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.

Results from an international phase 2 study of the anti-CD22 immunotoxin moxetumomab pasudotox in relapsed or refractory childhood B-lineage acute lymphoblastic leukemia.

BACKGROUND: In a multicenter phase 1 study of children with relapsed/refractory acute lymphoblastic leukemia (ALL), moxetumomab pasudotox, an anti-CD22 immunotoxin, demonstrated a manageable safety profile and preliminary evidence of clinical activity. A phase 2 study further evaluated efficacy. PROCEDURE: This international, multicenter, phase 2 study enrolled children with relapsed/refractory B-cell precursor ALL who received moxetumomab pasudotox 40 µg/kg intravenously every other day, for six doses per 21-day cycle. The primary objective was to evaluate the complete response (CR) rate. Secondary objectives included safety, pharmacokinetics, and immunogenicity evaluations. RESULTS: Thirty-two patients (median age, 10 years) were enrolled at 16 sites; 30 received study drug and were evaluable for safety; 28 were evaluable for response. The objective response rate was 28.6%, with three patients (10.7%) achieving morphologic CR, and five patients (17.9%) achieving partial response. Disease progression occurred in 11 patients (39.3%). Ten patients (33.3%) experienced at least one treatment-related serious adverse event, including capillary leak syndrome (CLS; n = 6), hemolytic uremic syndrome (HUS; n = 4), and treatment-related death (n = 1) from pulmonary edema. No differences were observed in inflammatory markers in patients who did or did not develop CLS or HUS. CONCLUSIONS: Despite a signal for clinical activity, this phase 2 study was terminated at interim analysis for a CR rate that did not reach the stage 1 target. Preclinical data suggest enhanced efficacy of moxetumomab pasudotox via continuous infusion or in combination regimens; thus, further studies designed to optimize the efficacy and safety of moxetumomab pasudotox in pediatric ALL may be warranted.

Phase 1 study of the anti-CD22 immunotoxin moxetumomab pasudotox for childhood acute lymphoblastic leukemia.

Novel therapies are needed to overcome chemotherapy resistance for children with relapsed/refractory acute lymphoblastic leukemia (ALL). Moxetumomab pasudotox is a recombinant anti-CD22 immunotoxin. A multicenter phase 1 study was conducted to determine the maximum-tolerated cumulative dose (MTCD) and evaluate safety, activity, pharmacokinetics, and immunogenicity of moxetumomab pasudotox in children, adolescents, and young adults with ALL (N = 55). Moxetumomab pasudotox was administered as a 30-minute IV infusion at doses of 5 to 50 µg/kg every other day for 6 (cohorts A and B) or 10 (cohort C) doses in 21-day cycles. Cohorts B and C received dexamethasone prophylaxis against capillary leak syndrome (CLS). The most common treatment-related adverse events were reversible weight gain, hepatic transaminase elevation, and hypoalbuminemia. Dose-limiting CLS occurred in 2 of 4 patients receiving 30 µg/kg of moxetumomab pasudotox every other day for 6 doses. Incorporation of dexamethasone prevented further dose-limiting CLS. Six of 14 patients receiving 50 µg/kg of moxetumomab pasudotox for 10 doses developed hemolytic uremic syndrome (HUS), thrombotic microangiopathy (TMA), or HUS-like events, exceeding the MTCD. Treatment expansion at 40 µg/kg for 10 doses (n = 11) exceeded the MTCD because of 2 HUS/TMA/HUS-like events. Dose level 6B (ie, 50 µg/kg × 6 doses) was the MTCD, selected as the recommended phase 2 dose. Among 47 evaluable patients, an objective response rate of 32% was observed, including 11 (23%) composite complete responses, 5 of which were minimal residual disease negative by flow cytometry. Moxetumomab pasudotox showed a manageable safety profile and evidence of activity in relapsed or refractory childhood ALL. This trial was registered at www.clinicaltrials.gov as #NCT00659425.

Reductive activation of type 2 ribosome-inactivating proteins is promoted by transmembrane thioredoxin-related protein.

Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.

Increasing stability and toxicity of Pseudomonas exotoxin by attaching an antiproteasic Peptide.

