Functional Contribution and Clinical Implication of Cancer-Associated Fibroblasts in Glioblastoma.

PURPOSE: The abundance and biological contribution of cancer-associated fibroblasts (CAF) in glioblastoma (GBM) are poorly understood. Here, we aim to uncover its molecular signature, cellular roles, and potential tumorigenesis implications. EXPERIMENTAL DESIGN: We first applied single-cell RNA sequencing (RNA-seq) and bioinformatics analysis to identify and characterize stromal cells with CAF transcriptomic features in human GBM tumors. Then, we performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant GBM cells. RESULTS: We found that CAF abundance was low but significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling using three large cohorts containing bulk RNA-seq data and clinical information. Proteomic analysis of a GBM-derived CAF line and its secretome revealed fibronectin (FN1) as a critical candidate factor mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF-conditioned media and recombinant FN1 could facilitate the migration and invasion of GBM cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of GBMs. Interestingly, cell lines resembling the proneural state responded to the CAF signaling better for the migratory and invasive phenotypes. CONCLUSIONS: Overall, this study characterized the molecular features and functional impacts of CAFs in GBM, alluding to novel cell interactions mediated by CAFs in the GBM microenvironment.

Phase IIa Study of SurVaxM Plus Adjuvant Temozolomide for Newly Diagnosed Glioblastoma.

PURPOSE: Despite intensive treatment with surgery, radiation therapy, temozolomide (TMZ) chemotherapy, and tumor-treating fields, mortality of newly diagnosed glioblastoma (nGBM) remains very high. SurVaxM is a peptide vaccine conjugate that has been shown to activate the immune system against its target molecule survivin, which is highly expressed by glioblastoma cells. We conducted a phase IIa, open-label, multicenter trial evaluating the safety, immunologic effects, and survival of patients with nGBM receiving SurVaxM plus adjuvant TMZ following surgery and chemoradiation (ClinicalTrials.gov identifier: NCT02455557). METHODS: Sixty-four patients with resected nGBM were enrolled including 38 men and 26 women, in the age range of 20-82 years. Following craniotomy and fractionated radiation therapy with concurrent TMZ, patients received four doses of SurVaxM (500 µg once every 2 weeks) in Montanide ISA-51 plus sargramostim (granulocyte macrophage colony-stimulating factor) subcutaneously. Patients subsequently received adjuvant TMZ and maintenance SurVaxM concurrently until progression. Progression-free survival (PFS) and overall survival (OS) were reported. Immunologic responses to SurVaxM were assessed. RESULTS: SurVaxM plus TMZ was well tolerated with no serious adverse events attributable to SurVaxM. Of the 63 patients who were evaluable for outcome, 60 (95.2%) remained progression-free 6 months after diagnosis (prespecified primary end point). Median PFS was 11.4 months and median OS was 25.9 months measured from first dose of SurVaxM. SurVaxM produced survivin-specific CD8+ T cells and antibody/immunoglobulin G titers. Apparent clinical benefit of SurVaxM was observed in both methylated and unmethylated patients. CONCLUSION: SurVaxM appeared to be safe and well tolerated. The combination represents a promising therapy for nGBM. For patients with nGBM treated in this manner, PFS may be an acceptable surrogate for OS. A large randomized clinical trial of SurVaxM for nGBM is in progress.

Publisher Correction: Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

KX2-361: a novel orally bioavailable small molecule dual Src/tubulin inhibitor that provides long term survival in a murine model of glioblastoma.

PURPOSE: A major challenge to developing new therapies for patients with malignant brain tumors is that relatively few small molecule anticancer drugs penetrate the blood-brain barrier (BBB) well enough to provide therapeutically effective concentrations in brain tissue before drug exposure in non-CNS tissues results in unacceptable toxicity. METHODS: KX2-361, a member of a novel family of compounds with Src-kinase and tubulin polymerization inhibitory activity, demonstrates good oral bioavailability and readily crosses the BBB in mice. The objective of this study was to investigate the activity of KX2-361 against human and murine glioma cells and assess its therapeutic effect in a syngeneic orthotopic model of glioblastoma. RESULTS: In addition to reducing the level of Src autophosphorylation in the GL261 murine glioblastoma cell line, KX2-361 binds directly to tubulin and disrupts microtubule architecture in glioma cells maintained in culture. CONCLUSIONS: The drug is active in vivo against orthotopic GL261 gliomas in syngeneic C57BL/6 mice. Long term survival is not observed in mice lacking an adaptive immune system, indicating that KX2-361 works in concert with the host immune system to control tumor growth and promote long-term survival in the GL261 glioma model.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.

