Epidermal Growth Factor Receptor Variant III Research Articles

Temozolomide Induces Endocytosis of EGFRvIII via p38-Mediated Non-canonical Phosphorylation in Glioblastoma Cells.

The ligand-induced internalization of epidermal growth factor receptor (EGFR) is generally considered to attenuate downstream signaling via its endosomal degradation. However, the endocytosis of an oncogenic EGFR variant III (EGFRvIII) is impaired, which leads to persistent signaling from the cell surface, thereby promoting the proliferation and survival of glioblastoma multiforme (GBM) cells. Cellular stress triggers the non-canonical endocytosis-recycling of EGFR by p38-mediated phosphorylation. In the present study, we used temozolomide (TMZ), the standard chemotherapeutic agent for the treatment of GBM patients, to examine whether EGFRvIII is controlled by a non-canonical mechanism. TMZ triggered the endocytic trafficking of serine phosphorylated EGFRvIII. Moreover, phosphorylation and endocytosis were abrogated by the selective p38 inhibitor SB203580, but not gefitinib, indicating that EGFRvIII is recruited to p38-mediated non-canonical endocytosis. The combination of TMZ and SB203580 also showed potential inhibitory effects on the proliferation and motility of glioblastoma cells.

High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms.

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood–brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice.

Rigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART) cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII). We first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T cells and evaluated the function of the CART cells both in vitro and in vivo. To inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208). Both RV- and Tg-CART cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28. Our new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.

A Rational Designed Novel Bispecific Antibody for the Treatment of GBM.

Epidermal growth factor receptor variant III (EGFRvIII) is highly and specifically expressed in a subset of lethal glioblastoma (GBM), making the receptor a unique therapeutic target for GBM. Recently, bispecific antibodies (BsAbs) have shown exciting clinical benefits in cancer immunotherapy. Here, we report remarkable results for GBM treatment with a BsAb constructed by the “BAPTS” method. The BsAb was characterized through LC/MS, SEC-HPLC, and SPR. Furthermore, the BsAb was evaluated in vitro for bioactivities through FACS, antigen-dependent T-cell-mediated cytotoxicity, and a cytokine secretion assay, as well as in vivo for antitumor activity and pharmacokinetic (PK) parameters through immunodeficient NOD/SCID and BALB/c mouse models. The results indicated that the EGFRvIII-BsAb eliminated EGFRvIII-positive GBM cells by recruiting and stimulating effector T cells secreting cytotoxic cytokines that killed GBM cells in vitro. The results demonstrated the antitumor potential and long circulation time of EGFRvIII-BsAb in NOD/SCID mice bearing de2–7 subcutaneously heterotopic transplantation tumors and BALB/c mice. In conclusion, our experiments in both in vitro and in vivo have shown the remarkable antitumor activities of EGFRvIII-BsAb, highlighting its potential in clinical applications for the treatment of GBM. Additional merits, including a long circulation time and low immunogenicity, have also made the novel BsAb a promising therapeutic candidate.

Opening of the Blood-Brain Barrier Using Low-Intensity Pulsed Ultrasound Enhances Responses to Immunotherapy in Preclinical Glioma Models.

