Bispecific Ligand-directed Toxin Research Articles

Bispecific ligand-directed toxin targeting CD22 and CD19 (DT2219) for refractory B-cell malignancies: Results of phase I-II trial.

e19066 Background: The novel bispecific toxin DT2219 consists of a fusion between the catalytic domain of diphtheria toxin (DT) and single-chain variable fragments (scFV) of antibodies targeting human CD19 and CD22. Methods: The primary objective of Phase I Dose was to determine DT2219 dose which is well tolerated on an 8 dose schedule (days 1, 3, 5, 8 and day 15, 17, 19 and 22). Phase II Extension aimed to establish a preliminary estimate of overall response at day 29 in patients stratified to Arm A (relapsed/refractory B-cell lymphoma) and Arm B (B-cell leukemia). Baseline serum level of neutralizing antibody (NA) against diphteria toxin below 50% was required for eligibility. Results: Eighteen patients (NHL n = 12; ALL n = 6) at median age 65 years (range 50-83 yrs) with relapse/refractory disease and confirmed expression of CD19 and/or CD22 were enrolled. Treatment was well tolerated at 60 mcg/kg x 8 doses and the most common adverse events included capillary leak sy, elevated AST/ALT, low albumin, weight gain and leukopenia. All were Gr 1-2 and resolved after 3-5 days allowing day 15 DT2219 administration. There were no neutropenic fever or immune mediated adverse events. Four patients experienced dose limiting toxicity at dose 80 μg/kg/day: Gr 4 capillary leak syndrome (n = 1), Gr 3 LFTs abnormalities (n = 2) and Gr 4 thrombocytopenia > 7 days duration (n = 1). Thirteen patients were evaluable for response and 3 experienced objective clinical benefit. One patient with primary refractory pre-B acute lymphoblastic leukemia achieved complete remission after 1st cycle. Two patients with transformed lymphoma demonstrated transient tumor shrinkage, however, DT2219 therapy was discontinued due to DLT and increased NA titer after 1st cycle (pre C1 28%, pre C2 108%). Correlative studies showed a low incidence of neutralizing antibody in NHL patients recently exposed to Rituximab. Conclusions: We have established the biologically active dose of DT2219 at 60 μg/kg/day ×8 doses. Further development of DT2219 includes combination with rituximab (to reduce NA formation) and synergistic combinations against B-cell malignancies. Clinical trial information: NCT02370160.

Targeting EGFR and uPAR on human rhabdomyosarcoma, osteosarcoma, and ovarian adenocarcinoma with a bispecific ligand-directed toxin.

PurposeHuman sarcomas are rare and difficult to treat cancerous tumors typically arising from soft tissue or bone. Conversely, carcinomas are the most common cancer subtype in humans and the primary cause of mortality across all cancer patients. While conventional therapeutic modalities can prolong disease-free intervals and survival in some cases, treatment of refractory or recurrent solid tumors is challenging, and tumor-related mortality remains unacceptably high. The identification of overexpressed cell surface receptors on sarcoma and carcinoma cells has provided a valuable tool to develop targeted toxins as an alternative anticancer strategy. Recent investigation of recombinant protein-linked toxins that specifically target these cancer receptors has led to the development of highly specific, cytotoxic, and deimmunized drugs that can kill cancer cells.MethodsThis study investigated a recombinant protein called epidermal growth factor bispecific angiotoxin (eBAT), which is designed to target the epidermal growth factor receptor (EGFR) on cancer cells and the urokinase plasminogen activator receptor (uPAR) on cancer cells and associated tumor vasculature. Both receptors are expressed by a variety of human sarcomas and carcinomas. Flow cytometry techniques were used to determine binding affinity of eBAT to cancer cells, and proliferation assays were performed to calculate tumor killing ability based on half-maximal inhibitory concentrations.ResultseBAT demonstrated cytotoxicity against a variety of sarcoma and carcinoma cells that overexpress EGFR and uPAR in vitro and showed greater cell killing ability and binding affinity to cancer cells compared with its monospecific counterparts.ConclusionThe results of our study are promising, and further studies will be necessary to confirm the applicability of eBAT as a supplementary therapy for a variety of sarcomas, carcinomas, and possibly other refractory malignancies that express EGFR and uPAR.