Survivin Monoclonal Antibodies Detect Survivin Cell Surface Expression and Inhibit Tumor Growth In Vivo.

Purpose: Survivin is an inhibitor of apoptosis protein (IAP) that is highly expressed in many cancers and represents an attractive molecule for targeted cancer therapy. Although primarily regarded as an intracellular protein with diverse actions, survivin has also been identified in association with circulating tumor exosomes.Experimental Design: We have reported that active, specific vaccination with a long peptide survivin immunogen leads to the development of survivin-specific CD8-mediated tumor cell lysis and prolongation of survival in tumor-bearing mice. In addition to cellular antitumor responses, circulating anti-survivin antibodies are detected in the serum of mice and human glioblastoma patients following vaccination with the survivin immunogen.Results: Here we demonstrate that survivin is present on the outer cell membrane of a wide variety of cancer cell types, including both murine and human glioma cells. In addition, antibodies to survivin that are derived from the immunogen display antitumor activity against murine GL261 gliomas in both flank and intracranial tumor models and against B16 melanoma as well.Conclusions: In addition to immunogen-induced, CD8-mediated tumor cell lysis, antibodies to the survivin immunogen have antitumor activity in vivo Cell-surface survivin could provide a specific target for antibody-mediated tumor immunotherapeutic approaches. Clin Cancer Res; 24(11); 2642-52. ©2018 AACR.

Circulating CD9+/GFAP+/survivin+ exosomes in malignant glioma patients following survivin vaccination.

Glioma cells release exosomes in culture and into the extracellular matrix in vivo. These nanobodies transport an array of biomolecules and are capable of mediating cell-cell communication. Circulating exosomes in cancer patients may be indicative of disease status and response to therapy. The inhibitor of apoptosis protein (IAP) survivin (SVN) promotes cancer cell proliferation, local immune suppression and resistance to chemotherapy and it is a potential cancer biomarker. We used imaging flow cytometry to perform quantitative measurements of circulating SVN+ exosomes in the serum of malignant glioma patients undergoing investigational treatment with an anti-survivin vaccine (SurVaxM). Serum from glioma patients contained abundant CD9+ exosomes with both SVN and glial fibrillary acidic protein (GFAP) on their surface. Survivin and GFAP were evaluated both independently and together as possible tumor markers on CD9+ exosomes. Patients with longer time to tumor progression generally exhibited a decrease in circulating CD9+/SVN+ and CD9+/GFAP+/SVN+ exosomes immediately following survivin vaccination; whereas, those with early tumor progression had an increase in exosomes, despite anti-survivin immunotherapy. Serum from non-cancer healthy control individuals had very few detectable CD9+/GFAP+/SVN+ exosomes, although CD9+/GFAP+ exosomes were detectable in small numbers. This study demonstrates that patients with malignant gliomas have CD9+/GFAP+/SVN+ and CD9+/SVN+ exosomes that are released into the circulation and that early reductions in their numbers following anti-survivin immunotherapy might be associated with longer progression-free survival.

Novel vaccines for glioblastoma: clinical update and perspective.

Glioblastoma is the most common primary brain cancer. Aggressive treatment with surgery, radiation therapy and chemotherapy provides limited overall survival benefit. Glioblastomas have a formidable tumor microenvironment that is hostile to immunological effector cells and these cancers produce profound systemic immunosuppression. However, surgical resection of these tumors creates conditions that favor the use of immunotherapeutic strategies. Therefore, extensive surgical resection, when feasible, will remain part of the equation to provide an environment in which active specific immunotherapy has the greatest chance of working. Toward that end, a number of vaccination protocols are under investigation. Vaccines studied to date have produced cellular and humoral antitumor responses, but unequivocal clinical efficacy has yet to be demonstrated. In addition, focus is shifting toward the prospect of therapies involving vaccines in combination with immune checkpoint inhibitors and other immunomodulatory agents so that effector cells remain active against their targets systemically and within the tumor microenvironment.