The blood-brain barrier (BBB) inhibits adequate dosing/penetration of therapeutic agents to malignancies in the brain. Low-intensity pulsed ultrasound (LIPU) is a safe therapeutic method of temporary BBB disruption (BBBD) to enhance chemotherapeutic delivery to the tumor and surrounding brain parenchyma for treatment of glioblastoma. We investigated if LIPU could enhance therapeutic efficacy of anti-PD-1 in C57BL/6 mice bearing intracranial GL261 gliomas, epidermal growth factor receptor variant III (EGFRvIII) chimeric antigen receptor (CAR) T cells in NSG mice with EGFRvIII-U87 gliomas, and a genetically engineered antigen-presenting cell (APC)-based therapy producing the T-cell attracting chemokine CXCL10 in the GL261-bearing mice. Mice treated with anti-PD-1 and LIPU-induced BBBD had a median survival duration of 58 days compared with 39 days for mice treated with anti-PD-1, and long-term survivors all remained alive after contralateral hemisphere rechallenge. CAR T-cell administration with LIPU-induced BBBD resulted in significant increases in CAR T-cell delivery to the CNS after 24 (P < 0.005) and 72 (P < 0.001) hours and increased median survival by greater than 129%, in comparison with CAR T cells alone. Local deposition of CXCL10-secreting APCs in the glioma microenvironment with LIPU enhanced T-cell glioma infiltration during the therapeutic window (P = 0.004) and markedly enhanced survival (P < 0.05). LIPU increases immune therapeutic delivery to the tumor microenvironment with an associated increase in survival and is an emerging technique for enhancing novel therapies in the brain.

Preclinical Assessment of AMG 596, a Bispecific T-cell Engager (BiTE) Immunotherapy Targeting the Tumor-specific Antigen EGFRvIII.

AMG 596 is a bispecific T-cell engager (BiTE) immuno-oncology therapy in clinical development for treatment of glioblastoma multiforme (GBM), the most common primary brain tumor in adults with limited therapeutic options. AMG 596 is composed of two single-chain variable fragments that simultaneously bind to the tumor-specific antigen, EGFR variant III (EGFRvIII), on GBM cells and to CD3 on T cells, thereby activating T cells to proliferate and secrete cytotoxic substances that induce lysis of the bound tumor cell. T-cell-redirected lysis by AMG 596 is very potent; in vitro studies revealed EC50 values in the low picomolar range, and in vivo studies showed that AMG 596 treatment significantly increased the overall survival of mice bearing EGFRvIII-expressing orthotopic tumors. In addition, AMG 596 activity is highly specific; no AMG 596-induced T-cell activity can be observed in assays with EGFRvIII-negative GBM cells, and no signs of toxicity and activity were observed in cynomolgus monkeys, which lack expression of EGFRvIII on normal tissues. With EGFRvIII-expressing GBM cells, we showed shedding of EGFRvIII-containing membrane vesicles, followed by vesicle uptake and EGFRvIII cell surface presentation by EGFRvIII noncoding GBM cells. Cell membrane presentation of EGFRvIII following microvesicle transfer allows engagement by AMG 596, resulting in T-cell activation and T-cell-dependent lysis of GBM cells. Together, these data show a compelling preclinical efficacy and safety profile of AMG 596, supporting its development as a novel immunotherapy for treatment of GBM.

Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer, for which effective therapies are urgently needed. Chimeric antigen receptor (CAR)-based immunotherapy represents a promising therapeutic approach, but it is often impeded by highly immunosuppressive tumor microenvironments (TME). Here, in an immunocompetent, orthotopic GBM mouse model, we show that CAR-T cells targeting tumor-specific epidermal growth factor receptor variant III (EGFRvIII) alone fail to control fully established tumors but, when combined with a single, locally delivered dose of IL-12, achieve durable anti-tumor responses. IL-12 not only boosts cytotoxicity of CAR-T cells, but also reshapes the TME, driving increased infiltration of proinflammatory CD4+ T cells, decreased numbers of regulatory T cells (Treg), and activation of the myeloid compartment. Importantly, the immunotherapy-enabling benefits of IL-12 are achieved with minimal systemic effects. Our findings thus show that local delivery of IL-12 may be an effective adjuvant for CAR-T cell therapy for GBM.