Trypsin-like activities are present within the endocytic pathway and allow cells to inactivate a fraction of incoming toxins, such as Pseudomonas exotoxin (PE), that require endocytic uptake before reaching the cytosol to inactivate protein synthesis. PE is a favorite toxin for building immunotoxins. The latter are promising molecules to fight cancer or transplant rejection, and producing more active toxins is a key challenge. More broadly, increasing protein stability is a potentially useful approach to improve the efficiency of therapeutic proteins. We report here that fusing an antiproteasic peptide (bovine pancreatic trypsin inhibitor, BPTI) to PE increases its toxicity to human cancer cell lines by 20-40-fold. Confocal microscopic examination of toxin endocytosis, digestion, and immunoprecipitation experiments showed that the fused antiproteasic peptide specifically protects PE from trypsin-like activities. Hence, the attached BPTI acts as a bodyguard for the toxin within the endocytic pathway. Moreover, it increased the PE elimination half-time in mice by 70%, indicating that the fused BPTI stabilizes the toxin in vivo. This BPTI-fusion approach may be useful for protecting other circulating or internalized proteins of therapeutic interest from premature degradation.

A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity.

Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.

Immunogenicity of Immunotoxins Containing Pseudomonas Exotoxin A: Causes, Consequences, and Mitigation.

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. Pseudomonas exotoxin A (PE) is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.

Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.

Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

Systematic urokinase-activated anthrax toxin therapy produces regressions of subcutaneous human non-small cell lung tumor in athymic nude mice.

The novel recombinant anthrax toxin, PrAgU2/FP59, composed of the urokinase-activated protective antigen and a fusion protein of Pseudomonas exotoxin and lethal factor was tested for anti-lung cancer efficacy in an in vivo human tumor model. Male athymic nude mice (age 4-6 weeks) were inoculated s.c. with 10 million H1299 non-small cell lung cancer (NSCLC) cells in the left flank. When tumor volumes reached 200 mm(3) (6-8 days), i.p. injection of 100 muL saline or different ratios and doses of PrAgU2/FP59 in 100 muL saline were given every 3 days for four doses and an additional dose at day 29. Animals were monitored twice daily and tumor measurements were made by calipers. The maximum tolerated doses of PrAgU2/FP59 differed dependent on the ratios of PrAgU2 to FP59 over the range of 3:1 to 25:1, respectively. At tolerated doses, tumor regressions were seen in all animals. Complete histologic remission lasting 60 days occurred in 30% of animals. PrAgU2/FP59 showed dramatic anti-NSCLC efficacy and warrants further clinical development for therapy of patients with advanced NSCLC.

Moxetumomab pasudotox for the treatment of relapsed and/or refractory hairy cell leukemia.

Introduction: Hairy cell leukemia is a rare indolent B-cell malignancy, characterized by pancytopenia, recurrent infections, and splenomegaly. After initial therapy with purine nucleoside analogs, up to 50% of patients relapse after several years of remission. The number of relapsed patients is increasing and, until recently, there was no approved therapy with durable responses for hairy cell leukemia patients in the relapsed setting, thus the need for new non-chemotherapy approach with significant efficacy and less myelosuppression. Areas covered: Moxetumomab pasudotox is a recombinant immunotoxin containing a Fv fragment of an anti-CD22 monoclonal antibody and truncated Pseudomonas exotoxin (PE38). The authors reviewed pre-clinical and clinical studies that led to the FDA approval of the drug in patients with relapsed and/or refractory hairy cell leukemia, who received at least two prior therapies, including at least one purine nucleoside analog. Expert opinion: Moxetumomab pasudotox demonstrated a durable complete remission rate of 30% in heavily pretreated patients with hairy cell leukemia, and MRD eradication in 85% of responding patients. Moxetumomab pasudotox got a global FDA approval in September 2018. The US prescribing information carries boxed warnings regarding the risk of capillary leak syndrome and hemolytic uremic syndrome. Long-term follow-up of the pivotal study is ongoing (NCT01829711).

Moxetumomab pasudotox for the treatment of hairy cell leukemia.

Introduction: Cladribine and pentostatin are the drugs of choice in the treatment of hairy cell leukemia (HCL). Recently, immunotoxin moxetumomab pasudotox has been introduced to improve the prognosis in relapsed and refractory HCL. Areas covered: This review discusses the mechanism of action, safety, and efficacy of moxetumomab pasudotox in HCL patients. A literature review of the MEDLINE database for articles in English concerning immunotoxins, moxetumomab pasudotox, and hairy cell leukemia was conducted via PubMed. Publications from 2000 through December 2018 were scrutinized. The search terms used were immunotoxins and moxetumomab pasudotox in conjunction with hairy cell leukemia. Conference proceedings from the previous five years of the American Society of Hematology, European Hematology Association and American Society of Clinical Oncology were searched manually. Additional relevant publications were obtained by reviewing the references from the chosen articles. Results/conclusion: Moxetumomab pasudotox, a novel recombinant anti-CD22 immunotoxin, was well tolerated and active in the previous phase 1 and 3 studies in patients with HCL. The drug has been approved in 2018 by the FDA for the treatment of patients with relapsed/refractory HCL who had at least two prior systemic therapies including at least one purine nucleoside analog. Expert opinion: The use of moxetumomab pasudotox is a promising new strategy for the treatment of HCL.