Clinical study of a survivin long peptide vaccine (SurVaxM) in patients with recurrent malignant glioma.

Survivin is an anti-apoptotic protein that is highly expressed in many cancers, including malignant gliomas. Preclinical studies established that the conjugated survivin peptide mimic SurVaxM (SVN53-67/M57-KLH) could stimulate an anti-tumor immune response against murine glioma in vivo, as well as human glioma cells ex vivo. The current clinical study was conducted to test safety, immunogenicity and clinical effects of the vaccine. Recurrent malignant glioma patients whose tumors were survivin-positive, and who had either HLA-A*02 or HLA-A*03 MHC class I allele-positivity, were given subcutaneous injections of SurVaxM (500 µg) in Montanide ISA 51 with sargramostim (100 µg) at 2-week intervals. SurVaxM was well tolerated with mostly grade one adverse events (AE) and no serious adverse events (SAE) attributable to the study drug. Six patients experienced local injection site reactions; three patients reported fatigue (grades 1 and 2), and 2 patients experienced myalgia (grade 1). Six of eight immunologically evaluable patients developed both cellular and humoral immune responses to vaccine. The vaccine also stimulated HLA-A*02, HLA-A*03 and HLA-A*24 restricted T cell responses. Three patients maintained a partial clinical response or stable disease for more than 6 months. Median progression-free survival was 17.6 weeks, and median overall survival was 86.6 weeks from study entry with seven of nine patients surviving more than 12 months.

Survivin co-ordinates formation of follicular T-cells acting in synergy with Bcl-6.

Follicular T helper (Tfh) cells are recognized by the expression of CXCR5 and the transcriptional regulator Bcl-6. Tfh cells control B cell maturation and antibody production, and if deregulated, may lead to autoimmunity. Here, we study the role of the proto-oncogene survivin in the formation of Tfh cells. We show that blood Tfh cells of patients with the autoimmune condition rheumatoid arthritis, have intracellular expression of survivin. Survivin was co-localized with Bcl-6 in the nuclei of CXCR5+CD4 lymphocytes and was immunoprecipitated with the Bcl-6 responsive element of the target genes. Inhibition of survivin in arthritic mice led to the reduction of CXCR5+ Tfh cells and to low production of autoantibodies. Exposure to survivin activated STAT3 and induced enrichment of PD-1+Bcl-6+ subset within Tfh cells. Collectively, our study demonstrates that survivin belongs to the Tfh cell phenotype and ensures their optimal function by regulating transcriptional activity of Bcl-6.

Survivin as a potential mediator to support autoreactive cell survival in myasthenia gravis: a human and animal model study.

The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular junction, associated with marked reduction of survivin-expressing circulating CD20+ cells. These data strongly suggest that survivin expression in cells with lymphocyte and plasma cell markers occurs in patients with myasthenia gravis and in two animal models of myasthenia gravis. Survivin expression may be part of a mechanism that inhibits the apoptosis of autoreactive B cells in myasthenia gravis and other autoimmune disorders.

Challenges in the development of a survivin vaccine (SurVaxM) for malignant glioma.

There is growing interest in immunotherapy for malignant gliomas. This interest stems from a number of immunological observations, together with the failure of conventional therapeutic agents to produce broad and clinically meaningful improvements in survival and quality of life. The challenges faced in translating laboratory-based immunological observations to Phase I and II clinical trials for immunotherapy of gliomas are substantial. Nevertheless, as our understanding of the effects of active specific vaccination in glioma patients grows, results support optimism that such methods may eventually prove useful as an adjunctive treatment for these cancers. This paper highlights a number of barriers encountered in the translational development of a survivin-targeted peptide vaccine (SurVaxM) for patients with malignant gliomas.

Metastasis suppressor NM23-H1 promotes repair of UV-induced DNA damage and suppresses UV-induced melanomagenesis.