ANO1 regulates the maintenance of stemness in glioblastoma stem cells by stabilizing EGFRvIII

Glioblastoma multiforme (GBM) or glioblastoma is the most deadly malignant brain tumor in adults. GBM is difficult to treat mainly due to the presence of glioblastoma stem cells (GSCs). Epidermal growth factor receptor variant III (EGFRvIII) has been linked to stemness and malignancy of GSCs; however, the regulatory mechanism of EGFRvIII is largely unknown. Here, we demonstrated that Anoctamin-1 (ANO1), a Ca2+-activated Cl- channel, interacts with EGFRvIII, increases its protein stability, and supports the maintenance of stemness and tumor progression in GSCs. Specifically, shRNA-mediated knockdown and pharmacological inhibition of ANO1 suppressed the self-renewal, invasion activities, and expression of EGFRvIII and related stem cell factors, including NOTCH1, nestin, and SOX2 in GSCs. Conversely, ANO1 overexpression enhanced the above phenomena. Mechanistically, ANO1 protected EGFRvIII from proteasomal degradation by directly binding to it. ANO1 knockdown significantly increased survival in mice and strongly suppressed local invasion of GSCs in an in vivo intracranial mouse model. Collectively, these results suggest that ANO1 plays a crucial role in the maintenance of stemness and invasiveness of GSCs by regulating the expression of EGFRvIII and related signaling molecules, and can be considered a promising therapeutic target for GBM treatment.

Multi-institutional noninvasive in vivo characterization of IDH, 1p/19q, and EGFRvIII in glioma using neuro-Cancer Imaging Phenomics Toolkit (neuro-CaPTk).

BackgroundGliomas represent a biologically heterogeneous group of primary brain tumors with uncontrolled cellular proliferation and diffuse infiltration that renders them almost incurable, thereby leading to a grim prognosis. Recent comprehensive genomic profiling has greatly elucidated the molecular hallmarks of gliomas, including the mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), loss of chromosomes 1p and 19q (1p/19q), and epidermal growth factor receptor variant III (EGFRvIII). Detection of these molecular alterations is based on ex vivo analysis of surgically resected tissue specimen that sometimes is not adequate for testing and/or does not capture the spatial tumor heterogeneity of the neoplasm.MethodsWe developed a method for noninvasive detection of radiogenomic markers of IDH both in lower-grade gliomas (WHO grade II and III tumors) and glioblastoma (WHO grade IV), 1p/19q in IDH-mutant lower-grade gliomas, and EGFRvIII in glioblastoma. Preoperative MRIs of 473 glioma patients from 3 of the studies participating in the ReSPOND consortium (collection I: Hospital of the University of Pennsylvania [HUP: n = 248], collection II: The Cancer Imaging Archive [TCIA; n = 192], and collection III: Ohio Brain Tumor Study [OBTS, n = 33]) were collected. Neuro-Cancer Imaging Phenomics Toolkit (neuro-CaPTk), a modular platform available for cancer imaging analytics and machine learning, was leveraged to extract histogram, shape, anatomical, and texture features from delineated tumor subregions and to integrate these features using support vector machine to generate models predictive of IDH, 1p/19q, and EGFRvIII. The models were validated using 3 configurations: (1) 70–30% training–testing splits or 10-fold cross-validation within individual collections, (2) 70–30% training–testing splits within merged collections, and (3) training on one collection and testing on another.ResultsThese models achieved a classification accuracy of 86.74% (HUP), 85.45% (TCIA), and 75.15% (TCIA) in identifying EGFRvIII, IDH, and 1p/19q, respectively, in configuration I. The model, when applied on combined data in configuration II, yielded a classification success rate of 82.50% in predicting IDH mutation (HUP + TCIA + OBTS). The model when trained on TCIA dataset yielded classification accuracy of 84.88% in predicting IDH in HUP dataset.ConclusionsUsing machine learning algorithms, high accuracy was achieved in the prediction of IDH, 1p/19q, and EGFRvIII mutation. Neuro-CaPTk encompasses all the pipelines required to replicate these analyses in multi-institutional settings and could also be used for other radio(geno)mic analyses.

PARK7 maintains the stemness of glioblastoma stem cells by stabilizing epidermal growth factor receptor variant III.

