to moxetumomab pasudotox, an immunotoxin with two functional domains

Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determination of ADA domain specificity is a competitive inhibition with domain-containing molecules. When developing a conventional domain specificity assay for moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin, comprised of two functional domains (CD22-binding fragment and truncated Pseudomonas exotoxin A (PE38), we encountered a bioanalytical challenge. The method was able to detect immunodominant anti-PE38 (ADA-PE) but generated false negative results for low abundant CD22-binding domain ADA (ADA-BD) in a polyclonal sample. Troubleshooting experiments using control samples with varying levels of each ADA subtype demonstrated that a major factor for successful ADA identification was the ratio of the ADA signals contributed by each ADA subtype. To overcome this unique bioanalytical challenge, we developed a novel approach, which ensures detection of a domain-specific ADA subtype regardless of its relative level in a polyclonal ADA sample by evaluating signal inhibition by a respective domain-containing molecule at the condition when signals from all other ADAs are fully blocked. The method has been used for characterization of ADA domain specificity in moxetumomab pasudotox clinical trials, including study 1053, the pivotal Phase III study in hairy cell leukemia patients. It allowed for successful detection of ADA-BD in the presence of immunodominant ADA-PE, enabling accurate determination of domain specificity for moxetumomab pasudotox. The results demonstrated that the method was superior than the conventional approach. The method could be applied broadly to other biologics with two or more domains when there is a need to detect a minor ADA subtype in polyclonal samples.

Contextualizing the Use of Moxetumomab Pasudotox in the Treatment of Relapsed or Refractory Hairy Cell Leukemia.

Hairy cell leukemia (HCL) is an indolent B-cell malignancy characterized by high initial sensitivity to purine analog chemotherapy, minimal residual disease (MRD) frequently accompanying complete remission (CR), and relapses requiring additional treatment. Repeat chemotherapy shows decreasing efficacy and increasing toxicity with each course. Newer therapies targeting BRAF/MEK or Bruton's tyrosine kinase are effective but generally leave MRD. Rituximab has modest activity as a single agent and can achieve MRD-negative CR in combination with purine analogs, but there is significant toxicity from the chemotherapy. Moxetumomab pasudotox-tdfk (Moxe) is a biologic containing an antibody fragment (Fv) binding to CD22, attached to a portion of Pseudomonas exotoxin A. Binding to CD22 enables the toxin to enter and kill cells. Moxe is administered by 30-minute infusions on days 1, 3, and 5 of up to six cycles spaced 4 weeks apart. In phase I testing, 64% of 33 patients at the highest dose level achieved CR, most without MRD. Lack of MRD correlated with prolonged CR duration; of 11 MRD-negative CRs, 10 were still in CR after a median of 42 months of observation. In pivotal testing, 75% of 80 patients had a hematologic response, 41% with CR; 82% (27/33) of CRs were MRD-negative, and only 4 of the 27 MRD-negative patients relapsed during the follow-up period. Hemolytic uremic syndrome and capillary leak syndrome were each observed in 9% of patients, all reversible. In September 2018, the U.S. Food and Drug Administration approved Moxe for the treatment of relapsed/refractory HCL after ≥2 prior therapies. Moxe is undergoing further development in combination with rituximab. IMPLICATIONS FOR PRACTICE: Hairy cell leukemia (HCL) has effective treatments including purine analogs with and without rituximab, and oral inhibitors of BRAF, MEK and Bruton's tyrosine kinase (BTK). Despite these therapies, relapse occurs, and moxetumomab pasudotox has an important role in relapsed and refractory HCL because of its ability to achieve high rates of complete remissions (CRs) without chemotherapy; most of these CRs are without minimal residual disease (MRD). CR duration is enhanced in patients who achieve eradication of MRD. To improve the efficacy of this recombinant immunotoxin, a phase I trial is underway in combination with rituximab to reduce tumor burden and decrease immunogenicity.