EGFR-positive Tumors Research Articles

ALK and EGFR expression by immunohistochemistry are associated with Merkel cell polyomavirus status in Merkel cell carcinoma.

Merkel cell carcinoma, a rare cutaneous neuroendocrine tumour of the skin, can be categorised into two groups according to Merkel cell polyomavirus (MCV) presence. MCV-negative tumours are more aggressive and frequently associated with gene mutations. Some of the genes are potential therapeutic targets. We have previously reported EGFR mutations in six of 27 MCC tumours and overexpression of ALK and EZH2 at mRNA level in MCC tumours. In this study, we sought to determine expression of ALK, EGFR and EZH2 in MCC samples and assess their correlation to MCV status and clinical parameters. Tissue microarrays were utilised and stained with primary antibodies. Staining data were statistically compared to patient sex, tumour location and development of metastasis and MCC-specific death; 112 tumours and their corresponding patient data were included. We found strong expression of ALK in 51% and strong expression of EZH2 in 76% of the tumours. There was evident correlation of ALK expression with MCV-positivity. Expression of EGFR was infrequent, presenting only in seven MCV-negative tumours. None of the proteins associated with development of metastasis or MCC specific death. ALK and EZH2 expression are frequent in MCC and ALK expression correlates to MCV positivity. EGFR positive tumours might respond to EGFR inhibiting treatment.

Modulating antibody-dependent cellular cytotoxicity of epidermal growth factor receptor-specific heavy-chain antibodies through hinge engineering.

Human IgG1 and IgG3 antibodies (Abs) can mediate Ab-dependent cellular cytotoxicity (ADCC), and engineering of the Ab Fc (point mutation; defucosylation) has been shown to affect ADCC by modulating affinity for FcRγIIIa. In the absence of a CH 1 domain, many camelid heavy-chain Abs (HCAbs) naturally bear very long and flexible hinge regions connecting their VH H and CH 2 domains. To better understand the influence of hinge length and structure on HCAb ADCC, we produced a series of hinge-engineered epidermal growth factor receptor (EGFR)-specific chimeric camelid VH H-human Fc Abs and characterized their affinities for recombinant EGFR and FcRγIIIa, their binding to EGFR-positive tumor cells, and their ability to elicit ADCC. In the case of one chimeric HCAb (EG2-hFc), we found that variants bearing longer hinges (IgG3 or camelid hinge regions) showed dramatically improved ADCC in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced CH 2 fucosylation. Conversely, an EG2-hFc variant bearing a truncated human IgG1 upper hinge region failed to elicit ADCC. However, there was no consistent association between hinge length and ADCC for four similarly engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC of some HCAbs can be modulated simply by varying the length of the Ab hinge. Although this effect appears to be heavily epitope-dependent, this strategy may be useful to consider during the design of VH H-based therapeutic Abs for cancer.

A radiopharmaceutical [89Zr]Zr-DFO-nimotuzumab for immunoPET with epidermal growth factor receptor expression in vivo

IntroductionThe potential of the positron-emitting zirconium-89 (89Zr) (t1/2 = 78.4 h) has been recently reported for immune positron emission tomography (immunoPET) radioimmunoconjugates design. In our work, we explored the optimized preparation of [89Zr]Zr-DFO-nimotuzumab, and evaluated 89Zr-labeled monoclonal antibody (mAb) construct for targeted imaging of epidermal growth factor receptor (EGFR) overexpressed in glioma. MethodsTo optimize the radiolabeling efficiency of 89Zr with DFO-nimotuzumab, multiple immunoconjugates and radiolabeling were performed. Radiolabeling yield, radiochemical purity, stability, and activity assay were investigated to characterize [89Zr]Zr-DFO-nimotuzumab for chemical and biological integrity. The in vivo behavior of this tracer was studied in mice bearing subcutaneous U87MG (EGFR-positive) tumors received a 3.5 ± 0.2 MBq/dose using PET/CT imaging. One group mice bearing subcutaneous U87MG (EGFR-positive) tumors received [89Zr]Zr-DFO-nimotuzumab (3.5 ± 0.2 MBq, ~3 μg) (nonblocking) for immunoPET; the other group had 30 μg predose (blocking) of cold nimotuzumab 24 h prior to [89Zr]Zr-DFO-nimotuzumab. Results[89Zr]Zr-DFO-nimotuzumab was prepared with high radiochemical yield (>90%), radiochemical purity (>99%), and specific activity (115 ± 0.8 MBq/mg). In vitro validation showed that [89Zr]Zr-DFO-nimotuzumab had an initial immunoreactive fraction of 0.99 ± 0.05 and remained active for up to 5 days. A biodistribution study revealed excellent stability of [89Zr]Zr-DFO-nimotuzumab in vivo compared with 89Zr as a bone seeker. High uptake in the liver and heart and modest penetration in the brain were observed, with no significant accumulation of activity in other organs. ImmunoPET studies also indicated prominent image contrast that remarkably high uptake up to ~20%ID/g for nonblocking and ~2%ID/g for blocking in tumor between 12 and 120 h after administration. ConclusionThese studies developed a radiopharmaceutical [89Zr]Zr-DFO-nimotuzumab with optimized synthesis. The potential utility of [89Zr]Zr-DFO-nimotuzumab in assessing EGFR status in glioma was demonstrated in this study.