A Deimmunized Bispecific Ligand-Directed Toxin That Shows an Impressive Anti–Pancreatic Cancer Effect in a Systemic Nude Mouse Orthotopic Model

The objective was to test a bispecific ligand-directed toxin (BLT), with reduced immunogenicity for enhanced efficacy in targeting orthotopic pancreatic cancer in vivo. A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single chain molecule with deimmunized pseudomonas exotoxin (dEGF4KDEL). Key amino acids dictating B-cell generation of neutralizing antitoxin antibodies were mutated. Bioassays were used to determine whether mutation reduced potency, and enzyme-linked immunosorbent assay studies were performed to determine whether antitoxin antibodies were reduced. A genetically altered luciferase MIA PaCa-2 xenograft model was used to image in real time and determine effects on systemic malignant human cancer. Bispecific ligand-directed toxins targeting B cells were used as specificity controls. Deimmunized EGF4KDEL was significantly effective after systemic injection against established orthotopic MIA PaCa-2 pancreatic cancer and selectively prevented metastasis. Mutagenesis significantly reduced antitoxin levels in vivo with no apparent activity loss in vitro. The drug was effective against 3 human pancreatic cancer lines in vitro, MIA PaCa-2, SW1990, and S2VP10. Despite the metastatic nature of the MIA PaCa-2 orthotopic tumor xenografted in nude mice, high percentages of tumors responded to extended dEGFKDEL treatment resulting in significant anticancer effects and disease-free survivors.

Evaluation of a bispecific biological drug designed to simultaneously target glioblastoma and its neovasculature in the brain

The authors of this study aimed to genetically design a bispecific targeted toxin that would simultaneously target overexpressed markers on glioma as well as the tumor vasculature, to mutate certain amino acids to reduce the immunogenicity of this new drug, and to determine whether the drug was able to effectively reduce aggressive human brain tumors in a rat xenograft model via a novel hollow fiber (HF) catheter delivery system. A new bispecific ligand-directed toxin (BLT) was created in which 2 human cytokines-epidermal growth factor ([EGF], targeting overexpressed EGF receptor) and amino acid terminal fragment ([ATF], targeting urokinase plasminogen activator receptor)-were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin with a terminal lysyl-aspartyl-glutamyl-leucine (KDEL) sequence. Site-specific mutagenesis was used to mutate amino acids in 7 key epitopic toxin regions that dictate the B cell generation of neutralizing antitoxin antibodies to deimmunize the drug, now called "EGFATFKDEL 7mut." Bioassays were used to determine whether mutation reduced the drug's potency, and enzyme-linked immunosorbent assay studies were performed to determine whether antitoxin antibodies were decreased. Aggressive brain tumors were intracranially established in nude rats by using human U87 glioma genetically marked with a firefly luciferase reporter gene (U87-luc), and the rats were stereotactically treated with 2 intracranial injections of deimmunized EGFATFKDEL via convection-enhanced delivery (CED). Drug was administered through a novel HF catheter to reduce drug backflow upon delivery. In vitro, EGFATFKDEL 7mut selectively killed the human glioblastoma cell line U87-luc as well as cultured human endothelial cells in the form of the human umbilical vein endothelial cells. Deimmunization did not reduce drug activity. In vivo, when rats with brain tumors were intracranially treated with drug via CED and a novel HF catheter to reduce backflow, there were significant tumor reductions in 2 experiments (p < 0.01). Some rats survived with a tumor-free status until 130 days post-tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The maximal tolerated dose of EGFATFKDEL 7mut was established at 2 μg/injection or 8.0 μg/kg, and data indicated that this dose was nontoxic. Antitoxin antibodies were reduced by at least 90%. First, data indicated that the BLT framework is effective for simultaneously targeting glioma and its neovasculature. Second, in the rodent CED studies, newly developed HF catheters that limit backflow are effective for drug delivery. Third, by mutating critical amino acids, the authors reduced the threat of the interference of neutralizing antibodies that are generated against the drug. The authors' experiments addressed some of the most urgent limitations in the targeted toxin field.

A novel bispecific ligand-directed toxin designed to simultaneously target EGFR on human glioblastoma cells and uPAR on tumor neovasculature

A bispecific ligand-directed toxin (BLT), called EGFATFKDEL, consisting of human epidermal growth factor, a fragment of urokinase, and truncated pseudomonas exotoxin (PE38) was assembled in order to target human glioblastoma. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGFATFKDEL 7mut. In vitro, the drug selectively killed several human glioblastoma cell lines. EGFATFKDEL is our first BLT designed to simultaneously target EGFR on solid tumors and uPAR on the tumor neovasculature. In vitro assays revealed that the agent is effective against glioblastoma cell lines as well as human umbilical vein endothelial cells (HUVEC). Additionally, the bispecific drug displayed enhanced binding to overexpressed epidermal growth factor receptor and urokinase receptor when compared to similar monospecific drugs, EGFKDEL and ATFKDEL. In vivo, an aggressive human glioblastoma cell line was genetically marked with a firefly luciferase reporter gene and administered to the flanks of nude mice. Treatment with intratumoral injections of EGFATFKDEL 7mut eradicated small tumors in over half of the treated mice, which survived with tumor free status at least 100days post tumor inoculation. ATFKDEL, which primarily targets the tumor neovasculature, prevented tumor growth but did not result in tumor-free mice in most cases. Specificity was shown by treating with an irrelevant BLT control which did not protect mice. Finally, immunization experiments in immunocompetent mice revealed significantly reduced anti-toxin production in EGFATFKDEL 7mut treated groups. Thus, EGFATFKDEL 7mut is an effective drug for glioblastoma therapy in this murine model and warrants further study.