Potent and specific fusion toxins consisting of a HER2­binding, ABD­derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A.

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain­derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA­derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half­life extension, can be used for specific killing of HER2­expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (KD 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (KD) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50­values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA­derivatives are promising agents for targeted cancer therapy.

Induction of hyperintense signal on T2-weighted MR images correlates with infusion distribution from intracerebral convection-enhanced delivery of a tumor-targeted cytotoxin.

OBJECTIVE: Convection-enhanced delivery is a promising approach to intracerebral drug delivery in which a fluid pressure gradient is used to infuse therapeutic macromolecules through an indwelling catheter into the interstitial spaces of the brain. Our purpose was to test the hypothesis that hyperintense signal changes on T2-weighted images produced by such infusions can be used to track drug distribution. SUBJECTS AND METHODS: Seven adults with recurrent malignant glioma underwent concurrent intracerebral infusions of the tumor-targeted cytotoxin cintredekin besudotox and 123I-labeled human serum albumin. The agents were administered through a total of 18 catheters among the seven patients. Adequacy of distribution of drug was determined by evidence of distribution of 123I-labeled human serum albumin on SPECT images coregistered with MR images. Qualitative analysis was performed by three blinded observers. Quantitative analysis also was performed. RESULTS: Infusions into 12 catheters produced intraparenchymal distribution as seen on SPECT images, but infusions into six catheters did not. At qualitative assessment of signal changes on MR images, reviewers correctly predicted which catheters would produce extraparenchymal distribution and which catheters would produce parenchymal distribution. Of the 12 infusions that produced intraparenchymal distribution, four catheters had been placed in regions of relatively normal signal intensity and produced regions of newly increased signal intensity, the volume of which highly correlated with the volume and geometry of distribution on SPECT (r2 = 0.9502). Eight infusions that produced intraparenchymal distribution were performed in regions of preexisting hyperintense signal. In these brains, additional signal changes were always produced, but quantitative correlations between areas of newly increased signal intensity and the volume and geometry of distribution on SPECT could not be established. CONCLUSION: Convection-enhanced infusions frequently do not provide intraparenchymal drug distribution, and these failures can be identified with MRI soon after infusion. When infusions are performed into regions of normal signal intensity, development of hyperintense signal change strongly correlates with the volume and geometry of distribution of infusate.

Real-time, image-guided, convection-enhanced delivery of interleukin 13 bound to pseudomonas exotoxin.

PURPOSE: To determine if the tumor-targeted cytotoxin interleukin 13 bound to Pseudomonas exotoxin (IL13-PE) could be delivered to the brainstem safely at therapeutic doses while monitoring its distribution in real-time using a surrogate magnetic resonance imaging tracer, we used convection-enhanced delivery to perfuse rat and primate brainstems with IL13-PE and gadolinium-bound albumin (Gd-albumin). EXPERIMENTAL DESIGN: Thirty rats underwent convective brainstem perfusion of IL13-PE (0.25, 0.5, or 10 microg/mL) or vehicle. Twelve primates underwent convective brainstem perfusion of either IL13-PE (0.25, 0.5, or 10 microg/mL; n = 8), co-infusion of 125I-IL13-PE and Gd-albumin (n = 2), or co-infusion of IL13-PE (0.5 microg/mL) and Gd-albumin (n = 2). The animals were permitted to survive for up to 28 days before sacrifice and histologic assessment. RESULTS: Rats showed no evidence of toxicity at all doses. Primates showed no toxicity at 0.25 or 0.5 microg/mL but showed clinical and histologic toxicity at 10 microg/mL. Quantitative autoradiography confirmed that Gd-albumin precisely tracked IL13-PE anatomic distribution and accurately showed the volume of distribution. CONCLUSIONS: IL13-PE can be delivered safely and effectively to the primate brainstem at therapeutic concentrations and over clinically relevant volumes using convection-enhanced delivery. Moreover, the distribution of IL13-PE can be accurately tracked by co-infusion of Gd-albumin using real-time magnetic resonance imaging.

Convection-enhanced and local delivery of targeted cytotoxins in the treatment of malignant gliomas.