Brain Distribution of a Panel of Epidermal Growth Factor Receptor Inhibitors Using Cassette Dosing in Wild-Type and Abcb1/Abcg2-Deficient Mice.

Tyrosine kinase inhibitors that target the epidermal growth factor receptor (EGFR) have had success in treating EGFR-positive tumors, including non-small-cell lung cancer (NSCLC). However, developing EGFR inhibitors that can be delivered to the brain remains a challenge. To identify optimal compounds for brain delivery, eight EGFR inhibitors [afatinib, 6-[4-[(4-ethylpiperazin-1-yl)methyl]phenyl]-N-(1-phenylethyl)-7H-pyrrolo[2,3-day]pyrimidin-4-amine (AEE788), [4-(3-chloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl] (2R)-2,4-dimethylpiperazine-1-carboxylate (AZD3759), erlotinib, dacomitinib, gefitinib, osimertinib, and vandetanib] were evaluated for distributional kinetics using cassette dosing with the ultimate goal of understanding the brain penetrability of compounds that share the same molecular target in an important oncogenic signaling pathway for both primary brain tumors (glioblastoma) and brain metastases (e.g., NSCLC). Cassette dosing was validated by comparing the brain-to-plasma ratios obtained from cassette-dosing to discrete-dosing studies. The brain-to-blood partition coefficients (Kp,brain) were calculated following cassette dosing of the eight EGFR inhibitors. The comparison of Kp,brain in wild-type and transporter-deficient mice confirmed that two major efflux transporters at the blood-brain barrier (BBB), P-glycoprotein and breast cancer resistance protein, play a crucial role in the brain distribution of seven out of eight EGFR inhibitors. Results show that the prediction of brain distribution based on physicochemical properties of a drug can be misleading, especially for compounds subject to extensive efflux transport. Moreover, this study informs the choice of EGFR inhibitors, i.e., determining BBB permeability combined with a known target potency, that may be effective in future clinical trials for brain tumors.

Zwitterionic near-infrared fluorophore-conjugated epidermal growth factor for fast, real-time, and target-cell-specific cancer imaging.

Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancers, which is associated with metastatic potential and poor prognosis in cancer patients. Therefore, development of EGFR-targeted sensitive imaging probes has been a challenge in tumor targeting, image-guided cancer surgery, patient-selective anti-EGFR therapy, and efficient targeted therapies.Methods: We synthesized a zwitterionic near-infrared fluorophore (ATTO655)-conjugated epidermal growth factor (EGF) as a novel activatable molecular probe. Fluorescence OFF/ON property and EGFR-targeting specificity of EGF-ATTO655 as well as its utility in real-time near-infrared (NIR) fluorescence imaging of EGFR-positive cancers were evaluated using in vitro and in vivo studies.Results: When conjugated to EGF, the fluorescence of ATTO655 quenched efficiently by photo-induced electron transfer (PET) mechanism between the conjugated dyes and nearby amino acid quenchers (tryptophan/tyrosine residues), which was stably maintained at physiological pH and in the presence of serum for at least 17 h. The fluorescence of EGF-ATTO655 turned on by receptor-mediated endocytosis and subsequent disintegration of EGF in EGFR-positive A431 cancer cells, thereby enabling specific and real-time fluorescence imaging of EGFR-positive cancer cells. Consequently, EGFR-positive tumors could be clearly visualized 3 h post-injection with a significantly high tumor-to-background ratio (TBR = 6.37).Conclusion: This PET mechanism-based OFF/ON type of EGF probe showed great potential for rapid, real-time, and target-cell-specific imaging of EGFR-overexpressing cancers in vitro and in vivo.

GE11-PDA-Pt@USPIOs nano-formulation for relief of tumor hypoxia and MRI/PAI-guided tumor radio-chemotherapy.