Reduced expression of the metastasis suppressor NM23-H1 is associated with aggressive forms of multiple cancers. Here, we establish that NM23-H1 (termed H1 isoform in human, M1 in mouse) and two of its attendant enzymatic activities, the 3'-5' exonuclease and nucleoside diphosphate kinase, are novel participants in the cellular response to UV radiation (UVR)-induced DNA damage. NM23-H1 deficiency compromised the kinetics of repair for total DNA polymerase-blocking lesions and nucleotide excision repair of (6-4) photoproducts in vitro. Kinase activity of NM23-H1 was critical for rapid repair of both polychromatic UVB/UVA-induced (290-400 nm) and UVC-induced (254 nm) DNA damage, whereas its 3'-5' exonuclease activity was dominant in the suppression of UVR-induced mutagenesis. Consistent with its role in DNA repair, NM23-H1 rapidly translocated to sites of UVR-induced (6-4) photoproduct DNA damage in the nucleus. In addition, transgenic mice hemizygous-null for nm23-m1 and nm23-m2 exhibited UVR-induced melanoma and follicular infundibular cyst formation, and tumor-associated melanocytes displayed invasion into adjacent dermis, consistent with loss of invasion-suppressing activity of NM23 in vivo. Taken together, our data show a critical role for NM23 isoforms in limiting mutagenesis and suppressing UVR-induced melanomagenesis.

Antitumor cytotoxic T-cell response induced by a survivin peptide mimic.

Survivin is a tumor-associated antigen with significant potential as a cancer vaccine target. We have identified a survivin peptide mimic containing human MHC class I epitopes and a potential class II ligand that induces a potent antitumor response in C57BL/6 mice with GL261 cerebral gliomas. This peptide is able to elicit both CD8+ CTL and T helper cell responses in C57BL/6 mice. The corresponding region of the human survivin molecule represented by peptide SVN53-67 is 100% homologous to the murine protein, but SVN53-67 is weakly immunogenic in man. We evaluated several amino acid substitutions in putative human MHC I anchor positions in SVN53-67 to identify potential peptide mimics that could provide an enhanced antitumor immune response against human glioma and primary central nervous system lymphoma (PCNSL) cells in culture. We evaluated survivin peptides with predicted binding to human HLA-A*0201 antigen using peptide-loaded dendritic cells from PBMC of patients with these malignancies. One alteration (M57) led to binding to HLA-A*0201 with significantly higher affinity. We compared the ability of autologous dendritic cells loaded with SVN53-67 peptide and SVN53-67/M57 in CTL assays against allomatched and autologous, survivin-expressing, human malignant glioma and PCNSL cells. Both SVN53-67 and SVN53-67/M57 produced CTL-mediated killing of malignant target cells; however, SVN53-67/M57 was significantly more effective than SVN53-67. Thus, SVN53-67/M57 may act as a peptide mimic to induce an enhanced antitumor CTL response in tumor patients. The use of SVN53-67/M57 as a cancer vaccine might have application for cancer vaccine therapy.

Radiosensitization of GL261 glioma cells by tetraiodothyroacetic acid (tetrac).

We studied effects of tetrac (tetraiodothyroacetic acid) on survival of GL261, a murine brain tumor cell line, following single doses of 250 kVp x-rays and on repair of damage (sublethal and potentially lethal damage repair; SLDR, PLDR) in both exponential and plateau phase cells. Cells were exposed to 2 muM tetrac (1 h at 37 degrees C) prior to x-irradiation. At varying times after irradiation, cells were re-plated in medium without tetrac. Two weeks later, colonies were counted and results analyzed using either the linear-quadratic (LQ) or single-hit, multitarget (SHMT) formalisms. Tetrac sensitized both exponential and plateau phase cells to x-irradiation, as shown by a decrease in the quasi-threshold dose (Dq), leading to an average tetrac enhancement factor (ratio of SF2 values) of 2.5. Tetrac reduced SLDR in exponential cells by a factor of 1.8. In plateau phase cells there was little expression of SLDR, but tetrac produced additional cell killing at 1-4 h after the first dose. For PLDR expression in exponential cells, tetrac inhibited PLDR by a factor of 1.9, and in plateau phase cells, tetrac decreased PLDR expression by a factor of 3.4. These data show that the decreased Dq value seen after single doses of x-rays with tetrac treatment is also accompanied by a significant decrease in recovery from sublethal and potentially lethal damage.

MRI-based characterization of vascular disruption by 5,6-dimethylxanthenone-acetic acid in gliomas.

