Production and quality control assessment of a GLP-grade immunotoxin, D2C7-(scdsFv)-PE38KDEL, for a phase I/II clinical trial.

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel recombinant Pseudomonas exotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. Initial pre-clinical testing demonstrated the anti-tumor efficacy of D2C7-IT against orthotopic glioblastoma xenograft models expressing EGFRwt, EGFRvIII, or both EGFRwt and EGFRvIII. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter in Escherichia coli BLR (λ DE3). D2C7-IT was produced by a 10-L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300 mg of purified protein. The final vialed batch of D2C7-IT for clinical testing was at a concentration of 0.12 ± 0.1 mg/mL, the pH was at 7.4 ± 0.4, and endotoxin levels were below the detection limit of 10 EU/mL (1.26 EU/mL). The stability of the vialed D2C7-IT has been monitored over a period of 42 months through protein concentration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, size exclusion chromatography, cytotoxicity, sterility, and pH measurements. The vialed D2C7-IT is currently being tested in a phase I/II clinical trial by intratumoral convection-enhanced delivery for 72 h in patients with recurrent glioblastoma (NCT02303678, D2C7 for Adult Patients with Recurrent Malignant Glioma; clinicaltrials.gov ).

Preclinical toxicity evaluation of a novel immunotoxin, D2C7-(scdsFv)-PE38KDEL, administered via intracerebral convection-enhanced delivery in rats.

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel immunotoxin that reacts with wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFRvIII proteins overexpressed in glioblastomas. This study assessed the toxicity of intracerebral administration of D2C7-IT to support an initial Food and Drug Administration Investigational New Drug application. After the optimization of the formulation and administration, two cohorts (an acute and chronic cohort necropsied on study days 5 and 34) of Sprague-Dawley (SD) rats (four groups of 5 males and 5 females) were infused with the D2C7-IT formulation at total doses of 0, 0.05, 0.1, 0.4 µg (the acute cohort) and 0, 0.05, 0.1, 0.35 µg (the chronic cohort) for approximately 72 h by intracerebral convection-enhanced delivery using osmotic pumps. Mortality was observed in the 0.40 µg (5/10 rats) and 0.35 µg (4/10 rats) high-dose groups of each cohort. Body weight loss and abnormal behavior were only revealed in the rats treated with high doses of D2C7-IT. No dose-related effects were observed in clinical laboratory tests in either cohort. A gross pathologic examination of systemic tissues from the high-dose and control groups in both cohorts exhibited no dose-related or drug-related pathologic findings. Brain histopathology revealed the frequent occurrence of dose-related encephalomalacia, edema, and demyelination in the high-dose groups of both cohorts. In this study, the maximum tolerated dose of D2C7-IT was determined to be between 0.10 and 0.35 µg, and the no-observed-adverse-effect-level was 0.05 µg in SD rats. Both parameters were utilized to design the Phase I/II D2C7-IT clinical trial.

Systemic administration of a bispecific antibody targeting EGFRvIII successfully treats intracerebral glioma.

Bispecific antibodies (bscAbs), particularly those of the bispecific T-cell engager (BiTE) subclass, have been shown to effectively redirect T cells against cancer. Previous efforts to target antigens expressed in both tumors and normal tissues have produced significant toxicity, however. Moreover, like other large molecules, bscAbs may be restricted from entry into the "immunologically privileged" CNS. A tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, is a constitutively activated tyrosine kinase not found in normal tissues but frequently expressed in glioblastomas and many other neoplasms. Because it is localized solely to tumor tissue, EGFRvIII presents an ideal target for immunotherapy. Here we report the preclinical evaluation of an EGFRvIII-targeted BiTE, bscEGFRvIIIxCD3. Our results show that bscEGFRvIIIxCD3 activates T cells to mediate potent and antigen-specific lysis of EGFRvIII-expressing gliomas in vitro (P < 0.001) at exceedingly low concentrations (10 ng/mL) and effector-to-target ratios (2.5:1). Treatment with i.v. bscEGFRvIIIxCD3 yielded extended survival in mice with well-established intracerebral tumors (P < 0.05) and achieved durable complete cure at rates up to 75%. Antitumor efficacy was significantly abrogated on blockade of EGFRvIII binding, demonstrating the need for target antigen specificity both in vitro and in vivo. These results demonstrate that BiTEs can be used to elicit functional antitumor immunity in the CNS, and that peptide blockade of BiTE-mediated activity may greatly enhance the safety profile for antibody-redirected T-cell therapies. Finally, bscEGFRvIIIxCD3 represents a unique advancement in BiTE technology given its exquisite tumor specificity, which enables precise elimination of cancer without the risk of autoimmune toxicity.

Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors.

Our study demonstrates the glioma tumor antigen podoplanin to be present at very high levels (>90%) in both glioblastoma (D2159MG, D08-0308MG and D08-0493MG) and medulloblastoma (D283MED, D425MED and DAOY) xenografts and cell line. We constructed a novel recombinant single-chain antibody variable region fragment (scFv), NZ-1, specific for podoplanin from the NZ-1 hybridoma. NZ-1-scFv was then fused to Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide (NZ-1-PE38KDEL). The immunotoxin (IT) was further stabilized by a disulfide (ds) bond between the heavy-chain and light-chain variable regions as the construct NZ-1-(scdsFv)-PE38KDEL. NZ-1-(scdsFv)-PE38KDEL exhibited significant reactivity to glioblastoma and medulloblastoma cells. The affinity of NZ-1-(scdsFv), NZ-1-(scdsFv)-PE38KDEL and NZ-1 antibody for podoplanin peptide was 2.1 × 10(-8) M, 8.0 × 10(-8) M and 3.9 × 10(-10) M, respectively. In a protein stability assay, NZ-1-(scdsFv)-PE38KDEL retained 33-98% of its activity, whereas that of NZ-1-PE38KDEL declined to 13% of its initial levels after incubation at 37°C for 3 days. In vitro cytotoxicity of the NZ-1-(scdsFv)-PE38KDEL was measured in cells isolated from glioblastoma xenografts, D2159MG, D08-0308MG and D08-0493MG, and in the medulloblastoma D283MED, D425MED and DOAY xenografts and cell line. The NZ-1-(scdsFv)-PE38KDEL IT was highly cytotoxic, with an 50% inhibitory concentration in the range of 1.6-29 ng/ml. Significantly, NZ-1-(scdsFv)-PE38KDEL demonstrated tumor growth delay, averaging 24 days (p < 0.001) and 21 days (p < 0.001) in D2159MG and D283MED in vivo tumor models, respectively. Crucially, in the D425MED intracranial tumor model, NZ-1-(scdsFv)-PE38KDEL caused a 41% increase in survival (p ≤ 0.001). In preclinical studies, NZ-1-(scdsFv)-PE38KDEL exhibited significant potential as a targeting agent for malignant brain tumors.

Affinity-matured anti-glycoprotein NMB recombinant immunotoxins targeting malignant gliomas and melanomas.

Glycoprotein NMB (GPNMB), a transmembrane glycoprotein highly expressed in high-grade gliomas (HGGs), is an attractive target in cancer immunotherapy. We isolated a GPNMB-specific scFv clone, G49, from a human synthetic phage-display library. To obtain mutant single-chain variable-fragment antibodies (scFvs) with improved affinity and immunotoxins with increased activity, we subjected G49 to in vitro affinity maturation by a complementarity-determining-region (CDR) random-mutagenesis technique. Using light-chain CDR3 mutagenesis, cell-based panning by phage display, subsequent heavy-chain CDR1 mutagenesis, and flow-cytometric selection by yeast-surface display, we generated the mutant scFv clone 902V, with an overall 11-fold increase in affinity for GPNMB. Clone 902V was further randomized throughout the whole scFv by error-prone PCR, and one mutant, F6V, was selected by yeast-surface display. F6V scFv, differing from 902V by one amino-acid change in the light-chain CDR2, exhibited an affinity for GPNMB of 0.30 nM. The F6V mutant scFv clone was fused with a truncated form of Pseudomonas exotoxin A to form the immunotoxin F6V-PE38. F6V-PE38 demonstrated significant protein-synthesis-inhibition activity on GPNMB-expressing glioma and malignant melanoma cells (IC(50) = 0.5 ng/ml [8 pM]), a 60-fold improvement over G49 activity, but no cytotoxicity on GPNMB-negative cells. Furthermore, F6V-PE38 exhibited significant antitumor activity against subcutaneous malignant glioma xenografts in two nude-mouse models and a melanoma neoplastic meningitis model in athymic rats. These GPNMB-specific scFv antibodies and immunotoxins hold promise as reagents in targeted therapy for HGGs and other GPNMB-expressing malignancies.

