Exotoxin Research Articles

Primary T cells for mRNA-mediated immunotoxin delivery.

Immune cells become increasingly attractive as delivery system for immunotoxins in cancer therapy to reduce the intrinsic toxicity and severe side effects of chimeric protein toxins. In this study, we investigated the potential of human primary T cells to deliver a secreted immunotoxin through transient messenger RNA (mRNA) transfection. The chimeric protein toxin was directed toward the neovasculature of cancer cells by fusing a truncated version of Pseudomonas exotoxin A (PE38) to human vascular endothelial growth factor (VEGF) and to the single chain variable fragment (scFv) of anti-Her2/neu. Protocols for the transient transfection of human embryonic kidney cells (HEK293) as well as activated primary human T cells were established. Transient transfection with mRNA coding for the immunotoxins e23-PE38, VEGF-PE38 and its attenuated variant VEGF-PE38D yielded efficient expression and secretion. Mass spectrometry analysis endorsed that a fraction of VEGF-PE38D was properly translocated into the endoplasmic reticulum. Furthermore, cytotoxic activity of immunotoxin secreting T cells toward cancer cells was confirmed in co-culture with ovarian adenocarcinoma cells in the presence of a bispecific antibody (bsAb), highlighting the potential of primary T cells for mRNA-mediated immunotoxin delivery.

The Effect of the Targeted Recombinant Toxin DARPin-PE40 on the Dynamics of HER2-Positive Tumor Growth

The development of targeted toxins based on non-immunoglobulin targeting molecules appears to be one of the most advanced approaches in the targeted therapy of malignant tumors with a high expression of the HER2 receptor. Earlier, we showed that the targeted toxin DARPin-PE40 consisting of the HER2-specific non-immunoglobulin polypeptide (the targeting module) and a fragment of Pseudomonas exotoxin A (the toxic module) exhibits an antitumor effect in vivo against the HER2-positive adenocarcinoma xenograft. In this work, an in-depth analysis of the effect of DARPin-PE40 on the growth dynamics of experimental xenograft tumors was carried out. DARPin-PE40 was shown to inhibit tumor growth at a dose of 25 and 50 μg/animal and to cause tumor node reduction at a dose of 80 μg/animal, followed by growth resumption at the end of therapy. An evaluation of the tumor growth dynamics revealed statistically significant differences in tumor volume in mice in the experimental groups compared to the control group. The results testify to the potential of using the created targeted toxin as an agent for the targeted therapy of HER2-overexpressing tumors.

Differences in Genetic Background Contribute to Pseudomonas Exotoxin A-Induced Hepatotoxicity in Rats

Pseudomonas aeruginosa exotoxin A (PEA) causes severe hepatotoxicity in experimental animals and is useful in investigations of immune-mediated liver injury. However, strain differences in the sensitivity to PEA-induced hepatotoxicity in rats remains be elucidated. In this study, we determined the severity of PEA-induced hepatotoxicity in six genetically different rat strains. Male LE (Long Evans), Wistar, F344, WKY, BN/SsN and LEW rats were administered a single intravenous injection of PEA (20 μg/kg). Significantly elevated serum ALT and AST levels, massive necrosis and hemorrhage, and numerous TUNEL-positive hepatocytes were observed in BN/SsN rats. In contrast, low levels of ALT and AST as well as mild changes in liver histopathology were observed in Wistar and F344 rats. Moderate levels of hepatic injuries were observed in LE, WKY, and LEW rats. Pro-inflammatory cytokines including TNF-α, IL-2 and IL-6 serum levels were markedly increased in BN/SsN rats compared to Wistar and F344 rats. However, the hepatic levels of low density lipoprotein receptor-related protein (LRP), which functions as the PEA receptor, were not significantly different in each strain. Taken together, we suggest that BN/SsN is the most sensitive rat strain, whereas Wistar and F344 were the most resistant rat strains to PEA-induced liver damage. The different genetic background of rat strains plays an important role in the susceptibility to PEA-induced epatotoxicity that may depend on immune-regulation but not LRP receptor levels.

