T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2–7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.

This article is featured in Highlights of This Issue, p. 1497

GBM is the most common brain cancer of the adult with still a dismal prognosis. Immunotherapy has shown good results in several solid tumors, but GBM remains recalcitrant to this type of therapies (1–3).

GBM is considered to be a noninflamed tumor with low level of CD8 T-cell infiltration, and this may hinder the efficacy of immunotherapies (4–6). In this sense, novel agents to redirect cytotoxic T cells to the tumor may hold promise to improve GBM therapies (7–13).

Among the different therapeutic approaches designed to engage the immune system against tumors, TCBs have been shown to elicit good antitumor responses by cross-linking T cells to target tumor cells (14–17). TCBs are antibodies engineered to include binding sites to the invariant CD3e chain of the TCR and to a tumor-associated antigen. TCBs then promote the recruitment of CD3 lymphocytes to the tumor expressing the tumor-associated antigen-inducing tumor cell killing. Promising clinical activity has been described with TCBs in hematologic malignancies (16). However, the clinical development of TCBs in solid tumors has been hampered by the lack of tumor-specific antigens that are not expressed in healthy tissue.

EGFR gene amplification is a common event in GBM and is generally accompanied by a gene rearrangement that leads to the EGFR variant III (EGFRvIII) mutation (18–21). EGFRvIII is characterized by a deletion in the extracellular domain of exons 2–7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion (20). EGFRvIII is expressed on the surface of tumor cells, and it is not expressed in normal tissues, making EGFRvIII an ideal target for TCBs. Several strategies have been designed to target EGFRvIII in GBM, including bispecific antibodies with dual specificities to CD3 and EGFRvIII (22–26). These antibodies were characterized by a short half-life due to a lack of an Fc portion.

We designed, developed, and characterized a novel 2+1 IgG-based EGFRvIII-TCB (RG6156) molecule and evaluated its antitumoral activity both in vitro and in vivo using patient-derived models of GBM. We observed EGFRvIII-specific T-cell activation resulting in cytotoxicity on EGFRvIII-expressing patient-derived tumoroids. In vivo, EGFRvIII-TCB induced T-cell infiltration and antitumor response including tumor regression in both subcutaneous and orthotopic humanized GBM PDXs. Globally, our findings indicate that EGFRvIII-TCBs can be considered as a promising therapeutic tool against GBM warranting clinical testing.

Cells

Adherent cells were maintained under sterile conditions at 37°C in a humidified incubator (5% CO2) and passaged regularly upon reaching 80% confluency. All cells were within 10 passages, and mycoplasma tests have been performed on all cell lines routinely (A8994.0050, VWR). The authentication of all cell lines has been performed by suppliers using a short tandem repeat analysis.

U87MG-huEGFRvIII sorted pool

Human brain–derived U87MG cells were purchased from ATCC (ATCC HTB-14) and stably transfected with huEGFRvIII (high EGFRvIII expression). Cells were grown adherent in DMEM containing 1% GlutaMax, 10% FBS, and 6 g/mL of fresh puromycin.

U87MG-huEGFRvIII clone 2

Clone 2 of human brain-derived U87MG cells was purchased from ECACC (ECACC 89081402) and stably transfected with huEGFRvIII; clone 2 expresses low levels of huEGFRvIII. Cells were grown adherent in DMEM containing 1% GlutaMax and 10% FBS.

DK-MG sorted pool

Human GBM-derived DK-MG cells were purchased from DMSZ (DMSZ, ACC 277) and sorted according to EGRFvIII expression. Cells were grown adherent in RPMI-1640 containing 10% FBS and 1% GlutaMax. The cells are fibroblast-like and duplicate rather slowly.

A431

Human epidermoid carcinoma cell lines expressing EGFRwt were purchased from ATCC (ATCCCRL-1555). Cells were grown adherent in RPMI-1640 containing 10% FBS and 1% GlutaMax.

Peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) were isolated from human blood collected from healthy donors by conventional Histopaque gradient.

Jurkat NFAT reporter cells

GloResponse Jurkat NFAT-RE-luc2P were purchased from Promega (Promega #CS176501) in a human acute lymphatic leukemia reporter cell line with a NFAT promoter, expressing human CD3. The cells grow in suspension and were cultured in RPMI-1640, 2 g/L glucose, 2 g/L NaHCO3, 10% FCS, 25 mmol/L HEPES, 2 mmol/L L-glutamin, 1× NEAA, 1× sodium-pyruvate at 0.1–0.5 Mio cells per mL. A final concentration of 200 μg per mL hygromycin B was added, whenever cells were passaged.

Human GBM-derived tumoroids

All studies were conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS). Human GBM specimens were collected the same day of the tumor resection from the Vall d'Hebron University Hospital and Clinic Hospital. The clinical protocol was approved by the Vall d'Hebron Institutional Review Board and Clinic Hospital (CEIC). In all cases, a written informed consent from the patients has been obtained before sample collection. GBM tumoroids were processed as follows. Minced pieces of human GBM samples were digested with the Human Tumor Dissociation kit (Miltenyi Biotec) in RPMI medium, for 40 minutes at 37°C with constant vigorous agitation. The single-cell suspension was filtered through a 70-μm cell strainer (BD Falcon) and washed with PBS. Finally, cells were resuspended and subsequently cultured in GBM medium which consists of Neurobasal medium (21103049) supplemented with B27 (A3582801), penicillin/streptomycin (15140148, all from Life Technologies), 20 ng/mL EGF (AF-100-15, PeproTech), and 20 ng/mL FGF-2 (100-18C-0100, PeproTech).

Coculture of human tumoroids with PBMCs

PBMCs were obtained from the whole blood of the same patient or from healthy donors by centrifuge density separation using Lymphosep (Biowest). PBMCs were cryopreserved in RPMI medium supplemented with 10% inactivated FBS and 10% DMSO until use. Human tumoroids were cocultured together with 1 × 106 PBMCs in a 1:10 ratio into a 12-well plate in RPMI medium supplemented with 10% inactivated FBS. 10 ng/mL of EGFRvIII-TCB or the control DP47-TCB were added for 48 hours.