PARK7 is involved in many key cellular processes, including cell proliferation, transcriptional regulation, cellular differentiation, oxidative stress protection, and mitochondrial function maintenance. Deregulation of PARK7 has been implicated in the pathogenesis of various human diseases, including cancer. Here, we aimed to clarify the effect of PARK7 on stemness and radioresistance of glioblastoma stem cells (GSCs). Serum differentiation and magnetic cell sorting of GSCs revealed that PARK7 was preferentially expressed in GSCs rather than differentiated GSCs. Immunohistochemical staining showed enhanced expression of PARK7 in glioma tissues compared to that in normal brain tissues. shRNA-mediated knockdown of PARK7 inhibited the self-renewal activity of GSCs in vitro, as evidenced by the results of neurosphere formation, limiting dilution, and soft-agar clonogenic assays. In addition, PARK7 knockdown suppressed GSC invasion and enhanced GSC sensitivity to ionizing radiation (IR). PARK7 knockdown suppressed expression of GSC signatures including nestin, epidermal growth factor receptor variant III (EGFRvIII), SOX2, NOTCH1, and OCT4. Contrarily, overexpression of PARK7 in CD133- non-GSCs increased self-renewal activities, migration, and IR resistance, and rescued the reduction of GSC factors under shPARK7-transfected and serum-differentiation conditions. Intriguingly, PARK7 acted as a co-chaperone of HSP90 by binding to it, protecting EGFRvIII from proteasomal degradation. Knockdown of PARK7 increased the production of reactive oxygen species, inducing partial apoptosis and enhancing IR sensitivity in GSCs. Finally, PARK7 knockdown increased mouse survival and IR sensitivity in vivo. Based on these data, we propose that PARK7 plays a pivotal role in the maintenance of stemness and therapeutic resistance in GSCs.

IMMU-36. HIGH AFFINITY CHIMERIC ANTIGEN RECEPTOR WITH CROSS-REACTIVITY TO CLINICALLY-RELEVANT EGFR MUTATED PROTEINS

Abstract Tumor heterogeneity is one of the key reasons for therapeutic failure in Glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell clinical trial (NCT02209376) against Epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood brain barrier into GBM active tumor sites. However, CART cell infiltration was associated with a selective loss of EGFRvIII+ tumor cells and upregulation of immunosuppressive molecules. Post-CAR T treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations. To study this further We generated two structurally different CARs by fusing the scFv of mAb806 to 4-1BB and Killer immunoglobulin like receptor (KIR) co-stimulatory domains. Both 4-1BB and KIR based EGFR806 CAR T cells specifically lysed tumor cells and secreted cytokines when co-cultured with U87-MG (expressing low levels of EGFR) and U87-MG EGFR (U87-MG transduced with EGFR wild type for amplified background) cell lines and engineered to ectopically express targeted EGFR-ECD missense mutations and EGFRvIII. Unlike EGFR-specific cetuximab based CAR, EGFR-806CART cells did not kill EGFR wild type expressing fetal brain primary astrocytes and keratinocytes in vitro. We further evaluated the in vivo efficacy of 806-CARs and EGFRvIII CART currently in clinic in NSG mice. While EGFRvIII CART cells cleared EGFRvIII+ tumors alone 806 CART cells controlled EGFRvIII, amplified EGFR and EGFRR108K-mutant bearing tumors. KIR-CAR treated tumors are GVHD free and enhanced survival compared to 4-1BB based CARs. The broad specificity of EGFR806 CART cells to amplified EGFR and its mutant variants gives us the potential to clear various forms of EGFR. The enhanced anti-tumor efficacy by KIR based CAR in vivo setting provides us with additional therapeutic options.