Abstract 614: VB4-845 tumor cell killing in a combination study with the anti-PD-1, Nivolumab

Abstract VB4-845 is a Targeted Protein Therapeutic (TPT) comprised of a scFv fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to a truncated form of Pseudomonas exotoxin A (ETA), via a flexible linker. Due to the inherent immunogenicity of the ETA moiety, VB4-845 is only used for the treatment of loco-regionally accessible tumors and is currently in a Phase III clinical trial for the treatment of high grade, non-muscle invasive bladder cancer. In a Phase I study in late stage squamous cell carcinoma of the head and neck, direct injection of VB4-845 led to shrinkage of the principal injected tumor, as well as non-targeted tumors in some patients, suggesting the activation of a T cell-mediated anti-tumor response through cross-priming. Only cells undergoing immunogenic cell death, or ICD, are capable of stimulating cross-priming. ICD is characterized by distinct “eat-me” signals such as the release of ATP and HMGB1, and cell surface translocation of calreticulin, an endoplasmic reticulum chaperone protein. To evaluate whether VB4-845-mediated cell killing results in ICD events, treated tumor cell lines were assessed for these distinct signaling molecules. In vitro studies using flow cytometry and ELISA showed both translocation of calreticulin and release of ATP and HMGB1 by VB4-845-treated tumor cells. Immune T cell activation via an ICD pathway suggests a complimentary outcome when combined with checkpoint inhibitors. A study was performed in PDX tumor-bearing NOG mice reconstituted with a human immune system to assess the combination of intratumoral injection of VB4-845 with an anti-PD1 antibody, Nivolumab, given i.p. VB4-845 showed growth suppression of the injected tumor whereas the growth delay of the contralateral, non-injected tumor was more pronounced with the addition of Nivolumab. In summary, the data presented suggests that VB4-845 mediates tumor cell killing by an ICD pathway and that the resulting cross-priming effect can enhance the anti-tumoral activity of immune checkpoint inhibitors. Citation Format: Rachelle Lee Dillon, Shilpa Chooniedass, Arjune Premsukh, Glen MacDonald, Jeannick Cizeau, Gregory A. Adams. VB4-845 tumor cell killing in a combination study with the anti-PD-1, Nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 614. doi:10.1158/1538-7445.AM2017-614

Abstract 58: Local intratumoral treatment with anti-mesothelin Immunotoxins overcomes resistance to anti-CTLA-4 therapy

Abstract Background: SS1P and RG7787 are immunotoxins composed of anti-mesothelin antibody fused to a fragment from pseudomonas exotoxin A. We previously observed delayed tumor responses in patients treated with SS1P leading us to hypothesis that immunotoxins can provoke anti-tumor immunity. We hypothesize that intra-tumoral injection of SS1P or RG7787 will produce immunologic death and convert living tumors to a source of antigens and boost the therapeutic effect of immune check point blockade. Method: A BALB/c breast cancer cell line was transfected with human mesothelin (66C14-M) and grown in BALB/c mice transgenic for human mesothelin. SS1P or RG7787 was injected directly into established tumors (80-100 mm3) and anti-CTLA-4 administered IP. Results: We observed total tumor regressions in 23 out of 38 mice (60%) treated with anti-CTLA-4 and either RG7787 or SS1P. No cure was achieved when the drugs were given as a monotherapy supporting the case for synergic anti-tumor activity. Pathologic evaluation of regressing tumors showed a massive inflammation of admixed eosinophils and mononuclear cells located in a collar surrounding the tumors. Tumor regression was associated with increased number of CD8+ cells and was blocked when CD8+ cells were depleted by an antibody. In addition, cured mice were resistant to re-implantation of 66C14 cells not expressing mesothelin indicating that a long-term anti-tumor immunity was formed. To determine if pathogen associated molecular patterns contributed to the response, we combined anti-CTLA-4 with an inactive RG7787 and observed a major reduction in anti-tumor activity (P<0.01) indicating that the effect is not mediated by recognition of a foreign bacterial protein. Conclusions: We suggest that injecting immunotoxins into tumors is a useful approach to boost the activity of anti-CTLA-4. Based on the synergistic anti-tumor effect demonstrated in this study we believe that there is a strong rational to explore such combination therapies in patients. Citation Format: Yasmin Leshem, Xiu-fen Liu, Tapan Bera, Masaki Terabe, Jay A. Berzofsky, Birgit Bossenmaier, Gerhard Niederfellner, James O'Brien, Yoram Reiter, Ira Pastan. Local intratumoral treatment with anti-mesothelin Immunotoxins overcomes resistance to anti-CTLA-4 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 58. doi:10.1158/1538-7445.AM2017-58