Subcutaneous in vivo humanized models

All animal experiments were approved by and performed according to the guidelines of the Institutional Animal Care Committee of the Vall d'Hebron Research Institute in agreement with the European Union and national directives. We used subcutaneous mouse models to assess the in vivo efficacy of EGFRvIII-TCB in Hu-CD34+ NSG mice. Tumor growth inhibition was the readout for the subcutaneous model. Briefly, Hu-CD34+ NSG mice (The Jackson Laboratory-JAX West) were inoculated with 1 × 106 U87-huEGFRvIII cells injected subcutaneously. Mice were maintained under specific pathogen-free conditions with daily cycles of 12 hours light/darkness according to guidelines (GV-SOLAS; FELASA), and food and water were provided ad libitum. Continuous health monitoring was carried out, and the experimental study protocol was reviewed and approved by the Veterinary Department of Kanton Zurich.

Mice were randomized into different treatment groups, and therapy started when tumors reached an average of 200 mm3 volume as measured by a caliper in the subcutaneous model. Tumor volume was calculated with the formula: length × width × depth × π × 4/3. All treatments were administered intravenously. In the subcutaneous model, tumor growth inhibition was used as readout and to test for significant differences in group means for multiple comparisons, the standard analysis of variance (ANOVA) was used with the Dunnett method. JMP statistic software program was used for analyses.

In the case of GBM PDXs in Hu-CD34+ NSG mice, EGFRvIII-positive or -negative GBM PDXs maintained after several passages in NSG mice were checked for the presence of EGFRvIII mutation by qRT-PCR before proceeding with the experiment in humanized mice models. Specimens were cut with a scalpel into rectangular blocks of 5–10 mm length and 1–2 mm width and transferred into a tube containing 100 μL of Matrigel (356237, Cultek). Each specimen was introduced subcutaneously into two flanks of 14–16-week-old Hu-CD34+NSG mice (The Jackson Laboratory-JAX West) generated from the same donor and checked for hCD45+, hCD19+, hCD3+ hCD33+ percentages in mice blood at the moment of the shipment. Once GBM PDXs reached 100 mm3 volume, tumors were randomized and treatments started. Mice were treated biweekly with 2.5 mg/kg of DP47-TCB or EGFRvIII-TCB subcutaneously. Tumor progression was monitored by measuring tumor volume by a digital caliper. Once tumors reached around 400–600 mm3 volume, animals were euthanized and tumors were collected for FACS analysis or embedded in paraffin for IHC analysis.

Orthotopic in vivo models

For the orthotopic PBMC-humanized GBM model, 1.5 × 105 U87MG or U87MG-EGFRvIII all expressing luciferase were stereotactically inoculated into the corpus striatum of the right brain hemisphere (1 mm anterior and 1.8 mm lateral to the lambda; 2.5 mm intraparenchymal) of 5-week-old NSG mice. After 3 days, 1 × 107 PBMCs together with 5 mg/kg of EGFRvIII-TCB or control DP47-TCB were i.v. inoculated into the mice and treatment continued twice a week subcutaneously. In the case of PDXs, 3 × 105 EGFRvIII-positive GBM-derived tumoroids expressing luciferase were orthotopically injected in 14–16-week-old Hu-CD34+NSG mice and the treatment with 5 mg/kg of EGFRvIII-TCB or control DP47-TCB was started at 10–22 days after inoculation and continued twice a week subcutaneously. In the orthotopic syngeneic GBM model, 5 × 105 U87MG-EGFRvIII nonluciferase-expressing cells were stereotactically inoculated (as described above) into the right brain hemisphere of Balb/c nude mice. After 18 days, animals received a single i.v. bolus of EGFRvIII-TCB or control DP47-TCB all labeled with Alexa Fluor 647 fluorescence dye. After 24 hours, whole brains were harvested and processed for 3D light-sheet fluorescence microscopy (3D LSFM). For luciferase-expressing tumors, tumor progression was monitored by bioluminescence measurements using the Xenogen IVIS Spectrum. Mice were euthanized by cervical dislocation when they exhibited clinical signs of disease or distress and tumors were collected for FACS analysis or embedded in paraffin for IHC analysis.

Statistical analysis

GraphPad Prism 5.0 software was used to analyze data. P value was calculated using Student t test (paired or unpaired) for parametric variables and Mann–Whitney test for nonparametric variables. P value is shown in figures as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Generation of a bispecific antibody against EGFRvIII and CD3

TCB molecules against EGFRvIII and CD3 were generated in the previously described IgG-based head-to-tail 2:1 heterodimeric format (two EGFRvIII binding Fabs and one CD3 binding Fab; refs. 14–17; Supplementary Table S1). Heterodimerization of these bispecific antibodies was achieved by using the “knob-into-hole” technology (Fig. 1A; refs. 14–17). EGFRvIII-TCB binding affinity was assessed at 25°C by surface plasmon resonance (SPR) using recombinant antigens. The kinetics of binding (monovalent interaction) was measured on histidine-tagged EGFRvIII and recombinant Fc-fused CD3 ε-δ heterodimer. EGFRvIII-TCB is bivalent for EGFRvIII binding (Fig. 1A), with an apparent avidity of 0.2 nmol/L and a monovalent affinity of 4 nmol/L (Table 1); EGFRvIII-TCB is monovalent for CD3 binding with an affinity of 4.3 nmol/L to prevent unspecific T-cell activation. Moreover, EGFRvIII-TCB shows no binding to histidine-tagged EGFRwt by SPR (Table 1). The molecule is based on the human IgG1 isotype with the Fc region bearing P329G LALA mutations to abrogate its binding to Fc gamma receptors (FcγRs), and prevent any FcγR-mediated coactivation of innate immune effector cells, including natural killer cells, monocytes/macrophages, and neutrophils, without changes in functional binding to FcRn (neonatal Fc receptor). Consequently, the EGFRvIII-TCB has a long half-life, and the presence of a silent Fc reduced the risk of FcgR-mediated infusion reactions (Fig. 1A). Charge exchanges and CrossMab technology was used to reduce antibody chain mispairing (27–29). We assessed the dose-dependent EGFRvIII-TCB binding to CD3 in Jurkat cells (Fig. 1B) and to EGFRvIII antigen in the GBM cell line U87MG engineered to express EGFRvIII (U87MG-EGFRvIII; Fig. 1C). The efficacy of the EGFRvIII-TCB was also tested on U87MG-EGFRvIII cells using a blocking experiment with recombinant antigen (Fig. 1D).

