Site-Specific PEGylation of Anti-Mesothelin Recombinant Immunotoxins Increases Half-life and 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.

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Site-Specific PEGylation Enhances the Pharmacokinetic Properties and Antitumor Activity of Interferon Beta-1b

Journal of Interferon & Cytokine Research ◽

10.1089/jir.2012.0148 ◽

2013 ◽

Vol 33 (12) ◽

pp. 769-777 ◽

Cited By ~ 12

Author(s):

Ji I. Lee ◽

Stephen P. Eisenberg ◽

Mary S. Rosendahl ◽

Elizabeth A. Chlipala ◽

Jacquelyn D. Brown ◽

...

Keyword(s):

Antitumor Activity ◽

Interferon Beta ◽

Site Specific ◽

Pharmacokinetic Properties

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9-Substituted acridine derivatives with long half-life and potent antitumor activity: synthesis and structure-activity relationships

Journal of Medicinal Chemistry ◽

10.1021/jm00017a006 ◽

1995 ◽

Vol 38 (17) ◽

pp. 3226-3235 ◽

Cited By ~ 52

Author(s):

Tsann-Long Su ◽

Ting-Chao Chou ◽

Joong Young Kim ◽

Jai-Tung Huang ◽

Grazyna Ciszewska ◽

...

Keyword(s):

Antitumor Activity ◽

Half Life ◽

Structure Activity Relationships ◽

Acridine Derivatives ◽

Structure Activity

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Tolerogenic nanoparticles restore the antitumor activity of recombinant immunotoxins by mitigating immunogenicity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1717063115 ◽

2018 ◽

Vol 115 (4) ◽

pp. E733-E742 ◽

Cited By ~ 23

Author(s):

Ronit Mazor ◽

Emily M. King ◽

Masanori Onda ◽

Nicolas Cuburu ◽

Selamawit Addissie ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Monoclonal Antibodies ◽

Antitumor Activity ◽

Regulatory T Cells ◽

Immune Tolerance ◽

Checkpoint Inhibitors ◽

Recombinant Immunotoxins ◽

Antidrug Antibodies

Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919038117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 6086-6091 ◽

Cited By ~ 2

Author(s):

Xui-Fen Liu ◽

Junxia Wei ◽

Qi Zhou ◽

Bruce A. Molitoris ◽

Ruben Sandoval ◽

...

Keyword(s):

Half Life ◽

Proximal Tubular Cells ◽

Proximal Tubule Cells ◽

Tubular Cells ◽

Two Photon Microscopy ◽

Two Photon ◽

Protein Toxin ◽

Mouse Imaging ◽

Proximal Tubular ◽

Recombinant Immunotoxins

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 ofl-lysine. Our data suggests thatl-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

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Development of a recombinant human IL-15·sIL-15Rα/Fc superagonist with improved half-life and its antitumor activity alone or in combination with PD-1 blockade in mouse model

Biomedicine & Pharmacotherapy ◽

10.1016/j.biopha.2019.108677 ◽

2019 ◽

Vol 112 ◽

pp. 108677 ◽

Cited By ~ 6

Author(s):

Meiqi Zhao ◽

Manyu Luo ◽

Yueqing Xie ◽

Hua Jiang ◽

Cedric Cagliero ◽

...

Keyword(s):

Antitumor Activity ◽

Mouse Model ◽

Half Life

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Site-Specific PEGylation of Factor VIII (PEG-FVIII) Preserves Full Clotting Activity and Extends Therapeutic Efficacy in HemophiliaA Dogs

Blood ◽

10.1182/blood.v112.11.511.511 ◽

2008 ◽

Vol 112 (11) ◽

pp. 511-511 ◽

Cited By ~ 3

Author(s):

Tongyao Liu ◽

David Lillicrap ◽

Xin Zhang ◽

Andrea Labelle ◽

Sandra Powell ◽

...

Keyword(s):