Abstract 4576: Targeted human cytolytic fusion proteins: an update

Abstract Targeted immunotherapeutics, such as antibody drug conjugates (ADCs) or immunotoxins (ITs) represent promising agents for treatment of cancer. Despite their encouraging performance in clinical trials, both ADCs and ITs suffer from disadvantages like stoichiometrically undefined chemical linkage of the cytotoxic payload and potential immunogenicity of bacteria- or plant-derived toxins in humans, respectively. Therefore, in contrast to depleting B cell epitopes from bacterial toxins, we designed a number of targeted recombinant cytolytic fusion proteins, replacing bacterial toxins like Pseudomonas exotoxin A by human enzymes like proteases, kinases, RNases or microtubuli-associated proteins. Binding and biological activities were shown in vitro, ex vivo and in different in vivo models. This presentation will summarize the latest results on the hCFPs generated and demonstrate the efficacy of treatment of CD64-, CSPG4-, and EpCAM-targeting constructs in direct comparison to the corresponding Pseudomonas exotoxin-based immunotoxins. The presentation will conclude with an outlook on future perspective and applications of the next generation of targeted fully human recombinant cytolytic fusion proteins. Citation Format: Stefan Barth. Targeted human cytolytic fusion proteins: an update [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2017-4576

Antileukemia Efficacy and Mechanisms of Action of SL-101, a Novel Anti-CD123 Antibody Conjugate, in Acute Myeloid Leukemia.

Purpose: The persistence of leukemia stem cells (LSC)-containing cells after induction therapy may contribute to minimal residual disease (MRD) and relapse in acute myeloid leukemia (AML). We investigated the clinical relevance of CD34+CD123+ LSC-containing cells and antileukemia potency of a novel antibody conjugate SL-101 in targeting CD123+ LSCs.Experimental Methods and Results: In a retrospective study on 86 newly diagnosed AML patients, we demonstrated that a higher proportion of CD34+CD123+ LSC-containing cells in remission was associated with persistent MRD and predicted shorter relapse-free survival in patients with poor-risk cytogenetics. Using flow cytometry, we explored the potential benefit of therapeutic targeting of CD34+CD38-CD123+ cells by SL-101, a novel antibody conjugate comprising an anti-CD123 single-chain Fv fused to Pseudomonas exotoxin A The antileukemia potency of SL-101 was determined by the expression levels of CD123 antigen in a panel of AML cell lines. Colony-forming assay established that SL-101 strongly and selectively suppressed the function of leukemic progenitors while sparing normal counterparts. The internalization, protein synthesis inhibition, and flow cytometry assays revealed the mechanisms underlying the cytotoxic activities of SL-101 involved rapid and efficient internalization of antibody, sustained inhibition of protein synthesis, induction of apoptosis, and blockade of IL3-induced p-STAT5 and p-AKT signaling pathways. In a patient-derived xenograft model using NSG mice, the repopulating capacity of LSCs pretreated with SL-101 in vitro was significantly impaired.Conclusions: Our data define the mechanisms by which SL-101 targets AML and warrant further investigation of the clinical application of SL-101 and other CD123-targeting strategies in AML. Clin Cancer Res; 23(13); 3385-95. ©2017 AACR.

Abstract 72: Induction of immune tolerance to recombinant immunotoxin LMB-100 using synthetic vaccine particles encapsulating rapamycin