Figure 1.

Structure of EGFRvIII-TCB and binding characteristics. A, Structure of EGFRvIII-TCB with EGFRvIII targeting Fabs in blue (bivalent) and the CD3 targeting Fab in orange (monovalent). B, Binding of EGFRvIII-TCB to CD3 on Jurkat cells. C, Binding of EGFRvIII-TCB to the GBM cell line U87MG-EGFRvIII. D, Blocking of EGFRvIII-TCB binding on U87MG-EGFRvIII cells. EGFRvIII antigen (5 μg/mL) or EGFRWT antigen (5 μg/mL) were preincubated with EGFRvIII-TCB (10 nmol/L, Ab in the figure) for 30 minutes and then added on U87MG-EGFRvIII cells for additional 30 minutes. A secondary antibody was added to detect the TCB by measuring fluorescence by flow cytometry. Triplicates are represented as mean ± SD.

Figure 1.

Structure of EGFRvIII-TCB and binding characteristics. A, Structure of EGFRvIII-TCB with EGFRvIII targeting Fabs in blue (bivalent) and the CD3 targeting Fab in orange (monovalent). B, Binding of EGFRvIII-TCB to CD3 on Jurkat cells. C, Binding of EGFRvIII-TCB to the GBM cell line U87MG-EGFRvIII. D, Blocking of EGFRvIII-TCB binding on U87MG-EGFRvIII cells. EGFRvIII antigen (5 μg/mL) or EGFRWT antigen (5 μg/mL) were preincubated with EGFRvIII-TCB (10 nmol/L, Ab in the figure) for 30 minutes and then added on U87MG-EGFRvIII cells for additional 30 minutes. A secondary antibody was added to detect the TCB by measuring fluorescence by flow cytometry. Triplicates are represented as mean ± SD.

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Table 1.

Affinity and avidity of EGFRvIII-TCB to EGFRvIII, EGFRwt, and affinity to CD3 ε-δ heterodimer.

AntigenKon (1/Ms)Koff (1/s)KD (nmol/L)Method
EGFRvIII 7 × 104 2.8 × 10−4 SPR, 25°C, Kinetic 
 (5 × 103(1 × 10−5(0.5) Monovalent binding 
    Affinity 
EGFRvIII 3 × 105 7 × 10−5 0.2 SPR, 25°C, Kinetic 
 (3 × 104(2 × 10−6(0.02) Bivalent binding 
    Apparent Avidity 
EGFRwt No binding No binding No binding SPR, 25°C, single 
    injection 
CD3ε/CD3δ 1.8 × 106 7.6 × 10−3 4.3 SPR, 25°C, Kinetic 
 (2 × 105(5 × 10−5(0.4) Monovalent binding 
    Affinity 
AntigenKon (1/Ms)Koff (1/s)KD (nmol/L)Method
EGFRvIII 7 × 104 2.8 × 10−4 SPR, 25°C, Kinetic 
 (5 × 103(1 × 10−5(0.5) Monovalent binding 
    Affinity 
EGFRvIII 3 × 105 7 × 10−5 0.2 SPR, 25°C, Kinetic 
 (3 × 104(2 × 10−6(0.02) Bivalent binding 
    Apparent Avidity 
EGFRwt No binding No binding No binding SPR, 25°C, single 
    injection 
CD3ε/CD3δ 1.8 × 106 7.6 × 10−3 4.3 SPR, 25°C, Kinetic 
 (2 × 105(5 × 10−5(0.4) Monovalent binding 
    Affinity 

Note: Average of triplicates with SD in parenthesis.

Abbreviations: CD3ε/CD3δ, CD3 ε-δ heterodimer; EGFRvIII, extracellular domain of epidermal growth factor receptor variant III; EGFRwt, extracellular domain of wild-type human epidermal growth factor receptor; KD, dissociation equilibrium constant; Koff, first-order dissociation rate constant; Kon, second-order association rate constant; SPR, surface plasmon resonance.

Functional characterization of the EGFRvIII-TCB

Several experiments were performed to functionally characterize the EGFRvIII-TCB. Jurkat NFAT reporter cell assays were performed with U87MG-EGFRvIII cells showing a dose-dependent CD3 downstream signaling induction following EGFRvIII-TCB treatment (Fig. 2A). We then coincubated U87MG-EGFRvIII cells with human PBMCs and different concentrations of EGFRvIII-TCB. CD8 and CD4 T-cell proliferation was induced in a dose-dependent manner (Fig. 2B). Moreover, lymphocytes induced cell killing of U87MG-EGFRvIII cells at increasing levels of EGFRvIII-TCB (Supplementary Fig. S1A). This phenomenon coincided with an increase in CD8 and CD4 T-cell activation, measured by the expression of CD25, and the release of the effector cytokines, interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and granzyme B (GZMB; Supplementary Fig. S1B and S1C). No effect was observed when U87MG-EGFRvIII cells were coincubated with human PBMCs in the presence of the control TCB, nor when A431 cells (a cell line with EGFR amplification but no EGFRvIII expression) were coincubated with human PBMCs and different concentrations of EGFRvIII-TCB (Supplementary Fig. S1A–S1C). Similar results were obtained with DK-MG cells (a cell line that expresses endogenous EGFRvIII) when coincubated with human PBMCs and different concentrations of EGFRvIII-TCB. Lymphocytes induced cell killing at increasing levels of EGFRvIII-TCB, and a subsequent increase in CD8 T-cell activation, and the release of the effector cytokine TNFα (Fig. 2CE) was observed.

Figure 2.