Novel Molecular Markers in Glioblastoma—Benefits of Liquid Biopsy

Glioblastoma is a primary Central Nervous System (CNS) malignancy with poor survival. Treatment options are scarce and despite the extremely heterogeneous nature of the disease, clinicians lack prognostic and predictive markers to characterize patients with different outcomes. Certain immunohistochemistry, FISH, or PCR-based molecular markers, including isocitrate dehydrogenase1/2 (IDH1/2) mutations, epidermal growth factor receptor variant III (EGFRvIII) mutation, vascular endothelial growth factor overexpression (VEGF) overexpression, or (O6-Methylguanine-DNA methyltransferase promoter) MGMT promoter methylation status, are well-described; however, their clinical usefulness and accuracy is limited, and tumor tissue samples are always necessary. Liquid biopsy is a developing field of diagnostics and patient follow up in multiple types of cancer. Fragments of circulating nucleic acids are collected in various forms from different bodily fluids, including serum, urine, or cerebrospinal fluid in order to measure the quality and quantity of these markers. Multiple types of nucleic acids can be analyzed using liquid biopsy. Circulating cell-free DNA, mitochondrial DNA, or the more stable long and small non-coding RNAs, circular RNAs, or microRNAs can be identified and measured by novel PCR and next-generation sequencing-based methods. These markers can be used to detect the previously described alterations in a minimally invasive method. These markers can be used to differentiate patients with poor or better prognosis, or to identify patients who do not respond to therapy. Liquid biopsy can be used to detect recurrent disease, often earlier than using imaging modalities. Liquid biopsy is a rapidly developing field, and similarly to other types of cancer, measuring circulating tumor-derived nucleic acids from biological fluid samples could be the future of differential diagnostics, patient stratification, and follow up in the future in glioblastoma as well.

Characterize brain tumor genetic signatures with focused ultrasound-enabled liquid biopsy

Although blood-based liquid biopsy is a promising noninvasive diagnostic technique, the blood-brain barrier (BBB) hinders the efficient transport of brain tumor biomarkers into the bloodstream. In our previous studies, we demonstrated the feasibility for focused ultrasound-enabled liquid biopsy (FUS-LBx) to increase BBB permeability and enhance the transport of glioblastoma (GBM)-specific biomarkers into the bloodstream of mice using mRNA as a model biomarker. The objective of this study was to demonstrate the clinical translatability of FUS-LBx by evaluating the enhanced transport of the GBM-specific gene mutation, epidermal growth factor receptor variant III (EGFRvIII). Rats were randomly assigned to the treated (n = 10) or control groups (n = 6) 10 days after F98-EGFRvIII tumor cell implantation. MRI scans guided the combined FUS and microbubble sonication to the tumor center and verified successful BBB disruption. Blood was collected after FUS sonication or sham treatment. Droplet digital polymerase chain reaction was used to analyze the EGFRvIII complementary DNA. Rat brains were collected for histological analysis of potential FUS-induced tissue damage. MRI scans confirmed successful FUS-induced BBB opening. The EGFRvIII mutation that was undetectable by conventional LBx (no FUS), was detected by FUS-LBx. Histological analysis showed no significant tissue damage caused by FUS. This study demonstrated that FUS-LBx has the clinical translational potential for the noninvasive characterization of the GBM genetic profiles.

ERK-mediated negative feedback regulation of oncogenic EGFRvIII in glioblastoma cells.

Epidermal growth factor receptor variant III (EGFRvIII) is the most common active EGFR mutant in glioblastoma multiforme (GBM). The expression of this mutant often correlates with a poor patient prognosis due to its ability to extend downstream signaling. The EGFR pathway is controlled by a negative feedback mechanism that restricts the extent and length of downstream signaling. To date, the role of negative feedback in the oncogenic EGFRvIII mutant remains undetermined. The present study indicated that activation of the MEK-ERK pathway led to the phosphorylation of Thr-402, a conserved negative feedback residue in the juxtamembrane domain corresponding to Thr-669 of wild-type EGFR (EGFRwt), which resulted in a rapid reduction in the tyrosine phosphorylation of EGFRvIII in U87MG human glioblastoma and 293 cells. Moreover, despite the incapability of EGFRvIII to bind ligands, EGF was indicated to downregulate the tyrosine phosphorylation of EGFRvIII by activating the EGFRwt-ERK pathway. These results demonstrated a conserved negative feedback mechanism in the activation of EGFRvIII, which presents a new aspect in functional interactions between EGFRvIII and EGFRwt in glioblastoma cells.