Abstract Recombinant Immunotoxins (RITs) are genetically engineered proteins designed for cancer therapy. LMB-100 is a second generation RIT that is composed of a humanized Fab targeting mesothelin and a de-immunized fragment of Pseudomonas Exotoxin A. Due to the bacterial origin of the toxin, LMB-100 is immunogenic, although less immunogenic than first generation immunotoxins. Almost all patients treated with LMB-100 made anti-drug antibodies (ADAs) after two or more cycles of treatment that neutralized the RIT and greatly lowered efficacy. Kishimoto et al. demonstrated that Synthetic Vaccine Particles containing Rapamycin (SVP-R) inhibited the formation of ADAs, when administered with a foreign protein such as KLH or Pegsiticase. The SVP-R are taken up by macrophages and dendritic cells and increase the number of regulatory T cells in treated mice. Here we evaluated the efficacy of combination therapy of LMB-100 and SVP-R to eradicate mesothelin expressing tumors while preventing ADA formation. To evaluate if SVP-R can prevent ADAs against LMB-100, we immunized immune-competent mice with a combination of SVP-R and RIT and measured ADAs titers by ELISA and by a functional neutralization assay. We found that the treatment reduced ADA titers by more than 99%. To determine if the mice were tolerized to LMB-100, we treated the mice with two doses of SVP-R and six doses of LMB-100 to induce tolerance; and followed by nine challenges of LMB-100 given over 6 weeks. We found that the ADAs were reduced by 98%, indicating development of immunological tolerance to LMB-100. To show that the tolerance is transferable, we tolerized the mice with six doses of SVP-R and LMB-100 and adoptively transferred their splenocytes to naïve recipient mice. The recipient mice were challenged with six doses of LMB-100 and ADA titers were measured. We found that splenocytes from tolerized mice induced a 66% decrease in ADA formation in the recipient mice indicating that the tolerance was mediated by cells of the immune system. To demonstrate that immune suppression is useful in treating tumors, we implanted mouse breast cancer cells expressing human mesothelin into Balb/c mice with a normal immune system and treated them with LMB-100. LMB-100 produced tumor regressions when given before ADAs developed, but was inactive in mice with pre-existing ADAs. However, when mice with preexisting ADAs were treated with LMB-100 and SVP-R, anti-tumor activity was restored and ADAs suppressed (P≤0.0001). SVP-R are being evaluated in humans to prevent ADA to Pegsiticase, an enzyme for refractory gout treatment. Our data indicates that combining SVP-R with LMB-100 should be useful in treating cancer by allowing more treatment cycles and better efficacy. This approach can be used to increase the efficacy of other immunogenic agents such as CAR-T cells, antibody drug conjugates and viral gene therapy vectors Citation Format: Ronit Mazor, Emily King, Takashi Kei Kishimoto, Ira Pastan. Induction of immune tolerance to recombinant immunotoxin LMB-100 using synthetic vaccine particles encapsulating rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 72. doi:10.1158/1538-7445.AM2017-72

Abstract 59: Engineering glypican-3 targeting immunotoxins for the treatment of liver cancer

Abstract The expression of glypican-3 (GPC3) in hepatocellular carcinoma offers a target with high tissue specificity and cell signaling implications. A human single domain antibody discovered by phage display technology, HN3, was fused to domain II and domain III of Pseudomonas exotoxin A. This protein (HN3-PE38) showed a high level of cytotoxic activity with a IC50 of 0.4 nM on Hep3B cells, but a relatively low maximum tolerated dose of 0.8 mg/kg in mice. In order to produce an immunotoxin with reduced toxicity, a new version was constructed that removed domain II and seven B cell epitopes from the Pseudomonas toxin. This deimmunized immunotoxin (HN3-mPE24) was shown to have a similar IC50 of 0.2 nM on Hep3B cells. To determine if further deimmunization was possible, three new versions have been generated with T cell epitopes or a series of both B and T cell epitopes removed. These include the HN3-T20 immunotoxin which had six T cell epitopes removed, HN3-T19 with 4 B cell, 4 T cell and 2 shared epitopes, and HN3-M11 with 5 B cell, 4 T cell and 2 shared epitopes. A comparative analysis of these immunotoxins was made using in vitro cell proliferation assays using Hep3B. Both the HN3-T20 (IC50 = 0.6 nM) and HN3-T19 (IC50 = 0.8 nM) immunotoxins had similar activity to HN3-mPE24 (IC50 = 0.7 nM) in a side by side comparison. The HN3-M11 variant had poor cytotoxicity and was excluded from in vivo examination. A Hep3B subcutaneous xenograft model was generated in athymic nude mice and was followed by nine rounds of intravenous immunotoxin treatments. The mPE24, T20 and T19 immunotoxins all showed an increase in average survival rate 70 days (mPE24), 76 days (T20) and 72 days (T19) when compared to the 55 days for PE38 and the 41 days for PBS alone. Additionally, the T20 and the T19 showed a maximum tolerated dose that was similar to that of the mPE24 with dosages as high as 10 mg/kg being well tolerated. This data would suggest that the HN3-T19 immunotoxins has potential clinical applications because it represents the most deimmunized immunotoxin to date. Citation Format: Bryan D. Fleming, Brittany Nixon, Ira Pastan, Mitchell Ho. Engineering glypican-3 targeting immunotoxins for the treatment of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 59. doi:10.1158/1538-7445.AM2017-59