Tumor cell lysis, T-cell activation, and cytokine release in vitro in response to EGFRvIII-TCB. A, Jurkat NFAT reporter cell assay with U87MG-EGFRvIII cells analyzing CD3 downstream signaling following EGFRvIII-TCB treatment. B, CD8 and CD4 T-cell proliferation after coculture of human PBMCs, U87MG-EGFRvIII cells with EGFRvIII-TCB for 5 days. C–E, Tumor cell lysis, late activation marker upregulation (CD25) on CD8 T cells, and TNFα release 48 hours after incubation of EGFRvIII-TCB with human PBMCs and DK-MG cells. F, Tumor cell lysis after 24 hours, T-cell activation, IFNγ, and GZMB release after 48 hours induced by EGFRvIII-TCB on EGFRvIII low expressing U87MG cells (∼6,000 antibody binding sites, dotted line) and on EGFRvIII high expressing U87MG cells (∼124,000 antibody binding sites, solid line).

Figure 2.

Tumor cell lysis, T-cell activation, and cytokine release in vitro in response to EGFRvIII-TCB. A, Jurkat NFAT reporter cell assay with U87MG-EGFRvIII cells analyzing CD3 downstream signaling following EGFRvIII-TCB treatment. B, CD8 and CD4 T-cell proliferation after coculture of human PBMCs, U87MG-EGFRvIII cells with EGFRvIII-TCB for 5 days. C–E, Tumor cell lysis, late activation marker upregulation (CD25) on CD8 T cells, and TNFα release 48 hours after incubation of EGFRvIII-TCB with human PBMCs and DK-MG cells. F, Tumor cell lysis after 24 hours, T-cell activation, IFNγ, and GZMB release after 48 hours induced by EGFRvIII-TCB on EGFRvIII low expressing U87MG cells (∼6,000 antibody binding sites, dotted line) and on EGFRvIII high expressing U87MG cells (∼124,000 antibody binding sites, solid line).

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These results indicate that the EGFRvIII-TCB induced cell death, activation of CD8 and CD4 T cells, and release of inflammatory cytokines in the presence of EGFRvIII-expressing tumor cells.

Moreover, we generated two versions of the U87MG-EGFRvIII, expressing different levels of EGFRvIII (low and high levels). The cell line expressing high levels of EGFRvIII exhibited an increased response to EGFRvIII-TCB by inducing cell death, CD8 T-cell activation, and IFNγ and GZMB, when compared with the cell line expressing low levels of EGFRvIII. This indicated that the response to the TCB was dependent on the level of EGFRvIII expression and cells with low levels can be still efficiently killed by the TCB (Fig. 2F).

Effect of EGFRvIII-TCB on patient-derived GBM models

We developed a qRT-PCR assay to exclusively detect EGFRvIII with specific probes. Using this approach, we analyzed 63 IDH wild-type GBMs, and we observed a wide range of expression (Fig. 3A; Table 2). EGFRvIII transcript was detected in 18 cases out of the 63 cases (28.6% of the cases). Interestingly, the differences in the levels of EGFRvIII expression were extremely large (Fig. 3A).

Figure 3.

Expression of EGFRvIII in human GBM and effect of EGFRvIII-TCB on GBM tumoroids. A, qRT-PCR analysis of EGFRvIII expression in 63 GBM specimens. EGFRvIII relative mRNA levels are represented as mean ± SD. The 14 patients studied in patient-derived models are shown. B, Representative images and quantification of EGFR, EGFRvIII, CD3, and CD8 staining in the selected 14 GBM specimens are shown. At least 3 fields of the staining were analyzed and quantified for each case. C, Schematic representation of the GBM patient-derived tumoroid model. D, CD8+CD25+ flow cytometry analysis of tumoroids cocultured with PBMCs in a 1:10 ratio together with DP47-TCB or EGFRvIII-TCB for 48 hours. Data are represented as a percentage of CD25+ cells within the CD8 T-cell population. Triplicates are represented as mean ± SD. E and F, ELISA of IFNγ and IL2 measured in the supernatants of tumoroids cocultured with PBMCs and EGFRvIII-TCB or DP47.

Figure 3.

Expression of EGFRvIII in human GBM and effect of EGFRvIII-TCB on GBM tumoroids. A, qRT-PCR analysis of EGFRvIII expression in 63 GBM specimens. EGFRvIII relative mRNA levels are represented as mean ± SD. The 14 patients studied in patient-derived models are shown. B, Representative images and quantification of EGFR, EGFRvIII, CD3, and CD8 staining in the selected 14 GBM specimens are shown. At least 3 fields of the staining were analyzed and quantified for each case. C, Schematic representation of the GBM patient-derived tumoroid model. D, CD8+CD25+ flow cytometry analysis of tumoroids cocultured with PBMCs in a 1:10 ratio together with DP47-TCB or EGFRvIII-TCB for 48 hours. Data are represented as a percentage of CD25+ cells within the CD8 T-cell population. Triplicates are represented as mean ± SD. E and F, ELISA of IFNγ and IL2 measured in the supernatants of tumoroids cocultured with PBMCs and EGFRvIII-TCB or DP47.

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Table 2.

Patient characteristics.

Patient features
n = 63
Female 33 (52.4%) 
Male 30 (47.6%) 
Mean age at diagnosis (years) 59.4 
Median age at diagnosis (years) 61 
Histopathologic features 
Primary tumor 55 (87.3%) 
Recurrent tumor 8 (12.7%) 
IDH1 status  
 Mutated 
 Wild-type 63 (100%) 
Ki-67 (mitotic index) 
 <10% 2 (3.3%)a 
 10%–30% 36 (59%)a 
 >30% 23 (37.7%)a 
p53 accumulation 
 Positive 17 (27.4%)a 
 Negative 45 (72.6%)a 
TERT status 
 Mutated 39 (83%)a 
 Wild-type 8 (17%)a 
MGMT status 
 Unmethylated 30 (57.7%)a 
 Methylated 22 (42.3)a 
EGFRvIII mutation 
 Yes 18 (28.6%) 
 No 45 (71.4%) 
Patient features
n = 63
Female 33 (52.4%) 
Male 30 (47.6%) 
Mean age at diagnosis (years) 59.4 
Median age at diagnosis (years) 61 
Histopathologic features 
Primary tumor 55 (87.3%) 
Recurrent tumor 8 (12.7%) 
IDH1 status  
 Mutated 
 Wild-type 63 (100%) 
Ki-67 (mitotic index) 
 <10% 2 (3.3%)a 
 10%–30% 36 (59%)a 
 >30% 23 (37.7%)a 
p53 accumulation 
 Positive 17 (27.4%)a 
 Negative 45 (72.6%)a 
TERT status 
 Mutated 39 (83%)a 
 Wild-type 8 (17%)a 
MGMT status 
 Unmethylated 30 (57.7%)a 
 Methylated 22 (42.3)a 
EGFRvIII mutation 
 Yes 18 (28.6%) 
 No 45 (71.4%) 

aData were not available for all patients.