Synergistic control of T cell development and tumor suppression by diacylglycerol kinase alpha and zeta.

Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK alpha and zeta synergistically promote T cell maturation in the thymus. Absence of both DGKalpha and zeta (DGKalpha(-/-)zeta(-/-)) results in a severe decrease in the number of CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGKalpha(-/-)zeta(-/-) mice. The developmental blockage in DGKalpha(-/-)zeta(-/-) mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.

Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis.

BACKGROUND: Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. RESULTS: B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. CONCLUSIONS: These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.

Recombinant single-chain variable fragment antibodies against extracellular epitopes of human multidrug resistance protein MRP3 for targeting malignant gliomas.

Multidrug resistance protein 3 (MRP3), a multidrug resistance protein identified by serial analysis of gene expression as a glioblastoma multiforme (GBM)-associated molecule, is highly expressed in GBM, but not in normal brain cells. Thus, MRP3 is a candidate for GBM immunotargeting, but to date, no monoclonal antibody has been isolated that can target an extracellular MRP3 epitope. By phage display, we have isolated 3 recombinant, fully human, single-chain Fv (scFv) antibodies, M25, M58 and M89, which specifically react with the extracellular N-terminus of human MRP3. In ELISA, these scFvs reacted only with the peptide used for screening and not with other MRP3-derived peptides. Flow cytometric analysis revealed that these scFv fragments bind specifically to viable human GBM cells displaying different MRP3 expression levels, but not to MRP3-null cells. Furthermore, these scFv antibodies failed to react with tumor cells overexpressing other MRP proteins, including MRP1, MRP2, MRP4 and MRP5. M25 and M58 also bound to viable neurospheres. Iodogen-labeled scFvs demonstrated a yield of 56-76%. The immunoreactive fractions of the radiolabeled M25, M58 and M89 scFvs were 32, 52 and 69%, respectively. M25 exhibited 20% internalization into D2159MG neurospheres, M58, 33% into D54MG cells and M89, 26% into D247MG. Immunohistochemical evaluation of human gliomas to determine the localization of MRP3 antigen using scFvs M25 and M58 showed a dense cytoplasmic and membranous staining pattern. These Fv-based recombinant antibodies, which possess superior tumor penetration capabilities and selectively target tumor cells that express MRP3, may potentially be used in immunotherapy and diagnosis for brain tumors and other cancers.

GMab-1, a high-affinity anti-3'-isoLM1/3',6'-isoLD1 IgG monoclonal antibody, raised in lacto-series ganglioside-defective knockout mice.

The lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1 have been identified as tumor-associated antigens whose formation is initiated by the Lc3-synthase. Until now, high-affinity IgG monoclonal antibodies (mAbs) against 3'-isoLM1 and 3',6'-isoLD1, which are highly expressed in gliomas, have not been developed, although mAbs against lacto-series gangliosides are powerful tools for functional studies. We previously produced the Lc3-synthase gene beta3Gn-T5 knockout mice. In this study, we immunized beta3Gn-T5 knockout mice with 3'-isoLM1/3',6'-isoLD1 and produced the anti-3'-isoLM1/3',6'-isoLD1 mAb GMab-1, of the IgG(3) subclass, which should be useful for functional analysis of lacto-series gangliosides and for antibody-based therapy of gliomas.

MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

BACKGROUND: Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). METHODS: We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. RESULTS: Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). CONCLUSIONS: Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.

A monoclonal antibody IMab-1 specifically recognizes IDH1R132H, the most common glioma-derived mutation.