Abstract 2438: Analysis of IL-13 signaling through IL-13Rα2 in human brain tumor specimens in situ

Abstract Previously, we have demonstrated that IL-13 receptor alpha2 (IL-13Rα2), a high affinity receptor for Th2 cytokine IL-13, is overexpressed in most glioblastoma multiforme (GBM) cell lines and approximately 78% of the patient-derived samples. We have also demonstrated that IL-13Rα2 can be targeted by a number of immunotherapeutic agents including chimeric antigen receptor modified T (CAR-T) cells, targeted lentivirus and adenovirus vectors, and a chimeric fusion immunotoxin consisting of IL-13 and truncated Pseudomonas exotoxin (IL-13-PE). However, the signal transduction initiated by IL-13 in GBM tumor is not known to occur through the IL13Rα2, which has a high affinity for IL-13. We have recently observed that IL-13 can signal through IL-13Rα2 by activating AP-1 transcription factors in human brain tumor cell lines. In this study, we have examined IL-13Rα2 expression in human brain tumor and normal brain specimens, and the subsequent signaling through the AP-1 pathway in situ. Using six human glioblastoma and three astrocytoma specimens, we evaluated the expression of AP-1 transcription factors by immunohistochemistry (IHC) and compared the extent of immunostaining and percent positive fields with three normal brain specimens. Six GBM specimens examined showed high degree of immunostaining for c-Fos, c-Jun, Jun D and Fra-1 (AP-1 family of transcription factors) and a high percentage of positive fields. These specimens also showed strong immunostaining for IL-13Rα2 (4+) in >70% fields (P<0.001 compared to normal brain). Three astrocytoma specimens showed staining for IL-13Rα2 (>2+ and 32% fields, P<0.01 compared to normal brain), but the extent of staining was lower compared to GBM. Similar to IL-13Rα2 expression, the extent of staining and percentage of positive fields for AP-1 transcription factors were highly statistically significant between tumors and normal brain (P<0.001 for GBM compared to normal brain and P<0.01 for astrocytoma compared to normal brain). The extent of immunostaining in GBM was highest for c-Fos (4+, 78% fields) followed by c-Jun (3+, 57% fields), Fra-1 (2+, 70% fields) and Jun-D (2+, 28% fields). Jun-B expression was the lowest among the AP-1 transcription factors (<1+, 7% fields) in GBM specimens. Astrocytoma specimens showed lesser extent of immunostaining for AP-1 members compared to GBM; c-Fos showed 2+ staining and 42% positive fields followed by c-Jun (2+, 12% fields), Fra-1 (2+, 48% fields) and Jun-D (<1+, 18% fields). Jun-B staining intensity was <1+ in only 6% fields. Normal brain specimens showed no immunostaining for AP-1 family members. Our results generally corroborate with data obtained from GBM cell lines and confirm that IL-13 can signal in IL-13Rα2 positive GBM tumors in-situ through the AP-1 pathway, thus indicating that this pathway may be an important target for therapeutic intervention of GBM in addition to targeting IL-13Rα2. Citation Format: Rukmini Bhardwaj, Akiko Suzuki, Pamela Leland, Bharat H. Joshi, Raj K. Puri. Analysis of IL-13 signaling through IL-13Rα2 in human brain tumor specimens in situ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2017-2438

Abstract 68: Methotrexate prevents primary immune responses against recombinant immunotoxin in murine models