Efficacy of osimertinib against EGFRvIII+ glioblastoma.

Epidermal Growth Factor Receptor variant III (EGFRvIII) is an active mutant form of EGFR that drives tumor growth in a subset of glioblastoma (GBM). It occurs in over 20% of GBMs, making it a promising receptor for small molecule targeted therapy. We hypothesize that poor penetration of the blood-brain barrier by previously tested EGFR-tyrosine kinase inhibitors (EGFR-TKIs) such as afateninb, erlotinib, gefitinib, and lapatinib played a role in their limited efficacy. The present study examined the effects of osimertinib (previously known as AZD9291) on EGFRvIII+ GBM models, both in vitro and in vivo. Therefore, a panel of six GBM stem cells (GSCs) expressing EGFRvIII+ was evaluated. The EGFRvIII+ GSC differed in the expression of EGFRvIII and other key genes. The GSC line D317, which expresses high levels of EGFRvIII and has robust tyrosine kinase activity, was selected for assessing osimertinib’s efficacy. Herein, we report that osimertinib inhibits the constitutive activity of EGFRvIII tyrosine kinase with high potency (<100 nM) while also inhibiting its downstream signaling. Further, osimertinib inhibited D317’s growth in vitro and in both heterotopic and orthotopic xenograft models. Additional preclinical studies are warranted to identify EGFRvIII+ GBM’s molecular signature most responsive to osimertinib.

Tailored therapy for recurrent glioblastoma: report of a personalized molecular approach.

Failure of clinical trials with targeted therapies in glioblastoma (GBM) is probably related to the enrollment of molecularly unselected patients. In this study we report the results of a precision medicine protocol in recurrent GBM. We prospectively evaluated 34 patients with recurrent GBM. We determined the expression of vascular endothelial growth factor (VEGF), epidermal growth factor receptor variant III (EGFRvIII), and phosphatase and tensin homolog (PTEN). According to the molecular pattern we administered bevacizumab alone in patients with VEGF overexpression, absence of EGFRvIII, and normal PTEN (group A; N.=16); bevacizumab + erlotinib in patients with VEGF overexpression, expression of EGFRvIII, and normal PTEN (group B; N.=14); and bevacizumab + sirolimus in patients with VEGF overexpression and loss of PTEN, irrespective of the EGFRvIII status (group C; N.=4). We evaluated the response rate, the clinical benefit rate, the 6-month progression-free survival (PFS-6), the 12-month PFS (PFS-12) and the safety profile of the treatment. Moreover, we compared our results with the ones of EORTC 26101 trial. Response rate was 50% in the whole cohort with the highest rate in group C (75%). Clinical benefit rate was 71% with the highest rate in group C (75%). PFS-6 was 56% in the whole cohort with the highest rate in group B (64%). PFS-12 was 21% in the whole cohort with the highest rate in group B (29%). When comparing our results with those from the combination arm of the EORTC 26101 trial we found a significantly higher PFS-6 and PFS-12 in our cohort. The precision medicine protocol for recurrent GBM is feasible and leads to improved results if compared with studies lacking molecular selection.

KHYG-1 Cells With EGFRvIII-specific CAR Induced a Pseudoprogression-like Feature in Subcutaneous Tumours Derived from Glioblastoma-like Cells.