IDH1 (isocitrate dehydrogenase 1) mutations have been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas as well as secondary glioblastomas. In contrast, primary glioblastomas very rarely contain IDH1 mutations, although primary and secondary glioblastomas are histologically indistinguishable. The IDH1 mutations are remarkably specific to a single codon in the conserved and functionally important Arg132 in IDH1. In gliomas, the most frequent IDH1 mutations (>90%) were G395A (R132H). In this study, we immunized mice with R132H-containing IDH1 (IDH1(R132H)) peptide. After cell fusion using Sendai virus envelope, the monoclonal antibodies (mAbs), which specifically reacted with IDH1(R132H), were screened in ELISA. One of the mAbs, IMab-1 reacted with the IDH1(R132H) peptide, but not with wild type IDH1 (IDH1(wt)) peptide in ELISA. In Western-blot analysis, IMab-1 reacted with only the IDH1(R132H) protein, not IDH1(wt) protein or the other IDH1 mutants, indicating that IMab-1 is IDH1(R132H)-specific. Furthermore, IMab-1 specifically stained the IDH1(R132H)-expressing cells in astrocytomas in immunohistochemistry, whereas it did not react with IDH1(R132H)-negative primary glioblastoma sections. In conclusion, we established an anti-IDH1(R132H)-specific monoclonal antibody IMab-1, which should be significantly useful for diagnosis and biological evaluation of mutation-bearing gliomas.

A Rationally Designed Fully Human EGFRvIII:CD3-Targeted Bispecific Antibody Redirects Human T Cells to Treat Patient-derived Intracerebral Malignant Glioma.

Purpose: Conventional therapy for malignant glioma fails to specifically target tumor cells. In contrast, substantial evidence indicates that if appropriately redirected, T cells can precisely eradicate tumors. Here we report the rational development of a fully human bispecific antibody (hEGFRvIII-CD3 bi-scFv) that redirects human T cells to lyse malignant glioma expressing a tumor-specific mutation of the EGFR (EGFRvIII).Experimental Design: We generated a panel of bispecific single-chain variable fragments and optimized design through successive rounds of screening and refinement. We tested the ability of our lead construct to redirect naïve T cells and induce target cell-specific lysis. To test for efficacy, we evaluated tumor growth and survival in xenogeneic and syngeneic models of glioma. Tumor penetrance following intravenous drug administration was assessed in highly invasive, orthotopic glioma models.Results: A highly expressed bispecific antibody with specificity to CD3 and EGFRvIII was generated (hEGFRvIII-CD3 bi-scFv). Antibody-induced T-cell activation, secretion of proinflammatory cytokines, and proliferation was robust and occurred exclusively in the presence of target antigen. hEGFRvIII-CD3 bi-scFv was potent and target-specific, mediating significant lysis of multiple malignant glioma cell lines and patient-derived malignant glioma samples that heterogeneously express EGFRvIII. In both subcutaneous and orthotopic models, well-engrafted, patient-derived malignant glioma was effectively treated despite heterogeneity of EGFRvIII expression; intravenous hEGFRvIII-CD3 bi-scFv administration caused significant regression of tumor burden (P < 0.0001) and significantly extended survival (P < 0.0001). Similar efficacy was obtained in highly infiltrative, syngeneic glioma models, and intravenously administered hEGFRvIII-CD3 bi-scFv localized to these orthotopic tumors.Conclusions: We have developed a clinically translatable bispecific antibody that redirects human T cells to safely and effectively treat malignant glioma. On the basis of these results, we have developed a clinical study of hEGFRvIII-CD3 bi-scFv for patients with EGFRvIII-positive malignant glioma. Clin Cancer Res; 24(15); 3611-31. ©2018 AACR.

Glycoprotein nonmetastatic melanoma protein B, a potential molecular therapeutic target in patients with glioblastoma multiforme.