Abstract Recombinant immunotoxins (rITs) are composed of a tumor antigen-targeting antibody fragment fused to a portion of Pseudomonas exotoxin A. rITs have been effective in clinical trials for patients with hematologic malignancies. The CD22 targeting rIT Moxetumomab Pasudotox has achieved overall response rates of 86% and complete remission rates of 46% in patients with relapsed-refractory Hairy Cell Leukemia. However, the therapeutic efficacy of rITs against solid tumors is limited by their immunogenicity in immune-competent patients. In clinical trials to treat mesothelioma patients with SS1P, a rIT targeting mesothelin, 90% of patients developed neutralizing antibodies against SS1P after one cycle of treatment. When immunosuppressive chemotherapy and SS1P were combined, more cycles of rIT could be given and several patients with advanced chemo-refractory mesothelioma had striking tumor regressions. This implicates high therapeutic potential for rITs against solid tumors once immunogenicity is surmounted. Methotrexate (MTX) is a folate antagonist which interferes with purine biosynthesis, and is used to treat osteosarcomas and other cancers. MTX also interferes with T cell responses and is used to treat autoimmune diseases. Based on its immunosuppressive properties, Joly et al. demonstrated that low-dose MTX prevented the formation of ADAs against recombinant human alglucosidase alfa in mice in an antigen-specific manner. We hypothesized that MTX would similarly prevent the formation of ADAs against rITs in an antigen-specific manner. To test our hypotheses, mice were treated with the mesothelin-targeting rIT RG7787 with or without MTX given 0, 24, and 48 hours after RG7787 treatment. Serum was collected and anti-RG7787 ADAs were measured by direct ELISA. We found that six doses of RG7787 combined with low dose MTX (1 mg/kg) inhibited the formation of ADAs against RG7787. This inhibition was sustained through six challenges with RG7787 without additional MTX. Further, we found that immunization with RG7787 plus MTX induced RG7787-specific tolerance, and had no effect on the ADA response against a second protein, ovalbumin. We conclude that combination of MTX and RG7787 is effective at preventing primary immune responses in a durable, antigen-specific manner. We propose to combine this agent in immune-competent cancer patients receiving rIT therapy to prevent rIT immunogenicity. Citation Format: Emily M. King, Ronit Mazor, Ira Pastan. Methotrexate prevents primary immune responses against recombinant immunotoxin in murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2017-68

Novel PSCA targeting scFv-fusion proteins for diagnosis and immunotherapy of prostate cancer.

Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA' comprising the PSCA(scFv) and a truncated version of Pseudomonas exotoxin A (PE, ETA') was generated. We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)-SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA'. The cytotoxic activity of PSCA(scFv)-ETA' was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa. Alexa Fluor® 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA' showed selective binding leading to internalization and efficient elimination of target cells. Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material.

Phase 1 single-center, dose escalation study of D2C7-IT administered intratumorally via convection-enhanced delivery for adult patients with recurrent malignant glioma.

e13532 Background: D2C7 immunotoxin (D2C7-IT) is a dual-specific recombinant immunotoxin consisting of EGFR-wt and EGFRvIII monoclonal antibodies with a genetically engineered Pseudomonas exotoxin, PE-38KDEL. The primary objective is to determine the maximum tolerated dose of D2C7-IT when delivered intratumorally by convection enhanced delivery (CED). Methods: Inclusion criteria includes subjects with a single, recurrent supratentorial WHO grade III or IV glioma, KPS ≥ 70 and a washout of chemotherapy, bevacizumab or study drug of ≥ 4 weeks. Prior to administration of D2C7-IT, recurrent tumor must be confirmed by histopathology. A minimum of 2 subjects are accrued by dose level. Results: Currently, 23 subjects have been treated (16 male, 7 female) with a median age of 54 years. Out of 9 dose levels, 2 subjects have been treated at every dose except for 4 at dose level 3 (120 ng/ml) and 5 at dose 6 (405ng/ml). Adverse events possibly, probably or definitely related to D2C7-IT are mostly grade 1 or 2 events consisting of, but not limited to: intracranial hemorrhage (n = 1), stroke (n = 2), headache (n = 15), seizure (n = 5), confusion (n = 4), paresthesia (n = 4), dysarthria (n = 1), dysphasia (n = 4), visual disturbances (n = 7), fatigue (n = 4), gait disturbance (n = 2), elevated transaminases (n = 5), decreased platelets (n = 3), decreased neutrophil count (n = 1), nausea (n = 3), vomiting (n = 1), and thromboembolic event (n = 1). There was 1 dose limiting toxicity (grade 4 seizure at dose level 3), 2 grade 3 headaches and 1 grade 3 elevated ALT. 14 subjects are still alive with 6 remaining on study. So far, the longest survival time from infusion is 18.2+ months. Conclusions: D2C7-IT infusion via CED is safe with encouraging results. This dose escalation Phase I study is ongoing and will set the stage for the Phase II trial. Clinical trial information: NCT02303678.