Despite advances in our knowledge about the genesis, molecular biology, and natural history of malignant gliomas and the use of a multi-disciplinary approach to their treatment, patients harboring this diagnosis continue to face a grim prognosis. At the time of diagnosis, patients typically undergo surgery for the establishment of a histologic diagnosis, the reduction of tumor burden, and the relief of mass effect, with the maintenance of the patient's neurological function in mind. This is followed by the administration of adjuvant therapeutics, including radiation therapy and chemotherapy. Many investigational agents with laboratory evidence of efficacy against malignant gliomas have not met their promise in the clinical setting, largely due to the barriers that they must overcome to reach the tumor at a therapeutically meaningful concentration for a durable period of time. The relevant aspects of the blood-brain barrier, blood-tumor barrier, and blood-cerebrospinal fluid barrier, as they pertain to the delivery of agents to the tumor, will be discussed along with the strategies devised to circumvent them. This discussion will be followed by a description of agents currently in preclinical and clinical development, many of which are the result of intense ongoing research into the molecular biology of gliomas.

A recombinant protein LHRH-PE40 for tumour therapy: preclinical safety studies.

LHRH-PE40, a recombinant DNA-derived protein composed of LHRH and Pseudomonas aeruginosa exotoxin A, is being developed for the treatment of malignant tumours. This experiment was designed to assess its preclinical safety. Reproductive toxicity studies, pharmacokinetic studies, single- and repeat-dose intraperitoneal or intravenous toxicity studies in mice, rats and monkeys were conducted to assess the toxicity of LHRH-PE40. In intravenous single-dose studies in mice, the LD50 was 731.26 microg/kg and 676.03 microg/kg in male and female mice respectively. In single-dose studies and repeat-dose range-finding studies in rats, dose-limited severe vascular leakage syndromes occurred. In repeat-dose long-term studies, except drug-related vascular leakage syndromes, other drug-related changes included decreased testis weight and testis atrophy. In single-dose and repeat-dose studies in monkeys, dose-limited acute tubular necrosis of the kidneys was the chief finding. In reproductive studies, drug-related changes were decreased food intakes, decreased testis weight and uterus weight, decreased foetus weight and increased foetus mortality, increased maternal and F1 offspring mortality and decreased maternal and F1 offspring body weight. Pharmacokinetic studies showed a similar half-time of distribution and clearance in mice and monkeys. Tissue distribution showed a high concentration in the kidneys and a low concentration in the brain. LHRH-PE40 induced vascular leak syndromes in rats and acute tubular necrosis in monkeys. It also led to testicle atrophy in rats and overt productive toxicity to parents and F1 generations in mice. Because of these findings, it should be monitored carefully in human clinical trials for things such as respiratory, urinary and reproductive toxicities.

Accumulation of a recombinant immunotoxin in a tumor in vivo: fewer than 1000 molecules per cell are sufficient for complete responses.

Recombinant immunotoxins have been shown to cure human tumor xenografts in mice, but their biodistribution to both tumors and normal organs has not been reported. Anti-Tac(Fv)-PE38 is a single-chain recombinant immunotoxin composed of the variable heavy and light domains of the anti-Tac monoclonal antibody that reacts with the primate interleukin 2 (IL2) receptor alpha subunit (IL2R alpha or CD25) fused to a truncated form of Pseudomonas exotoxin (PE). 125I-labeled anti-Tac(Fv)-PE38 was given i.v. to immunodeficient mice each bearing two A431 tumors, one that expresses IL2R alpha (ATAC-4) and one that does not (A431, parental). A single i.v. dose of 4 microg/mouse caused complete regression of the IL2R alpha + tumor. At 6 h, over 6% of the injected dose/g was found in the ATAC-4 tumor, and 2% was in the A431 tumor. Uptake in the ATAC-4 tumor was higher than in any other tissue. Sections of tumor examined by autoradiography indicated that anti-Tac(Fv)-PE38 was distributed throughout the entire tumor, with some portions having higher uptake than others. By subtracting uptake in tumors without receptor (A431) from uptake in receptor-containing tumors (ATAC-4), we calculated that at least 400 molecules/cell specifically bound to IL2R alpha-positive tumor cells at 90 min and 750 molecules/cell bound at 360 min. This is similar to the 400-870 molecules/cell required for >99.9% killing of ATAC-4 cells growing as a monolayer. The results show that solid tumors in mice can be eradicated like cells in tissue culture, and that delivery of less than 1000 molecules/cell is sufficient to cause complete tumor regressions.

Polyethylene glycol-modified chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin.