We selected 14 cases out of these 63 IDH wild-type GBMs based on the availability of patient-derived models and the expression of diverse levels of EGFRvIII. We first characterized the expression of EGFRvIII and T-cell infiltration in the cohort of 14 GBM tumors. IHC was performed using two antibodies, one specific for EGFR and another specific for EGFRvIII. We observed that patient 2, patient 8, and patient 12 were positive for EGFRvIII (Fig. 3B). As expected, these were the cases with the highest expression of EGFRvIII transcript as measured by qRT-PCR (see Fig. 3A). We also performed CD3 and CD8 staining in the same 14 cases to assess the degree of T-cell infiltration. A generally low, although detectable, T-cell infiltration was observed in most of the cases including those expressing EGFRvIII (Fig. 3B).

Based on these data we decided to evaluate the effect of EGFRvIII-TCB on patient-derived models. We confronted patient-derived tumoroids with EGFRvIII-TCB and PBMCs (Fig. 3C) and determined the activation of CD8 T cells. Of the 14 patient-derived GBM tumoroids, only in three cases (patients 2, 8, and 12) a statistically significant increase of CD25+CD8+ T cells was observed (Fig. 3D). These three cases were precisely the only ones that expressed EGFRvIII (see Fig. 3A). We did not observe significant changes in the remaining cases (Fig. 3D). In addition, we analyzed the conditioned medium of the patient-derived tumoroid cocultures and observed an increase in the inflammatory cytokines IL2 and IFNγ only in the same three EGFRvIII-expressing patients but not in the other cases (Fig. 3E and F).

In vivo efficacy of EGFRvIII-TCB

To test the in vivo efficacy of EGFRvIII-TCB, a subcutaneous model of U87MG-EGFRvIII tumors was generated in humanized mouse models (Hu-CD34+ NSG). Once tumors reached an average of 200 mm3, mice were randomized and treated with EGFRvIII-TCB at different doses of 0.5, 2.5, or 12.5 mg/kg, and tumor volume was monitored. EGFRvIII-TCB inhibited tumor growth and promoted tumor regression in all conditions (Fig. 4A; Supplementary Fig. S2). CD4 and CD8 T-cell numbers and the percentage of GZMB-positive CD8 T cells in the tumor of sentinel mice increased upon two EGFRvIII-TCB treatments (Fig. 4B and C; Supplementary Fig. S2).

Figure 4.

In vivo efficacy of EGFRvIII-TCB. A, EGFRvIII-TCB was tested in the s.c. U87MG-EGFRvIII model in Hu-CD34+ NSG mice. EGFRvIII-TCB was administered i.v. twice per week for 4 weeks starting at day 12 (n = 12 mice per group). B and C, CD4 and CD8 T-cell numbers and percentage of GZMB-positive CD8 T cells in the tumor were analyzed 3 days after the second therapy. D, Tumor growth of subcutaneous tumors of humanized PDX (patient 2) treated with DP47-TCB or EGFRvIII-TCB is shown. Arrow shows the time of the start of the treatment when all tumors reached approximately 100 mm3. Waterfall graph is reported at endpoint. E, Bar graphs represent percentage of CD45+, CD8+, CD4+, CD8+CD69+, and CD4+CD69+ cells, and percentage of cell death in the CD45 population in subcutaneous tumors of humanized PDX (patient 2). Data are represented as mean ± SEM. F, Representative CD3 and CD8 staining images of tumors of humanized PDX (patient 2) treated with DP47-TCB or EGFRvIII-TCB at 46 dpi. G, Tumor growth of subcutaneous tumors of PDX (patient 8) treated with DP47-TCB or EGFRvIII-TCB is shown. Arrow shows the time of the start of the treatment when all tumors reached approximately 100 mm3. Waterfall graph is reported at endpoint. H, Bar graphs represent percentage of CD45+, CD8+, CD4+, CD8+CD69+, and CD4+CD69+ cells, and percentage of cell death in the CD45 population in subcutaneous tumors of humanized PDX (patient 8). Data are represented as mean ± SEM. I, Representative CD3 and CD8 staining images of tumors of humanized PDX (patient 8) treated with DP47-TCB or EGFRvIII-TCB at 46 dpi.

Figure 4.

In vivo efficacy of EGFRvIII-TCB. A, EGFRvIII-TCB was tested in the s.c. U87MG-EGFRvIII model in Hu-CD34+ NSG mice. EGFRvIII-TCB was administered i.v. twice per week for 4 weeks starting at day 12 (n = 12 mice per group). B and C, CD4 and CD8 T-cell numbers and percentage of GZMB-positive CD8 T cells in the tumor were analyzed 3 days after the second therapy. D, Tumor growth of subcutaneous tumors of humanized PDX (patient 2) treated with DP47-TCB or EGFRvIII-TCB is shown. Arrow shows the time of the start of the treatment when all tumors reached approximately 100 mm3. Waterfall graph is reported at endpoint. E, Bar graphs represent percentage of CD45+, CD8+, CD4+, CD8+CD69+, and CD4+CD69+ cells, and percentage of cell death in the CD45 population in subcutaneous tumors of humanized PDX (patient 2). Data are represented as mean ± SEM. F, Representative CD3 and CD8 staining images of tumors of humanized PDX (patient 2) treated with DP47-TCB or EGFRvIII-TCB at 46 dpi. G, Tumor growth of subcutaneous tumors of PDX (patient 8) treated with DP47-TCB or EGFRvIII-TCB is shown. Arrow shows the time of the start of the treatment when all tumors reached approximately 100 mm3. Waterfall graph is reported at endpoint. H, Bar graphs represent percentage of CD45+, CD8+, CD4+, CD8+CD69+, and CD4+CD69+ cells, and percentage of cell death in the CD45 population in subcutaneous tumors of humanized PDX (patient 8). Data are represented as mean ± SEM. I, Representative CD3 and CD8 staining images of tumors of humanized PDX (patient 8) treated with DP47-TCB or EGFRvIII-TCB at 46 dpi.