Synergistic Anti-tumor Activity of Local SS1P with Systemic CTLA-4 Blockade Causes Complete Regression of Both Injected and Un-injected Tumors in Mice

Abstract SS1P consists of an anti-mesothelin Fv attached to Pseudomonas exotoxin A and kills tumor cells by inhibiting protein synthesis. Because of its unique mechanism of cell killing, SS1P does not inhibit the immune system like other anti-cancer drugs. Here we determined if SS1P can cooperate with anti-CTLA-4 to induce anti-tumor immunity and cause complete tumor regressions. A BALB/c breast cancer cell line was transfected with human mesothelin (66C14-M) and implanted in two different locations. SS1P was injected directly into one tumor and anti-CTLA-4 given IP. In mice treated with anti-CTLA-4 and SS1P, tumor regressions occurred in 11 out of 12 SS1P injected tumors (91%) and in 7 out of 12 un-injected tumors (53%). No tumor regressions occured with drugs given separately. Pathological evaluation showed a large sterile central abscess in injected tumors surrounded by a collar of inflammation containing a mixture of lymphocytes and other mononuclear cells. Analysis of responding tumors showed a time dependent increase in CD8+ cells located mainly in the inflammation collar. RNA analysis of responding tumors showed that 385/768 immune related gene transcripts were elevated by twofold or more including CD8a, PDL2 and IL1 alpha (13, 21 and 27-fold). Altogether, combining local SS1P with anti-CTLA-4 promotes massive inflammation that cures multi-centered cancer disease providing a strong rational to explore this combination therapy in patients.

Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR.

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 μg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956-65. ©2017 AACR.

Ligand Selectivity between the ADP-Ribosylating Toxins: An Inverse-Docking Study for Multitarget Drug Discovery.

Bacterial adenosine 5’-diphosphate-ribosylating toxins are encoded by several human pathogens, such as Pseudomonas aeruginosa (exotoxin A (ETA)), Corynebacterium diphtheriae (diphtheria toxin (DT)), and Vibrio cholerae (cholix toxin (CT)). The toxins modify eukaryotic elongation factor 2, an essential human enzyme in protein synthesis, thereby causing cell death. Targeting external virulence factors, such as the above toxins, is a promising alternative for developing new antibiotics, while at the same time avoiding drug resistance. This study aims to establish a reliable computational methodology to find a “silver bullet” able to target all three toxins. Herein, we have undertaken a detailed analysis of the active sites of ETA, DT, and CT, followed by the determination of the most appropriate selection of the size of the docking sphere. Thereafter, we tested two different approaches for normalizing the docking scores and used these to verify the best target (toxin) for each ligand. The results indicate that the methodology is suitable for identifying selective as well as multitoxin inhibitors, further validating the robustness of inverse docking for target-fishing experiments.

Essential role for Bim in mediating the apoptotic and antitumor activities of immunotoxins.

Protein synthesis is crucial for regulating cell homeostasis and, when unrestricted, it can lead to tumorigenesis. Immunotoxins derived from Pseudomonas exotoxin are antibody-toxin fusion proteins that inhibit protein synthesis of mammalian cells via ADP-ribosylation of the eukaryotic elongation factor-2. Here we investigate the role of the Bcl-2 family proteins in the response of cancer cells to immunotoxin challenge. Besides the well-known reduction of the prosurvival Bcl-2 family member, Mcl-1, following inhibition of protein synthesis, we show for the first time that immunotoxins also reduce the levels of selected proapoptotic BH-3-only proteins. Among these, only Bim protein levels correlated with the ability of immunotoxins to induce an apoptotic response. To support our findings, we verified that a Bim knockout completely abolished immunotoxin-mediated apoptosis. Further, mice bearing either wild-type or Bid knockout tumors responded to immunotoxin treatment with a decrease in growth kinetics, whereas mice engrafted with Bim knockout tumors showed no reduction in tumor size or prolongation of survival following immunotoxin treatment. From these results, we conclude that Bim expression is a major susceptibility factor for tumor cell death and, as such, constitutes a potential biomarker that could be evaluated before immunotoxin treatment. In support of this hypothesis, clinically, we analyzed patient cells before immunotoxin treatment and report that samples of hairy cell leukemia with high levels of Bim protein responded with a greater decrease in leukemic cell count compared with those samples expressing a low level of Bim.

Anti-tumor activity of an immunotoxin (TGFα-PE38) delivered by attenuated Salmonella typhimurium.