A New Drug Delivery Method of Bispecific Ligand-Directed Toxins, Which Reduces Toxicity and Promotes Efficacy in a Model of Orthotopic Pancreatic Cancer

Biologicals targeting epidermal growth factor (EGF) and interleukin 13 receptors not only react with overexpressed markers on cancer cells but also react with receptors on normal cells. Because we developed novel bispecific ligand-directed toxins synthesized by cloning EGF and interleukin 13 on the same molecule with toxin, our objective was to determine whether we could block normal receptors while still targeting receptors overexpressed on cancer cells, thereby decreasing toxicity while maintaining efficacy. A method, toxicity blocking (ToxBloc), was developed in which a bolus intraperitoneal dose of recombinant EGF13 (without toxin) was given to mice approximately 15 to 20 minutes before DTEGF13. Experiments were then performed to determine whether the maximal tolerated dose (MTD) was reduced and whether we were still able to eliminate progression of aggressive human, metastatic, pancreatic cancer induced by orthotopic injection (OT) in nude mice. ToxBloc permitted us to safely exceed the DTEGF13 maximal tolerated dose by 15-fold. This approach permitted repetitive high dosing with the bispecific ligand-directed toxin resulting in tumor regression (P < 0.01). Tumor effects were documented using a tumor imaging model in which OT tumor growth was monitored noninvasively in real time. ToxBloc was selective because other bispecific peptides did not block. ToxBloc represents a new method of drug delivery and a potential solution to the problem of toxicity.

Bioengineering a Unique Deimmunized Bispecific Targeted Toxin That Simultaneously Recognizes Human CD22 and CD19 Receptors in a Mouse Model of B-Cell Metastases

A drug of high potency and reduced immunogenicity is needed to develop a targeted biological drug that when injected systemically can penetrate to malignant B cells. Therefore, a novel deimmunized bispecific ligand-directed toxin targeted by dual high-affinity single-chain Fvs (scFv) spliced to PE38 with a KDEL COOH-terminus was genetically engineered. The aims were to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit antitoxin antibody responses, and show that mutated drug was effective against systemic B-cell lymphoma in vivo. Both human anti-CD22 scFv and anti-CD19 scFv were cloned onto the same single-chain molecule with truncated pseudomonas exotoxin (PE38) to create the drug. Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISAs were done to determine whether antitoxin antibodies were reduced. Finally, a powerful genetically altered luciferase xenograft model was used that could be imaged in real time to determine the effect on systemic malignant human B-cell lymphoma, Raji-luc. Patient B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B lymphoma were high in CD22 and CD19 expression. 2219KDEL7mut was significantly effective against systemic Raji-luc in mice and prevented metastatic spread. Mutagenesis reduced neutralizing antitoxin antibodies by approximately 80% with no apparent loss in in vitro or in vivo activity. Because 2219KDEL7mut immunogenicity was significantly reduced and the drug was highly effective in vivo, we can now give multiple drug treatments with targeted toxins in future clinical trials.

A Novel Reduced Immunogenicity Bispecific Targeted Toxin Simultaneously Recognizing Human Epidermal Growth Factor and Interleukin-4 Receptors in a Mouse Model of Metastatic Breast Carcinoma

To develop a targeted biological drug that when systemically injected can penetrate to metastatic breast cancer tumors, one needs a drug of high potency and reduced immunogenicity. Thus, we bioengineered a novel bispecific ligand-directed toxin (BLT) targeted by dual high-affinity cytokines with a PE(38)KDEL COOH terminus. Our purpose was to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit B-cell antitoxin antibody responses, and show that mutated drug was effective against systemic breast cancer in vivo. A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin (PE(38)). Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were done to determine whether antitoxin antibodies were reduced. Finally, a genetically altered luciferase xenograft model was used; this model could be imaged in real time to determine the effect on the systemic malignant human breast cancer MDA-MB-231. EGF4KDEL 7mut was significantly effective against established systemic human breast cancer and prevented metastatic spread. Mutagenesis reduced immunogenicity by approximately 90% with no apparent loss in in vitro or in vivo activity. Because EGF4KDEL 7mut was highly effective even when we waited 26 days to begin therapy and because immunogenicity was significantly reduced, we can now give multiple drug treatments for chemotherapy-refractory breast cancer in clinical trials.