The well-vascularized nature of gliomas has generated a lot of interest in antiangiogenic therapies. However, the potential of vascular disrupting agents (VDAs) against gliomas has not been investigated extensively. In this study, we examined the in vivo efficacy of the tumor-VDA 5,6-dimethylxanthenone-4-acetic acid (DMXAA) against gliomas. Contrast-enhanced magnetic resonance imaging (MRI) and diffusion-weighted MRI were used to characterize the vascular and cellular responses of GL261 and U87 gliomas to DMXAA treatment. Therapeutic efficacy was assessed by Kaplan-Meier survival analysis. Before VDA treatment, minimal enhancement was detected within the tumor in both models. Longitudinal relaxation rate (R1=1/T1) maps acquired 24 h after treatment showed marked extravasation and accumulation of the contrast agent in the tumor indicative of treatment-induced vascular disruption. Normalized change in relaxation rate (DeltaR1) values of the tumor showed a significant increase (P<0.01 GL261; P<0.05 U87) after therapy compared with baseline estimates. Mean apparent diffusion coefficient (ADC) values were significantly increased (P=0.015) 72 h after therapy in GL261 but not in U87 gliomas. Vascular disrupting agent therapy resulted in a significant (P<0.01) increase in median survival in both models evaluated. The results highlight the potential of VDAs against gliomas and the utility of MRI in the assessment of glioma response to VDA therapy.

Expression of HMP/AN2, a melanoma associated antigen, in murine cerebral gliomas: potential for radioimmunotargeting.

Human melanoma proteoglycan (HMP), a melanoma-associated antigen, is expressed in both human melanomas and gliomas. We used HMP-specific monoclonal antibody (mAb) VT68.2 to investigate whether murine GL261 cerebral gliomas express the HMP homologue AN2 and to determine whether AN2 could be targeted for selective delivery of radiation in vivo. HMP-specific mAb VT68.2 stained murine GL261 glioma cells grown in culture and intracerebrally in syngeneic C57BL/6 mice. Positron emission tomography with radiolabeled mAb VT68.2 showed high-contrast, positive images of gliomas against a negative background. At 96 h after injection, glioma uptake of radiolabeled mAb VT68.2 was 10x greater than that of the isotype control mAb and 20x greater than that detected in normal cerebral tissue. Our results show murine GL261 cerebral gliomas express AN2 and HMP-specific mAb VT68.2 accumulates selectively and specifically at high concentration and is retained within murine cerebral gliomas. Thus, HMP is a potential target for antibody-mediated selective delivery of radiation to cerebral gliomas in vivo.

Therapeutic effect of a T helper cell supported CTL response induced by a survivin peptide vaccine against murine cerebral glioma.

Survivin is a tumor-associated antigen (TAA) that has significant potential for use as a cancer vaccine target. To identify survivin epitopes that might serve as targets for CTL-mediated, anti-tumor responses, we evaluated a series of survivin peptides with predicted binding to mouse H2-K(b) and human HLA-A*0201 antigens in peptide-loaded dendritic cell (DC) vaccines. H2-K(b)-positive, C57BL/6 mice were vaccinated using syngeneic, peptide-loaded DC2.4 cells. Splenocytes from vaccinated mice were screened by flow cytometry for binding of dimeric H2-K(b):Ig to peptide-specific CD8+ T cells. Two survivin peptides (SVN(57-64) and SVN(82-89)) generated specific CD8+ T cells. We chose to focus on the SVN(57-64) peptide because that region of the molecule is 100% homologous to human survivin. A larger peptide (SVN(53-67)), containing multiple class I epitopes, and a potential class II ligand, was able to elicit both CD8+ CTL and CD4+ T cell help. We tested the SVN(53-67) 15-mer peptide in a therapeutic model using a peptide-loaded DC vaccine in C57BL/6 mice with survivin-expressing GL261 cerebral gliomas. This vaccine produced significant CTL responses and helper T cell-associated cytokine production, resulting in a significant prolongation of survival. The SVN(53-67) vaccine was significantly more effective than the SVN(57-64) core epitope as a cancer vaccine, emphasizing the potential benefit of incorporating multiple class I epitopes and associated cytokine support within a single peptide.

Molecular targeting and treatment of composite EGFR and EGFRvIII-positive gliomas using boronated monoclonal antibodies.