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We then generated subcutaneous PDX models in Hu-CD34+ NSG mice to assess the effect of EGFRvIII-TCB on patient-derived GBMs in vivo. Once tumors reached 100 mm3, mice were treated with EGFRvIII-TCB or a control TCB for at least three weeks. EGFRvIII-TCB was able to induce complete tumor regression in both tumors expressing EGFRvIII (patient 2 and patient 8; Fig. 4DI). Cytometry analysis revealed that EGFRvIII-TCB increased CD4 and CD8 T-cell tumor infiltration as well as their activation (measured by CD69) only in EGFRvIII-positive GBMs (Fig. 4DI). This result was confirmed by CD3 and CD8 IHC (Fig. 4DI; Supplementary Fig. S3A and S3B). Interestingly, tumor scars that remained after EGFRvIII-TCB treatment exhibited a much higher T-cell tumor infiltration than growing control tumors demonstrating the relevance of the T-cell engagement in the context of the EGFRvIII-TCB antitumor response (Supplementary Fig. S3A and S3B). Importantly, we did not detect EGFRvIII and nestin (both markers of tumor cells) staining in tumor scars, indicating that EGFRvIII-TCB was able to eradicate the tumor. The only two tumors that remained after treatment with EGFRvIII-TCB still retained the expression of EGFRvIII and nestin (Supplementary Fig. S3A and S3B). As expected, we observed an increase in cell death in tumors treated with EGFRvIII-TCB in EGFRvIII-positive GBMs (Fig. 4DI).

As a negative control, we treated an EGFRvIII-negative GBM PDX in Hu-CD34+ NSG (patient 6) with EGFRvIII-TCB and we did not observe any effect of the treatment on tumor growth (Supplementary Fig. S4A). Moreover, no T-cell infiltration or cell death was observed in the EGFRvIII-negative GBM PDX (Supplementary Fig. S4B).

Effect of EGFRvIII-TCB on intracranial tumors

One of the questions we wanted to address is whether EGFRvIII-TCB could facilitate T-cell infiltration into tumors expressing EGFRvIII. To this end, we performed two types of experiments in subcutaneous and intracranial GBM models. First, we implanted U87MG and U87MG-EGFRvIII cells not expressing luciferase in the flanks or the brain of NSG mice. CD3 T cells isolated from PBMCs from healthy donors were engineered to express luciferase and were inoculated intravenously (i.v.) in mice bearing U87MG or U87MG-EGFRvIII tumors in the flank or the brain of mice. Concomitantly, we treated the mice with EGFRvIII-TCB. Bioluminescence allowed monitoring of the distribution of the CD3 T cells in the mice. Three days after treatment, we observed that CD3 T cells were exclusively infiltrating the subcutaneous U87MG-EGFRvIII tumors and not the U87MG tumors implanted at the other flank of the same mouse (Fig. 5A). Interestingly, a similar result was observed in the orthotopic U87MG-EGFRvIII model and CD3 T-cell tumor infiltration was observed 14 days after treatment with EGFR-vIII TCB in the brain of mice (Fig. 5B). Importantly, we used fluorophore-labeled versions of the EGFRvIII-TCB and control TCB and observed the presence of the antibodies within the brain tumors 24 hours after i.v. injection using 3D light-sheet fluorescence microscopy (Fig. 5C). Fluorescence histologic analysis verified the membranous binding of EGFRvIII-TCB to tumor cells as compared with control TCB.

Figure 5.

Effect of EGFRvIII-TCB on GBM orthotopic models. A, Representative images of CD3 T cells expressing luciferase in U87MG xenografts on right flanks and U87MG-EGFRvIII on left flanks treated with DP47-TCB or EGFRvIII-TCB for 96 hours. Bar graph represents T-cell infiltration measured as total flux (p/s) within the tumor. Data are mean ± SEM. B, Representative images of CD3 T cells expressing luciferase in intracranial U87MG or U87MG EGFRvIII xenografts treated with EGFRvIII-TCB for 14 days. Bar graph represents T-cell infiltration measured as total flux (p/s) within the tumor. Data are mean ± SEM. C, 3D light-sheet fluorescence microscopy of brain tumors showing fluorophore-labeled versions of the EGFRvIII-TCB and DP47-TCB 24 hours after i.v. injection. D, Tumor growth of intracranial tumors of U87MG-EGFRvIII treated with DP47-TCB or EGFRvIII-TCB at 3 dpi. The graph indicates total flux (p/s) at 9 dpi. Data are represented as mean ± SEM. Representative bioluminescence images are shown. E, Tumor growth of intracranial tumors of humanized PDX (patient 2) treated with DP47-TCB or EGFRvIII-TCB at 22 dpi. The graph indicates total flux (p/s) at 42 dpi. Data are represented as mean ± SEM. Representative bioluminescence images are shown. F, Tumor growth of intracranial tumors of humanized PDX (patient 8) treated with DP47-TCB or EGFRvIII-TCB at 10 dpi. The graph indicates total flux (p/s) at 38 dpi. Data are represented as mean ± SEM. Representative bioluminescence images are shown.

Figure 5.