We previously established a novel type of epidermal growth factor receptor variant III (EGFRvIII)-specific chimeric antigen receptor (CAR)-expressing natural killer (NK) cell line, designated EvCAR-KHYG-1, which inhibited the growth of glioblastoma (GBM) cells in vitro via apoptosis. We investigated the cytokine-producing effect of EvCAR-KHYG-1 cells on GBM-like cell lines and their antitumour effect using in vivo xenograft assays. EvCAR-KHYG-1 cells produced interleukin-2, interferon-γ, and tumour necrosis factor-α on EGFRvIII-expressing U87MG cells. In vivo xenograft assays showed that EvCAR-KHYG-1 cells did not reduce the volume of subcutaneous tumours derived from EGFRvIII-expressing U87MG cells but did reduce tumour cell occupancy. EvCAR-KHYG-1 cells led to expression of cellular immunity-related cytokines on EGFRvIII-expressing U87MG in vitro but did not inhibit tumour progression due to the induction of a pseudo progression-like pathological feature. Future studies investigating the effect of different conditions in vivo are required to study the inhibition of tumour progression in GBM.

Characterization of a novel type NK cell line KHYG-1 carrying EGFRvIII-specific CAR in glioblastoma cells

Abstract Background Natural killer (NK) cells are considered potential antitumor effector cells. The aim of this study was to establish a novel type of a chimeric antigen receptor (CAR)-NK cell line (CAR-KHYG-1) specific for epidermal growth factor receptor variant III (EGFRvIII)-expressing glioblastoma (GBM) cells and investigate the antitumor effects of EGFRvIII-specific-CAR-expressing KHYG-1 (EvCAR-KHYG-1) in vitro and in vivo. Materials and Methods EvCAR-KHYG-1 was established by lentiviral-based transduction of the EvCAR gene and magnetic bead-based purification of EvCAR-expressing NK cells. The antitumor effects of EvCAR-KHYG-1 were evaluated using growth inhibition and apoptosis detection assays on GBM cell lines (EGFRvIII-expressing and non-expressing U87MG glioma cells). Cytokine production was determined by cytometric beads array. In vivo xenograft assays were performed in NOG mice subcutaneously implanted with EGFRvIII-expressing U87MG (Ev-U87MG). Results EvCAR-KHYG-1 inhibited GBM cell-growth via apoptosis in an EGFRvIII-expression specific manner in vitro. In addition, EvCAR-KHYG-1 produced IL-2, INF-γ and TNF-α on Ev-U87MG cells. In vivo xenograft assays showed that EvCAR-KHYG-1 did not decrease the volume of subcutaneous tumors but inhibited the occupancy of cancer cells in the subcutaneous tumor. Discussion EvCAR-KHYG-1 enhanced antitumor effects and produced Th1-type cytokines on EGFRvIII-expressing GBM cells, but did not inhibit progression of subcutaneous GBM cell-derived tumors to induce a pseudo progression pathological phenotype. Conclusion A novel type CAR-NK cell line was established. In vivo experiments are required to determine ways it can be used to inhibit tumor progression.

First in human dose calculation of a single-chain bispecific antibody targeting glioma using the MABEL approach

BackgroundFirst-in-human (FIH) clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal...

Transformation of recombinant plasmid pJ404-EGFRvIII-bfp into an Escherichia coli NiCo21(DE3) expression host and its characterization

Epidermal growth factor receptor variant III (EGFRvIII) is a mutant of epidermal growth factor receptor (EGFR) that lacks 267 amino acids (exons 2–7) within its extracellular domain that results in the formation of a new epitope as a tumor specific target. A synthetic gene of EGFRvIII has been constructed by previous researchers to encode a fusion protein as a marker in targeted cancer therapies. This research was conducted to transform the recombinant plasmid pJ404-EGFRvIII into Escherichia coli NiCo21(DE3) host cells and characterize the E. coli NiCo21(DE3) transformants. Recombinant plasmid pJ404-EGFRvIII was isolated with an alkali lysis method and transformed into E. coli NiCo21(DE3) by heat-shock method. The transformants were grown on LB medium containing100 μg/ml ampicillin and characterized by colony PCR method. The results showed that the pJ404-EGFRvIII recombinant plasmid was transformed successfully into E. coli NiCo21(DE3). With the result that, EGFRvIII gene might be express by E. coli NiCo21(DE3) for further analysis of protein expression and purification in tumor terapy.