PURPOSE: More brain tumor markers are required for prognosis and targeted therapy. We have identified and validated promising molecular therapeutic glioblastoma multiforme (GBM) targets: human transmembrane glycoprotein nonmetastatic melanoma protein B (GPNMB(wt)) and a splice variant form (GPNMB(sv), a 12-amino-acid in-frame insertion in the extracellular domain). EXPERIMENTAL DESIGN: We have done genetic and immunohistochemical evaluation of human GBM to determine incidence, distribution, and pattern of localization of GPNMB antigens in brain tumors as well as survival analyses. RESULTS: Quantitative real-time PCR on 50 newly diagnosed GBM patient tumor samples indicated that 35 of 50 GBMs (70%) were positive for GPNMB(wt+sv) transcripts and 15 of 50 GBMs (30%) were positive for GPNMB(sv) transcripts. Normal brain samples expressed little or no GPNMB mRNA. We have isolated and characterized an anti-GPNMB polyclonal rabbit antiserum (2640) and two IgG2b monoclonal antibodies (mAb; G11 and U2). The binding affinity constants of the mAbs ranged from 0.27 x 10(8) to 9.6 x 10(8) M(-1) measured by surface plasmon resonance with immobilized GPNMB, or 1.7 to 2.1 x 10(8) M(-1) by Scatchard analyses with cell-expressed GPNMB. Immunohistochemical analysis detected GPNMB in a membranous and cytoplasmic pattern in 52 of 79 GBMs (66%), with focal perivascular reactivity in approximately 27%. Quantitative flow cytometric analysis revealed GPNMB cell surface molecular density of 1.1 x 10(4) to 7.8 x 10(4) molecules per cell, levels sufficient for mAb targeting. Increased GPNMB mRNA levels correlated with elevated GPNMB protein expression in GBM biopsy samples. Univariate and multivariate analyses correlated expression of GPNMB with survival of 39 GBM patients using RNA expression and immunohistochemical data, establishing that patients with relatively high mRNA GPNMB transcript levels (wt+sv and wt), >3-fold over normal brain, as well as positive immunohistochemistry, have a significantly higher risk of death (hazard ratios, 3.0, 2.2, and 2.8, respectively). CONCLUSIONS: Increased mRNA and protein levels in GBM patient biopsy samples correlated with higher survival risk; as a detectable surface membrane protein in glioma cells, the data indicate that GPNMB is a potentially useful tumor-associated antigen and prognostic predictor for therapeutic approaches with malignant gliomas or any malignant tumor that expresses GPNMB.

Antiepidermal growth factor variant III scFv fragment: effect of radioiodination method on tumor targeting and normal tissue clearance.

INTRODUCTION: MR1-1 is a single-chain Fv (scFv) fragment that binds with high affinity to epidermal growth factor receptor variant III, which is overexpressed on gliomas and other tumors but is not present on normal tissues. The objective of this study was to evaluate four different methods for labeling MR1-1 scFv that had been previously investigated for the radioiodinating of an intact anti-epidermal growth factor receptor variant III (anti-EGFRvIII) monoclonal antibody (mAb) L8A4. METHODS: The MR1-1 scFv was labeled with (125)I/(131)I using the Iodogen method, and was also radiohalogenated with acylation agents bearing substituents that were positively charged--N-succinimidyl-3-[*I]iodo-5-pyridine carboxylate and N-succinimidyl-4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB)--and negatively charged--N-succinimidyl-3-[*I]iodo-4-phosphonomethylbenzoate ([*I]SIPMB). In vitro internalization assays were performed with the U87MGDeltaEGFR cell line, and the tissue distribution of the radioiodinated scFv fragments was evaluated in athymic mice bearing subcutaneous U87MGDeltaEGFR xenografts. RESULTS AND CONCLUSION: As seen previously with the anti-EGFRvIII IgG mAb, retention of radioiodine activity in U87MGDeltaEGFR cells in the internalization assay was labeling method dependent, with SGMIB and SIPMB yielding the most prolonged retention. However, unlike the case with the intact mAb, the results of the internalization assays were not predictive of in vivo tumor localization capacity of the labeled scFv. Renal activity was dependent on the nature of the labeling method. With MR1-1 labeled using SIPMB, kidney uptake was highest and most prolonged; catabolism studies indicated that this uptake primarily was in the form of epsilon-N-3-[*I]iodo-4-phosphonomethylbenzoyl lysine.

In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9.