Effect of EGFRvIII-TCB on GBM orthotopic models. A, Representative images of CD3 T cells expressing luciferase in U87MG xenografts on right flanks and U87MG-EGFRvIII on left flanks treated with DP47-TCB or EGFRvIII-TCB for 96 hours. Bar graph represents T-cell infiltration measured as total flux (p/s) within the tumor. Data are mean ± SEM. B, Representative images of CD3 T cells expressing luciferase in intracranial U87MG or U87MG EGFRvIII xenografts treated with EGFRvIII-TCB for 14 days. Bar graph represents T-cell infiltration measured as total flux (p/s) within the tumor. Data are mean ± SEM. C, 3D light-sheet fluorescence microscopy of brain tumors showing fluorophore-labeled versions of the EGFRvIII-TCB and DP47-TCB 24 hours after i.v. injection. D, Tumor growth of intracranial tumors of U87MG-EGFRvIII treated with DP47-TCB or EGFRvIII-TCB at 3 dpi. The graph indicates total flux (p/s) at 9 dpi. Data are represented as mean ± SEM. Representative bioluminescence images are shown. E, Tumor growth of intracranial tumors of humanized PDX (patient 2) treated with DP47-TCB or EGFRvIII-TCB at 22 dpi. The graph indicates total flux (p/s) at 42 dpi. Data are represented as mean ± SEM. Representative bioluminescence images are shown. F, Tumor growth of intracranial tumors of humanized PDX (patient 8) treated with DP47-TCB or EGFRvIII-TCB at 10 dpi. The graph indicates total flux (p/s) at 38 dpi. Data are represented as mean ± SEM. Representative bioluminescence images are shown.

Close modal

These results showed that EGFRvIII-TCB was able to promote T-cell tumor infiltration both in subcutaneous tumors and, importantly, in intracranial tumors.

We next investigated the impact of EGFRvIII-TCB on the tumor growth of orthotopic GBM xenografts. U87MG-EGFRvIII cells were implanted in the brain of NSG mice and treated with control TCB or EGFRvIII-TCB concomitantly with human PBMCs. EGFRvIII-TCB but not control TCB inhibited tumor growth of U87MG-EGFRvIII but not of U87MG (Fig. 5D) showing that EGFRvIII-TCB was efficacious in the intracranial setting.

Finally, we generated intracranial GBM PDXs in Hu-CD34+ NSG mice from patients 2 and 8. Once the tumors were established, mice were treated with EGFRvIII-TCB or control TCB for at least two weeks (Fig. 5E and F). We observed a decrease in tumor growth and tumor regression after treatment with EGFRvIII-TCB showing an antitumor response in the orthotopic EGFRvIII GBM PDXs (Fig. 5E and F). This reconfirmed the efficacy of EGFRvIII-TCB in the context of patient-derived intracranial GBM and demonstrated the EGFRvIII-TCB efficacy in the context of brain tumors.

Several bispecific therapeutic antibodies have been developed for the treatment of cancer and some of them have been approved, such as the CD3xCD19 bispecific antibody blinatumomab approved for the treatment of acute lymphoblastic leukemia. However, the extension of this type of therapies for the treatment of other forms of cancers, including solid tumors, has been limited. One of the challenges for the development of T-cell bispecific antibodies is the lack of tumor-specific antigens that allow the specific targeting of tumor cells sparing healthy cells. In this sense, the tumor-specific expression of a mutant truncated version of EGFR, EGFRvIII, representing a neoantigen exclusively expressed in tumor cells opens an opportunity to develop EGFRvIII-targeting T-cell bispecific antibodies.

EGFRvIII is mainly expressed in GBM. This tumor type is one of the most aggressive tumors and the most common primary tumor of the brain with dismal prognosis and a great medical unmet need. It is then crucial to develop novel therapeutic therapies to tackle GBM. Although immunotherapy is not very effective in a relatively noninflamed tumor such as GBM, the possibility to redirect T cells toward tumor cells can be a promising approximation for the treatment of these malignancies. Here, we study the effect of a novel and improved T-cell bispecific antibody, EGFRvIII-TCB, on GBM.

The EGFRvIII-TCB has some improvements over previous TCBs. It has the head-to-tail 2:1 heterodimeric format with 2 EGFRvIII Fabs + 1 CD3 Fabs format in order to increase avidity for the tumor target. It contains an Fc part with the goal to achieve IgG-like pharmacokinetics, but lacking Fc effector functions due to the insertion of specific mutations and importantly it has no crossreactivity to EGFRWT. We were able to show the efficacy of the TCB in cell lines cocultured with PBMCs observing T-cell activation, release of effector cytokines, and tumor cell death.

We decided to test the EGFRvIII-TCB in patient-derived models. We found a wide range of EGFRvIII expression in human GBM and were able to observe a potent response to EGFRvIII-TCB in tumoroids expressing EGFRvIII. This is of relevance when thinking about translating these results into the clinical context since they indicate that patients should be prescreened based on EGFRvIII expression prior to being enrolled in the trial.

Importantly, when using orthotopic models of GBM, we showed that the EGFRvIII-TCB upon i.v. injection was able to access the tumor in the brain and induce the infiltration of the CD3 T cells. Thus, we observed that EGFRvIII-TCB could increase T-cell tumor infiltration and hence promote tumor inflammation in a relatively noninflamed tumor such as GBM.

In order to test the antibody in the context of human GBM, we used CD34+ humanized mice to test EGFRvIII-TCB in vivo in a mouse model with a reconstituted human immune system. These mouse models have been shown to recapitulate compartments of the human immune system most relevant for T-cell redirection-based therapies and, although they show some limitations, these are nowadays one of the best models to test T-cell bispecific antibodies targeting human cancers.

Other EGFRvIII-targeted therapies have been developed. Among them, rindopepimut, a vaccine targeting EGFRvIII, is one of the most well-developed EGFRvIII-targeted compounds. Rindopepimut was tested in a phase III trial and, unfortunately, no significant differences in overall survival were observed in newly diagnosed GBM treated with rindopepimut (30). Some of the proposed explanations for this lack of success were the intratumor heterogeneity of EGFRvIII expression, changes in EGFRvIII expression at relapse, and potential immunoediting that promoted the decrease in EGFRvIII expression (31). These are all factors that could also affect the efficacy of the EGFRvIII-TCB. A study of these phenomena in the context of the clinical testing of the EGFvIII TCB is warranted, as well as the assessment of combinatory approaches to prevent acquired therapeutic resistance.