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

Background:Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy.Methods:To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut.Results:In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (P<0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14–16 days post tumour innoculation.Conclusion:These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.

Intracranial elimination of human glioblastoma brain tumors in nude rats using the bispecific ligand-directed toxin, DTEGF13 and convection enhanced delivery

A bispecific ligand-directed toxin (BLT) consisting of human interleukin-13, epithelial growth factor, and the first 389 amino acids of diphtheria toxin was assembled in order to target human glioblastoma. In vitro, DTEGF13 selectively killed the human glioblastoma cell line U87-luc as well as other human glioblastomas. DTEGF13 fulfilled the requirement of a successful BLT by having greater activity than either of its monospecific counterparts or their mixture proving it necessary to have both ligands on the same single chain molecule. Aggressive brain tumors established intracranially (IC) in nude rats with U87 glioma genetically marked with a firefly luciferase reporter gene were treated with two injections of DTEGF13 using convection enhanced delivery resulting in tumor eradication in 50% of the rats which survived with tumor free status at least 110 days post tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The bispecific DTEGF13 MTD dose was measured at 2 microg/injection or 0.5 microg/kg and toxicity studies indicated safety in this dose. Combination of monospecific DTEGF and DTIL13 did not inhibit tumor growth. ELISA assay indicated that anti-DT antibodies were not generated in normal immunocompetent rats given identical intracranial DTEGF13 therapy. Thus, DTEGF13 is safe and efficacious as an alternative drug for glioblastoma therapy and warrants further study.

Genetic alteration of a bispecific ligand-directed toxin targeting human CD19 and CD22 receptors resulting in improved efficacy against systemic B cell malignancy

A bispecific ligand-directed toxin (BLT) called DT2219ARL consisting of two scFv ligands recognizing CD19 and CD22 and catalytic DT 390 was genetically enhanced for superior in vivo anti-leukemia activity. Genetic alterations included reverse orienting VH-VL domains and adding aggregation reducing/stabilizing linkers. In vivo, these improvements resulted in previously unseen long-term tumor-free survivors measured in a bioluminescent xenograft imaging model in which the progression of human Raji Burkitt's lymphoma could be tracked in real time and in a Daudi model as well. Studies showed DT2219ARL was potent (IC 50s 0.06–0.2 nM range) and selectively blockable. Imaging studies indicated the highly invasive nature of this B cell malignancy model and showed it likely induced pre-terminal hind limb paralysis because of metastasis to spinal regions prevented by DT2219ARL. DT2219ARL represents a new class of bispecific biological that can be continually improved by genetic mutation.

POTENT AND PROLONGED THERAPEUTIC ACTIVITY OF RECOMBINANT BISPECIFIC LIGAND-DIRECTED TOXIN (BLT), DTEGF13 AGAINST ORTHOTOPIC PANCREATIC CANCER IN NUDE MICE

A new recombinant bispecific ligand-directed toxin (BLT), DTEGF13 was genetically engineered consisting of human EGF and IL-13 on the same single chain molecule with truncated diphtheria toxin (DT390). DTEGF13 had a far greater activity than its monospecific counterparts and targeted overexpressed epithelial growth factor (EGF) receptors and interleukin 13 (IL13) receptors on pancreatic tumor cell lines MiaPaCa-2 and SW1990. Since the BLT had greater activity against human pancreatic tumor lines than a mixture of the monospecific agents, there was a clear advantage to delivering the two ligands with toxin on the same molecule. DTEGF13 was studied in an ontarget animal model since it reacted with native mouse EGFR and IL13R, which limited its maximal tolerated dose. To provide a new method of delivering drug so as to exceed the MTD by 15-fold, we developed profectitious drug delivery (PDD) in which we synthesized EGF13 devoid of toxin and used excessive amounts to block to EGF and IL13 receptors on normal cells. Interestingly, when DTEGF13 was given IP using PDD, multiple courses prevented the progression of lethal orthotopic pancreatic cancer in nude mice as measured by bioluminescent imaging in real time. This new recombinant protein shows potential as an important alternative agent in managing the spread of metastatic drug refractory pancreatic cancer. Project Funding: Pancreatic SPORE P20 CA101955-01, Randy Shaver Foundation