The ability to selectively target disease-related tissues with molecules is critical to the design of effective therapeutic and diagnostic reagents. Recognizing the differences between the in vivo environment and in vitro conditions, we employed an in vivo selection strategy to identify RNA aptamers (targeting motifs) that could localize to tumor in situ. One of the selected molecules is an aptamer that binds to the protein DHX9, an RNA helicase that is known to be upregulated in colorectal cancer. Upon systemic administration, the aptamer preferentially localized to the nucleus of cancer cells in vivo and thus has the potential to be used for targeted delivery.

EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss.

PURPOSE: Chimeric antigen receptor (CAR) transduced T cells represent a promising immune therapy that has been shown to successfully treat cancers in mice and humans. However, CARs targeting antigens expressed in both tumors and normal tissues have led to significant toxicity. Preclinical studies have been limited by the use of xenograft models that do not adequately recapitulate the immune system of a clinically relevant host. A constitutively activated mutant of the naturally occurring epidermal growth factor receptor (EGFRvIII) is antigenically identical in both human and mouse glioma, but is also completely absent from any normal tissues. EXPERIMENTAL DESIGN: We developed a third-generation, EGFRvIII-specific murine CAR (mCAR), and performed tests to determine its efficacy in a fully immunocompetent mouse model of malignant glioma. RESULTS: At elevated doses, infusion with EGFRvIII mCAR T cells led to cures in all mice with brain tumors. In addition, antitumor efficacy was found to be dependent on lymphodepletive host conditioning. Selective blockade with EGFRvIII soluble peptide significantly abrogated the activity of EGFRvIII mCAR T cells in vitro and in vivo, and may offer a novel strategy to enhance the safety profile for CAR-based therapy. Finally, mCAR-treated, cured mice were resistant to rechallenge with EGFRvIII(NEG) tumors, suggesting generation of host immunity against additional tumor antigens. CONCLUSION: All together, these data support that third-generation, EGFRvIII-specific mCARs are effective against gliomas in the brain and highlight the importance of syngeneic, immunocompetent models in the preclinical evaluation of tumor immunotherapies.

Rational design and generation of recombinant control reagents for bispecific antibodies through CDR mutagenesis.

Developments in the field of bispecific antibodies have progressed rapidly in recent years, particularly in their potential role for the treatment of malignant disease. However, manufacturing stable molecules has proven to be costly and time-consuming, which in turn has hampered certain aspects of preclinical evaluation including the unavailability of appropriate "negative" controls. Bispecific molecules (e.g., bispecific tandem scFv) exhibit two specificities, often against a tumor antigen as well as an immune-activation ligand such as CD3. While for IgG antibodies, isotype-matched controls are well accepted, when considering smaller antibody fragments it is not possible to adequately control for their biological activity through the use of archetypal isotypes, which differ dramatically in affinity, size, structure, and design. Here, we demonstrate a method for the rapid production of negative control tandem scFvs through complementarity determining region (CDR) mutagenesis, using a recently described bispecific T-cell engager (BiTE) targeting a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII) as an example. Four independent control constructs were developed by this method through alteration of residues spanning individual CDR domains. Importantly, while target antigen affinity was completely impaired, CD3 binding affinity was conserved in each molecule. These results have a potential to enhance the sophistication by which bispecific antibodies can be evaluated in the preclinical setting and may have broader applications for an array of alternative antibody-derived therapeutic platforms.

Affinity-matured recombinant immunotoxin targeting gangliosides 3'-isoLM1 and 3',6'-isoLD1 on malignant gliomas.

About 60 percent of glioblastomas highly express the gangliosides 3'-isoLM1 and 3',6'-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3'-isoLM1 and 3',6'-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3'-isoLM1 and 3',6'-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3'-isoLM1 and 3',6'-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the KD of DmAb14m-IT for gangliosides 3'-isoLM1 and 3',6'-isoLD1 was 2.6 × 10(-9)M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3'-isoLM1 and 3',6'-isoLD1. The DmAb14m-IT IC 50 was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3'-isoLM1 and 3',6'-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3'-isoLM1 and 3',6'-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and eventually in the clinical setting.

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

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, or both.

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.