Factor Viii ◽

Whole Blood ◽

Half Life ◽

Therapeutic Efficacy ◽

Neutralizing Antibodies ◽

Hemophilia A ◽

Volume Of Distribution ◽

Pharmacokinetic Analysis ◽

Site Specific ◽

Intravenous Injections

Abstract To improve the effectiveness of Factor VIII replacement therapy for Hemophilia A, we sought to develop a PEGylated Factor VIII that would effectively treat bleeding episodes, while reducing the frequency of intravenous injections required for prophylaxis. Previously, we found that the site-specific PEGylation of Factor VIII (PEG-FVIII) preserves full clotting activity, prolongs circulating half-life and extends therapeutic efficacy in a number of bleeding models in hemophilic mice. To further characterize its activity, four naïve Hemophilia A dogs were treated with either PEG-FVIII or unmodified BDD-FVIII in a cross-over study design. All treated dogs showed normalized Whole Blood Clotting Time (WBCT), whole blood Thromboelastograph (TEG) profile, and Cuticle Bleeding Time within 30 min from dosing. Pharmacokinetic analysis of the decay of plasma FVIII activity and antigen levels showed that PEG-FVIII achieved 2-fold longer half-life and reduced clearance and volume of distribution relative to BDD-FVIII. Consistently, PEG-FVIII also demonstrated significantly prolonged efficacy relative to BDD-FVIII by measurement of WBCT and TEG. Both BDD-FVIII and PEG-FVIII were well tolerated in naïve HemA dogs, normal hematology and serum chemistry values were observed following administration. However, two naive dogs that received BDD-FVIII and one naive dog that received PEG-FVIII developed detectable neutralizing antibodies to human FVIII as early as on day 9 post-treatment. In summary, consistent with our previously reported findings in hemophilic mice, in comparison to BDD-FVIII, PEG-FVIII demonstrated superior half-life, full activity in stopping acute bleeding and prolonged efficacy in hemophilia A dogs. Taken together, the results support the use of site-specific PEGylation to create a homogeneous therapeutic for both prophylactic and on-demand treatment of patients with Hemophilia A.

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Phase 1 dose escalation study of MEDI-565, a bispecific T-cell engager that targets human carcinoembryonic antigen (CEA), in patients with advanced gastrointestinal (GI) adenocarcinomas.

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.4_suppl.320 ◽

2016 ◽

Vol 34 (4_suppl) ◽

pp. 320-320

Author(s):

Michael J. Pishvaian ◽

Michael Morse ◽

Jennifer T. McDevitt ◽

Song Ren ◽

Gabriel Robbie ◽

...

Keyword(s):

T Cell ◽

Antitumor Activity ◽

Dose Escalation ◽

Half Life ◽

High Titer ◽

Phase 1 ◽

Single Chain ◽

Dose Escalation Study ◽

Human T Cell ◽

Grade 3

320 Background: MEDI-565, a bispecific single-chain antibody, targets human CEA on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex. In murine models, MEDI-565 showed antitumor activity in CEA-expressing tumors (J Immunother 2009;34:341-52). Methods: This phase 1, multicenter, open-label, dose-escalation study enrolled adults with GI adenocarcinomas (including esophageal, gastric, small intestine, colorectal, biliary tract, and pancreatic). MEDI-565 was given intravenously over 3 h on days 1–5 in 28-day cycles, with 4 single-patient (pt) (0.75–20 μg) and 5 standard 3+3 escalation (60 μg–3 mg; 1.5–7.5 mg with dexamethasone [dex]) cohorts. Primary objective was to determine the maximum tolerated dose (MTD); secondary objectives were to evaluate pharmacokinetics (PK), antidrug antibody (ADA), and antitumor activity. Results: Study enrolled39 pts: mean age 59 y; 56% male; 28 (72%) colorectal, 6 (15%) pancreatic, 5 (13%) other. Dose-limiting toxicities (grade ≥ 3 nonhematologic) were seen in 4 pts (2 at 3-mg; 2 at 7.5-mg + dex): hypoxia (n = 2), diarrhea, and cytokine release syndrome (CRS). Grade 3 treatment-related adverse events (AEs) seen in 5 pts: diarrhea, CRS, increased alanine aminotransferase, hypertension (all n = 1), and hypoxia (n = 2). Treatment-related serious AEs seen in 6 pts: diarrhea, vomiting, pyrexia, CRS (all n = 1), and hypoxia (n = 2). Five pts discontinued treatment due to AEs: diarrhea, CRS, central nervous system metastases, and hypoxia (n = 2). MEDI-565 exposures increased in approximately dose-proportional manner, with clearance (35–77 L/d) and half-life (2–7 h) typical of drug class. ADA had minor impact; 19 pts (48.7%) had ADAs, 5/39 (12.8%) with high titer, with decreased MEDI-565 concentrations in 2 pts. Plasma inflammatory cytokines were elevated posttreatment in several pts at 1.5- and 3-mg (no dex) dose levels. No objective responses were observed; 11 (28%) pts had stable disease as best response. Conclusions: The MTD of MEDI-565 in pts with GI adenocarcinomas was 5 mg with dex. PK was linear, with fast clearance and short half-life. No objective responses were observed. Clinical trial information: NCT01284231.

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