Using the humanized models, we were able to assess the antitumor response to the EGFRvIII-TCB in the context of a human immune system in vivo. The EGFRvIII-TCB showed antitumor response and tumor regression in orthotopic humanized models, U87MG-EGFRvIII and PDX models.

In summary, our results show that EGFRvIII-TCB can exert a strong antitumor response in EGFRvIII-positive GBM and support further evaluation of this molecule in a first-in-human clinical trial (NCT05187624).

R. Iurlaro reports grants and personal fees from Roche Glycart AG during the conduct of the study. I. Waldhauer reports a patent for WO 2020/127619 pending. E. Bonfill-Teixidor reports grants from F. Hoffmann-La Roche AG during the conduct of the study. V.G. Nicolini reports other support from Roche outside the submitted work; in addition, V.G. Nicolini has a patent for antibody binding CD3 issued and licensed to Roche. A. Freimoser-Grundschober reports a patent for WO 2020/127619 pending. T. Pöschinger reports personal fees from Roche Diagnostics GmbH during the conduct of the study and personal fees from Roche Diagnostics GmbH outside the submitted work. M. Richard reports personal fees from Roche Glycart AG during the conduct of the study. S. Briner reports grants, personal fees, and nonfinancial support from Roche during the conduct of the study; and grants, personal fees, and nonfinancial support from Roche outside the submitted work. S. Colombetti reports Dr Sara Colombetti is an employee of F. Hoffmann-La Roche AG. M. Bacac reports other support from Roche during the conduct of the study. C. Klein reports other support from Roche during the conduct of the study; other support from Roche outside the submitted work; in addition, C. Klein has a patent for Roche pending, issued, and with royalties paid. P.G. Nuciforo reports personal fees from Targos Molecular Pathology and personal fees from AstraZeneca and ONA Therapeutics outside the submitted work. J. Carles reports other support from Astellas Pharma, AstraZeneca, Bayer, Bristol-Myers Squibb, Johnson & Johnson, MSD Oncology, Novartis (AAA), Pfizer, Roche, Sanofi, Astellas Pharma, Bayer, BMS, Ipsen, Roche, and AstraZeneca outside the submitted work. M. Vieito reports grants and nonfinancial support from Roche during the conduct of the study; and personal fees and nonfinancial support from Roche outside the submitted work. J. Tabernero reports personal fees from Array Biopharma, AstraZeneca, Bayer, Boehringer Ingelheim, Chugai, Daiichi Sankyo, F. Hoffmann-La Roche Ltd, Genentech Inc, HalioDX SAS, Hutchison MediPharma International, Ikena Oncology, Inspirna Inc, IQVIA, Lilly, Menarini, Merck Serono, Merus, MSD, Mirati, Neophore, Novartis, ONA Therapeutics, Orion Biotechnology, Peptomyc, Pfizer, Pierre Fabre, Samsung Bioepis, Sanofi, Seattle Genetics, Scandion Oncology, Servier, Sotio Biotech, Taiho, Tessa Therapeutics, and TheraMyc and personal fees from Imedex, Medscape Education, MJH Life Sciences, PeerView Institute for Medical Education, and Physicians Education Resource (PER) outside the submitted work. P. Umaña reports a patent for WO2020/127619 pending. J. Seoane reports grants from Roche/Glycart during the conduct of the study; and grants from Hoffmann-la Roche, AstraZeneca, Northern Biologics, and Mosaic Biomedicals outside the submitted work. No disclosures were reported by the other authors.

The data generated in this study are available within the article and its supplementary data files.

R. Iurlaro: Investigation, visualization, methodology, writing–review and editing. I. Waldhauer: Conceptualization, supervision, visualization, methodology, writing–original draft, writing–review and editing. E. Planas-Rigol: Investigation, methodology. E. Bonfill-Teixidor: Investigation, methodology. A. Arias: Investigation, methodology. V.G. Nicolini: Supervision, visualization, methodology, writing–original draft, writing–review and editing. A. Freimoser-Grundschober: Conceptualization, supervision, investigation, visualization, methodology, writing–review and editing. I. Cuartas: Investigation, methodology. A. Martínez-Moreno: Investigation, methodology. F. Martínez-Ricarte: Investigation, methodology. E. Cordero: Investigation, methodology. M. Cicuendez: Investigation, methodology. S. Casalino: Formal analysis, investigation. X. Guardia: Investigation, methodology. L. Fahrni: Investigation, visualization, writing–review and editing. T. Pöschinger: Investigation, visualization, methodology. V. Steinhart: Investigation. M. Richard: Investigation. S. Briner: Investigation. J.P. Mueller: Investigation. F. Osl: Investigation. J. Sam: Investigation. S. Colombetti: Investigation. M. Bacac: Supervision. C. Klein: Conceptualization, writing–review and editing. E. Pineda: Investigation, methodology. L. Reyes-Figueroa: Investigation. A. Di Somma: Investigation, methodology. J. Gonzalez: Investigation. P.G. Nuciforo: Investigation, methodology. J. Carles: Investigation, methodology. M. Vieito: Investigation, methodology. J. Tabernero: Investigation. P. Umaña: Conceptualization, writing–review and editing. J. Seoane: Conceptualization, supervision, visualization, writing–original draft, writing–review and editing.

The study was undertaken with the support of the Fundación Asociación Española contra el Cáncer (AECC), FERO (EDM), Ramón Areces Foundation, Cellex Foundation, BBVA (CAIMI), the ISCIII, FIS (PI19/00318), NIH grant U12AB123456 NIH grant R01AB123456, William K. Bowes Jr Foundation, German Research Foundation grant AB 1234/1-1, Office of Biological and Environmental Research of the U.S. Department of Energy Atmospheric System Research Program Interagency Agreement grant DE-SC0000001, National Institute of Health Research UK, UK-China Research and Innovation Partnership Fund through the Met Office Climate Science for Service Partnership (CSSP) China as part of the Newton Fund.

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

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Supplementary data