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Construction of an immunotoxin, D2C7-(scdsFv)-PE38KDEL, targeting EGFRwt and EGFRvIII for brain tumor therapy.

ABSTRACT: The EGF receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583-637 AAs) and EGFRvIII (292-346 AAs) proteins.The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII were measured by surface-plasmon resonance and flow cytometry. In vitro cytotoxicity of D2C7-(scdsFv)-PE38KDEL was measured by inhibition of protein synthesis in human EGFRwt-transfected NR6 (NR6W), human EGFRvIII-transfected NR6 (NR6M), EGFRwt-overexpressing A431-epidermoid-carcinoma, and glioblastoma xenograft cells (43, D08-0493MG, D2159MG, and D270MG). In vivo antitumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated using 43, NR6M, and D270MG orthotopic tumor models.The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6×10(-9) mol/L and 1.3×10(-9) mol/L, respectively. Flow cytometry with NR6W and NR6M cells confirmed the specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII. The D2C7-(scdsFv)-PE38KDEL IC50 was 0.18 to 2.5 ng/mL on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was approximately 0.25 ng/mL on EGFRvIII-expressing cells (NR6M) and on EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells (D2159MG and D270MG). Significantly, in intracranial tumor models of 43, NR6M, and D270MG, treatment with D2C7-(scdsFv)-PE38KDEL by convection-enhanced delivery prolonged survival by 310% (P=0.006), 28% (P=0.002), and 166% (P=0.001), respectively.In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, or both.

SUBMITTER: Chandramohan V

PROVIDER: S-EPMC3766439 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Construction of an immunotoxin, D2C7-(scdsFv)-PE38KDEL, targeting EGFRwt and EGFRvIII for brain tumor therapy.

Chandramohan Vidyalakshmi V Bao Xuhui X Keir Stephen T ST Pegram Charles N CN Szafranski Scott E SE Piao Hailan H Wikstrand Carol J CJ McLendon Roger E RE Kuan Chien-Tsun CT Pastan Ira H IH Bigner Darell D DD

Clinical cancer research : an official journal of the American Association for Cancer Research 20130715 17

<h4>Purpose</h4>The EGF receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583-637 AAs) and EGFRvIII (292-346 AAs) proteins.<h4>Experimental design</h4>The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and ...[more]

PMID: 23857604

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Project description:BackgroundD2C7-IT is a novel immunotoxin (IT) targeting wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins in glioblastoma. In addition to inherent tumoricidal activity, immunotoxins induce secondary immune responses through the activation of T cells. However, glioblastoma-induced immune suppression is a major obstacle to an effective and durable immunotoxin-mediated antitumor response. We hypothesized that D2C7-IT-induced immune response could be effectively augmented in combination with αCTLA-4/αPD-1/αPD-L1 therapies in murine models of glioma.MethodsTo study this, we overexpressed the D2C7-IT antigen, murine EGFRvIII (dmEGFRvIII), in established glioma lines, CT-2A and SMA560. The reactivity and therapeutic efficacy of D2C7-IT against CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII cells was determined by flow cytometry and in vitro cytotoxicity assays, respectively. Antitumor efficacy of D2C7-IT was examined in immunocompetent, intracranial murine glioma models and the role of T cells was assessed by CD4+ and CD8+ T cell depletion. In vivo efficacy of D2C7-IT/αCTLA-4/αPD-1 monotherapy or D2C7-IT+αCTLA-4/αPD-1 combination therapy was evaluated in subcutaneous unilateral and bilateral CT-2A-dmEGFRvIII glioma-bearing immunocompetent mice. Further, antitumor efficacy of D2C7-IT+αCTLA-4/αPD-1/αPD-L1/αTim-3/αLag-3/αCD73 combination therapy was evaluated in intracranial CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII glioma-bearing mice. Pairwise differences in survival curves were assessed using the generalized Wilcoxon test.ResultsD2C7-IT effectively killed CT-2A-dmEGFRvIII (IC50 = 0.47 ng/mL) and SMA560-dmEGFRvIII (IC50 = 1.05 ng/mL) cells in vitro. Treatment of intracranial CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII tumors with D2C7-IT prolonged survival (P = 0.0188 and P = 0.0057, respectively), which was significantly reduced by the depletion of CD4+ and CD8+ T cells. To augment antitumor immune responses, we combined D2C7-IT with αCTLA-4/αPD-1 in an in vivo subcutaneous CT-2A-dmEGFRvIII model. Tumor-bearing mice exhibited complete tumor regressions (4/10 in D2C7-IT+αCTLA-4 and 5/10 in D2C7-IT+αPD-1 treatment groups), and combination therapy-induced systemic antitumor response was effective against both dmEGFRvIII-positive and dmEGFRvIII-negative CT-2A tumors. In a subcutaneous bilateral CT-2A-dmEGFRvIII model, D2C7-IT+αCTLA-4/αPD-1 combination therapies showed dramatic regression of the treated tumors and measurable regression of untreated tumors. Notably, in CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII intracranial glioma models, D2C7-IT+αPD-1/αPD-L1 combinations improved survival, and in selected cases generated cures and protection against tumor re-challenge.ConclusionsThese data support the development of D2C7-IT and immune checkpoint blockade combinations for patients with malignant glioma.