Modification of proteins with monomethoxy-polyethylene glycol (mPEG) has been shown to prolong circulation time and to reduce immunogenicity. To make a mPEG-modified recombinant toxin that retained cytotoxic activity but had a longer residence time in circulation, we have constructed an altered form of TGF alpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGF alpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell-binding domain. In the newly designed protein, termed TGF alpha R29-L2-CH2-PE38QQ delta (TCP), there are no lysine residues in the TGF alpha and PE38 portions. Human IgG4 constant region CH2 and a tetradecapeptide linker; L2, are inserted between TGF alpha and PE38. Together, L2 and CH2 contain 13 lysine residues as potential modification sites for mPEG. mPEG conjugates of TCP (PEG-TCP) were generated and the products were resolved by ion exchange chromatography. Two PEG-TCP species termed B4 and B6 retained 15 and 4% of cytotoxicity, respectively, and 26% of their receptor binding activity compared with the unmodified TCP. Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in animals. The mean residence times of B4 and B6 were 37 and 68 min, respectively, compared to 7 min for TCP. When administered i.v. to tumor bearing mice, both B4 and B6 produced marked antitumor effects whereas the unmodified TCP had none. Also, the immunogenicity of PEG-TCP was 5-10 times less than that of TCP. We suggest that the prolonged circulating time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.

Preclinical development of a recombinant toxin containing circularly permuted interleukin 4 and truncated Pseudomonas exotoxin for therapy of malignant astrocytoma.

Effective treatment is lacking for malignant glioblastoma/astrocytoma. We have identified interleukin-4 receptors (IL-4R) on human malignant astrocytoma. We demonstrate that 16 of 21 surgical samples of high-grade astrocytoma and glioblastoma but not normal brain tissues expressed IL-4R as assessed by reverse transcriptase PCR. We further demonstrate that human malignant astrocytoma cell lines express high-affinity IL-4R. Using a chimeric protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin A, we observed that this toxin IL4(38-37)-PE38KDEL) is highly cytotoxic to IL-4R-bearing glioblastoma cells. Compared with a previously reported IL4-PE chimeric protein (IL-PE4E), IL4(38-37)-PE38KDEL bound with higher affinity and was 3-30-fold more cytotoxic to glioblastoma cell lines. Upon intrathecal administration in monkeys, high cerebrospinal fluid IL4(38-37)-PE38KDEL levels were achieved using 2- and 6-microg/kg doses without any central nervous system or other abnormalities. IL4(38-37)-PE38KDEL levels were not detectable in the serum of any monkey studied. When IL4(38-37)-PE38KDEL was injected into the right frontal cortex of rats, localized necrosis was observed at 1000-ng/ml doses but not at < or = 100-ng/ml doses. We conclude that by localized administration, nontoxic levels of IL4(38-37)-PE38KDEL can be achieved, which may have significant cytotoxic activity against malignant astrocytoma.

Increased antitumor activity of a circularly permuted interleukin 4-toxin in mice with interleukin 4 receptor-bearing human carcinoma.

We reported previously that circularly permuted interleukin-4 (IL4), composed of amino acids 38-129 of IL4 connected by a linker peptide GGNGG to amino acids 1-37, is preferable to native IL4 for fusing to the amino terminus of truncated Pseudomonas exotoxin (PE) to make a recombinant toxin, because the new ligand-toxin junction results in improved IL4 receptor (IL4R)-binding (R. J. Kreitman et al., Proc. Natl. Acad. Sci. USA, 91: 6889-6893, 1994). We now report that the improved binding of circularly permuted IL4-toxin is associated with improved antitumor activity in tumor-bearing mice. For in vivo testing, we made an improved circularly permuted IL4-toxin, termed IL4(38-37)-PE38KDEL. It contains an N38D mutation at the amino terminus, allowing improved expression and large-scale production in Escherichia coli. It also contains the truncated toxin PE38KDEL, which is composed of amino acids 253-364 and 381-608 of PE, followed by KDEL. To evaluate antitumor activity, nude mice carrying s.c. tumors composed of IL4R-bearing human A431 epidermoid carcinoma cells were injected with recombinant toxins i.v. every other day for three doses. IL4(38-37)-PE38KDEL induced complete remissions in 80% of mice receiving 50 micrograms/kg x 3 and 100% of mice receiving 100 micrograms/kg x 3, while only 70% of mice receiving 200 micrograms/kg x 3 of the native IL4-toxin IL4-PE38KDEL obtained complete remission. Disease-free survival after obtaining complete remissions was higher in mice treated with IL4(38-37)-PE38KDEL 50 micrograms/Kg QOD x 3 than with IL4-PE38KDEL 200 micrograms/Kg QOD x 3 (P < 0.03). IL4(38-37)-PE38KDEL and IL4-PE38KDEL exhibited similar toxicity and pharmacokinetics in the mice, indicating that the improved antitumor activity of the circularly permuted IL4-toxin was due to its improved binding to the IL4R on the target cells.