The anticancer strategy underlying the use of immunotoxins is as follows: the cancer-binding domain delivers the toxin to a cancer cell, after which the toxin enters and kills the cell. TGFα-PE38 is an immunotoxin comprising transforming growth factor alpha (TGFα), a natural ligand of epidermal growth factor receptor (EGFR), and a modified Pseudomonas exotoxin A (PE38) lacking N terminal cell-binding domain, a highly potent cytotoxic protein moiety. Tumor cells with high level of EGFR undergo apoptosis upon treatment with TGFα-PE38. However, clinical trials demonstrated that this immunotoxin delivered by an intracerebral infusion technique has only a limited inhibitory effect on intracranial tumors mainly due to inconsistent drug delivery. To circumvent this problem, we turned to tumor-seeking bacterial system. Here, we engineered Salmonella typhimurium to selectively express and release TGFα-PE38. Engineered bacteria were administered to mice implanted with mouse colon or breast tumor cells expressing high level of EGFR. We observed that controlled expression and release of TGFα-PE38 from intra-tumoral Salmonellae by either an engineered phage lysis system or by a bacterial membrane transport signal led to significant inhibition of solid tumor growth. These results demonstrated that delivery by tumor-seeking bacteria would greatly augment efficacy of immunotoxin in cancer therapeutics.

Combination Therapy to Enhance the Efficacy of Recombinant Immunotoxins: Evaluating the Role of Mitochondrial Translation in Toxin‐Mediated Cell Death

Recombinant immunotoxins (RITs) based on Pseudomonas exotoxin A (PE) are targeted cytotoxic proteins currently in clinical trials for the treatment of cancer. PE covalently modifies and inhibits elongation factor 2 (EF2), which halts protein synthesis within the cytosol and ultimately induces apoptotic cell death. Although PE inhibits translation in the cytoplasm, eukaryotes also have a translation system in mitochondria that is similar to the translation system in bacteria. Recent findings have highlighted the importance of mitochondrial stability and function in tumorigenesis, sustaining cancer cell proliferation, and maintaining cancer cell survival. By inhibiting protein synthesis within the mitochondria through the usage of antibiotics, mitochondria may become unable to properly function, resulting in the inability to meet the energy requirements of the cell and the activation of apoptotic cell death. I proposed that combination treatment with both PE‐based RITs and antibiotics that inhibit mitochondrial protein synthesis will reduce cancer cell survival compared to independent treatments of either therapeutic alone. To test this hypothesis, combination treatment of RITs and seven different antibiotics (tetracycline, chloramphenicol, streptomycin, linezolid, fusidic acid, doxycycline, and kanamycin) have been applied to CA46 and HEK293 cell lines followed by the assessment of cell viability through WST‐8 assay. No statistically relevant effects were observed for combination treatments in HEK293 cells, but the addition of a nontoxic level of chloramphenicol to RIT‐treated CA46 cells showed a ~50% enhancement in cell killing compared to RIT treatment alone (p=.05). In response, we are continuing to explore the usage of chloramphenicol in combination therapies with RIT. Though we conclude that targeting mitochondrial translation with antibiotics is not a general strategy for RIT combination therapy, combination therapy may be applicable in specific instances.Support or Funding InformationThis project was supported by funds from Towson University's Office of Undergraduate Research and the Fisher College of Science and Mathematics, as well as an undergraduate research award from the American Society for Biochemistry and Molecular Biology.

HER2-specific recombinant immunotoxin 4D5scFv-PE40 passes through retrograde trafficking route and forces cells to enter apoptosis

Immunotoxin 4D5scFv-PE40 is a recombinant protein that comprises 4D5scFv antibody as a targeting module and fragment of Pseudomonas exotoxin A as an effector (toxic) one. The immunotoxin has shown pronounced antitumor effect on cancer cells overexpressing HER2 receptor in vitro and on HER2-positive experimental tumors in vivo. We clarified the mechanism of 4D5scFv-PE40 activity that is of particular importance in the case of targeted therapeutic agent aimed at personalizing treatment of disease in relation to molecular genetic characteristics of each patient. After specific binding to HER2 on the cell surface and clathrin-mediated endocytosis the immunotoxin passes through retrograde trafficking route. During this route the immunotoxin molecule is supposed to undergo enzymatic processing that ends in separation of C-terminal and N-terminal fragments of the immunotoxin. Finally, C-terminal functionally active fragment of 4D5scFv-PE40 arrests protein synthesis in cytoplasm followed by cell death via apoptosis.