Project description:Glioblastoma is the most aggressive malignant brain tumor among all primary brain and central nervous system tumors. The median survival time for glioblastoma patients given the current standard of care treatment (surgery, radiation, and chemotherapy) is less than 15 months. Thus, there is an urgent need to develop more efficient therapeutics to improve the poor survival rates of patients with glioblastoma. To address this need, we have developed a novel tumor-targeted immunotoxin (IT), D2C7-(scdsFv)-PE38KDEL (D2C7-IT), by fusing the single chain variable fragment (scFv) from the D2C7 monoclonal antibody (mAb) with the Pseudomonas Exotoxin (PE38KDEL). D2C7-IT reacts with both the wild-type epidermal growth factor receptor (EGFRwt) and EGFR variant III (EGFRvIII), two onco-proteins frequently amplified or overexpressed in glioblastomas. Surface plasmon resonance and flow cytometry analyses demonstrated a significant binding capacity of D2C7-IT to both EGFRwt and EGFRvIII proteins. In vitro cytotoxicity data showed that D2C7-IT can effectively inhibit protein synthesis and kill a variety of EGFRwt-, EGFRvIII-, and both EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells and human tumor cell lines. Furthermore, D2C7-IT exhibited a robust anti-tumor efficacy in orthotopic mouse glioma models when administered via intracerebral convection-enhanced delivery (CED). A preclinical toxicity study was therefore conducted to determine the maximum tolerated dose (MTD) and no-observed-adverse-effect-level (NOAEL) of D2C7-IT via intracerebral CED for 72 hours in rats. Based on this successful rat toxicity study, an Investigational New Drug (IND) application (#116855) was approved by the Food and Drug Administration (FDA), and is now in effect for a Phase I/II D2C7-IT clinical trial (D2C7 for Adult Patients with Recurrent Malignant Glioma, https://clinicaltrials.gov/ct2/show/NCT02303678). While it is still too early to draw conclusions from the trial, results thus far are promising.

Project description:Conventional therapies for malignant gliomas (MGs) fail to target tumor cells exclusively, such that their efficacy is ultimately limited by non-specific toxicity. Immunologic targeting of tumor-specific gene mutations, however, may allow more precise eradication of neoplastic cells. The epidermal growth factor receptor variant III (EGFRvIII) is a consistent tumor-specific mutation that is widely expressed in MGs and other neoplasms. This mutation encodes a constitutively active tyrosine kinase that enhances tumorgenicity and migration and confers radiation and chemotherapeutic resistance. This in-frame deletion mutation splits a codon resulting in the creation of a novel glycine at the fusion junction between normally distant parts of the molecule and producing a sequence re-arrangement which creates a tumor-specific epitope for cellular or humoral immunotherapy in patients with MGs. We have previously shown that vaccination with a peptide that spans the EGFRvIII fusion junction is an efficacious immunotherapy in syngeneic murine models, but patients with MGs have a profound immunosuppression that may inhibit the ability of antigen presenting cells (APCs), even those generated ex vivo, to induce EGFRvIII-specific immune responses. In this report, we summarize our results in humans targeting this mutation in two consecutive and one multi-institutional Phase II immunotherapy trials. These trials demonstrated that vaccines targeting EGFRvIII are capable of inducing potent T- and B-cell immunity in these patients, and lead to an unexpectedly long survival time. Most importantly, vaccines targeting EGFRvIII were universally successful at eliminating tumor cells expressing the targeted antigen without any evidence of symptomatic collateral toxicity. These studies establish the tumor-specific EGFRvIII mutation as a novel target for humoral- and cell-mediated immunotherapy in a variety of cancers. The recurrence of EGFRvIII-negative tumors in our patients, however, highlights the need for targeting a broader repertoire of tumor-specific antigens.

Project description:BackgroundGlioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors.MethodsGBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7.ResultsImmunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD.ConclusionsThese findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

Dataset Information

Construction of an immunotoxin, D2C7-(scdsFv)-PE38KDEL, targeting EGFRwt and EGFRvIII for brain tumor therapy.

Publications

Construction of an immunotoxin, D2C7-(scdsFv)-PE38KDEL, targeting EGFRwt and EGFRvIII for brain tumor therapy.

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