PURPOSE: The purpose of the present study was to evaluate the anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), cetuximab, (IMC-C225) and the anti-EGFRvIII mAb, L8A4, used in combination as delivery agents for boron neutron capture therapy (BNCT) of a rat glioma composed of a mixture of cells expressing either wild-type (F98(EGFR)) or mutant receptors(F98(npEGFRvIII)). EXPERIMENTAL DESIGN: A heavily boronated polyamidoamine dendrimer (BD) was linked by heterobifunctional reagents to produce the boronated mAbs, BD-C225 and BD-L8A4. For in vivo biodistribution and therapy studies, a mixture of tumor cells were implanted intracerebrally into Fischer rats. Biodistribution studies were carried out by administering (125)I-labeled bioconjugates via convection-enhanced delivery (CED), and for therapy studies, nonradiolabeled bioconjugates were used for BNCT. This was carried out 14 days after tumor implantation and 24 h after CED at the Massachusetts Institute of Technology nuclear reactor. RESULTS: Following CED of a mixture of (125)I-BD-C225 and (125)I-BD-L8A4 to rats bearing composite tumors, 61.4% of the injected dose per gram (ID/g) was localized in the tumor compared with 30.8% ID/g for (125)I-BD-L8A4 and 34.7% ID/g for (125)I-BD-C225 alone. The corresponding calculated tumor boron values were 24.4 mug/g for rats that received both mAbs, and 12.3 and 13.8 mug/g, respectively, for BD-L8A4 or BD-C225 alone. The mean survival time of animals bearing composite tumors, which received both mAbs, was 55 days (P < 0.0001) compared with 36 days for BD-L8A4 and 38 days for BD-C225 alone, which were not significantly different from irradiated controls. CONCLUSIONS: Both EGFRvIII and wild-type EGFR tumor cell populations must be targeted using a combination of BD-cetuximab and BD-L8A4. Although in vitro C225 recognized both receptors, in vivo it was incapable of delivering the requisite amount of (10)B for BNCT of EGFRvIII-expressing gliomas.

Molecular targeting and treatment of an epidermal growth factor receptor-positive glioma using boronated cetuximab.

PURPOSE: The purpose of the present study was to evaluate the anti-epidermal growth factor monoclonal antibody (mAb) cetuximab (IMC-C225) as a delivery agent for boron neutron capture therapy (BNCT) of a human epidermal growth factor receptor (EGFR) gene-transfected rat glioma, designated as F98(EGFR). EXPERIMENTAL DESIGN: A heavily boronated polyamidoamine dendrimer was chemically linked to cetuximab by means of the heterobifunctional reagents N-succinimidyl 3-(2-pyridyldithio)-propionate and N-(k-maleimido undecanoic acid)-hydrazide. The bioconjugate, designated as BD-C225, was specifically taken up by F98(EGFR) glioma cells in vitro compared with receptor-negative F98 wild-type cells (41.8 versus 9.1 microg/g). For in vivo biodistribution studies, F98(EGFR) cells were implanted stereotactically into the brains of Fischer rats, and 14 days later, BD-C225 was given intracerebrally by either convection enhanced delivery (CED) or direct intratumoral (i.t.) injection. RESULTS: The amount of boron retained by F98(EGFR) gliomas 24 h following CED or i.t. injection was 77.2 and 50.8 microg/g, respectively, with normal brain and blood boron values <0.05 mug/g. Boron neutron capture therapy was carried out at the Massachusetts Institute of Technology Research Reactor 24 h after CED of BD-C225, either alone or in combination with i.v. boronophenylalanine (BPA). The corresponding mean survival times (MST) were 54.5 and 70.9 days (P = 0.017), respectively, with one long-term survivor (more than 180 days). In contrast, the MSTs of irradiated and untreated controls, respectively, were 30.3 and 26.3 days. In a second study, the combination of BD-C225 and BPA plus sodium borocaptate, given by either i.v. or intracarotid injection, was evaluated and the MSTs were equivalent to that obtained with BD-C225 plus i.v. BPA. CONCLUSIONS: The survival data obtained with BD-C225 are comparable with those recently reported by us using boronated mAb L8A4 as the delivery agent. This mAb recognizes the mutant receptor, EGFRvIII. Taken together, these data convincingly show the therapeutic efficacy of molecular targeting of EGFR using a boronated mAb either alone or in combination with BPA and provide a platform for the future development of combinations of high and low molecular weight delivery agents for BNCT of brain tumors.