Radio-chemo combination therapy has synergetic therapeutic effects on tumors. However, the tumor microenvironment, e.g. hypoxia and elevated H2S levels, limits its treatment efficacy. In this study, we developed a cisplatin-loaded, poly dopamine-coated and GE11 peptide-conjugated multi-functional theranostic system (GE11-PDA-Pt@USPIOs) based on poly acrylic acid-coated ultra-small superparamagnetic iron oxide nanoparticles (PAA@USPIOs) for modulation of the tumor hypoxic microenvironment and magnetic resonance imaging/photoacoustic imaging (MRI/PAI) guided radio-chemotherapy of tumors. The thick PAA coating on the USPIOs allowed highly efficient cisplatin loading by complexing the carboxylic groups on PAA with activated cisplatin. A subsequent thin layer of polydopamine (PDA) encapsulation following drug loading provided a means of further surface functionalization; it endowed the particles with photo-thermal properties but did not impede release of the drug or iron ions. GE11-PDA-Pt@USPIOs had high specificity for EGFR-positive tumor cells, could catalyze decomposition of H2O2 to oxygen and exhibited radio-chemo synergetic therapeutic effects under hypothermia conditions in vitro. Once administered intravenously, MRI and PA imaging revealed that the probes were able to accumulate in tumors with high efficiency; this relieved the tumor hypoxic conditions, sensitizing the tumors to radiation therapy. As a result, radio-chemo combination therapy significantly inhibited tumor growth. Our study illustrates for the first time that USPIOs can relieve tumor hypoxia and that GE11-PDA-Pt@USPIOs are highly effective for radio-chemotherapy of EGFR-positive tumors.

Synthesis and evaluation of 64Cu-radiolabeled NOTA-cetuximab (64Cu-NOTA-C225) for immuno-PET imaging of EGFR expression.

ObjectiveEpidermal growth factor receptor (EGFR) is overexpressed in a wide variety of solid tumors, serving as a well-characterized target for cancer imaging or therapy. In this study, we aimed to design and synthesize a radiotracer, 64Cu-NOTA-C225, targeting EGFR for tumor positron emission tomography (PET) imaging. MethodsCetuximab (C225) was conjugated to a bifunctional chelator, p-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and further radiolabeled with copper-64 for PET imaging. 64Cu-NOTA-IgG and Cy5.5-C225 were also synthesized as control probes. A431 and A549 mouse models were established for micro-PET and/or near-infrared fluorescence (NIRF) imaging. Results64Cu-NOTA-C225 exhibited stability in vivo and in vitro up to 24 h and 50 h post-injection, respectively. A431 tumors with average standard uptake values (SUVs) of 5.61±0.69, 6.68±1.14, 7.80±1.51 at 6, 18 and 36 h post-injection, respectively, which were significantly higher than that of moderate EGFR expressing tumors (A549), with SUVs of 0.89±0.16, 4.70±0.81, 2.01±0.50 at 6, 18 and 36 h post-injection, respectively. The expression levels of A431 and A549 were confirmed by western blotting. Additionally, the tracer uptake in A431 tumors can be blocked by unlabeled cetuximab, suggesting that tracer uptake by tumors was receptor-mediated. Furthermore, NIRF imaging using Cy5.5-C225 showed that the fluorescence intensity in tumors increased with time, with a maximal intensity of 8.17E+10 (p/s/cm2/sr)/(μW/cm2) at 48 h post-injection, which is consistent with the paradigm from micro-PET imaging in A431 tumor-bearing mice. ConclusionsThe 64Cu-NOTA-C225 PET imaging may be able to specifically and sensitively differentiate tumor models with different EGFR expression levels. It offers potentials as a PET radiotracer for imaging of tracer EGFR-positive tumors.

Radiolabeling, Characteristics and NanoSPECT/CT Imaging of 188Re-cetuximab in NCI-H292 Human Lung Cancer Xenografts.

Cetuximab has exhibited high EGFR-targeting specificity and clinical promise in previous studies. In this study, we formulated unit dose kits for preparation of high specific activity 188Re-cetuximab for imaging and treatment of EGFR-positive cancer. 188Re-cetuximab was prepared by adding 0.37-0.74 GBq/0.5 ml of 188Re-perrhenate for 4 h at 37°C. Cell surface expression of EGFR, cell binding and cytotoxic effects were evaluated in vitro using both EGFR-positive (NCI-H292, A431) and EGFR-negative (BT483) tumors. A nanoSPECT/CT imaging study was performed in mice bearing EGFR-expressing NCI-H292 tumors. 188Re-cetuximab bound specifically to EGFR-expressing cells and labeling of radionuclides to cetuximab preserved the binding ability of the antibody. Besides, the cytotoxic effect of 188Re-cetuximab was increased dose-dependently. NanoSPECT/CT imaging revealed that 188Re-cetuximab could continually target the tumor region for at least 48 h. The highly specific targeted property of 188Re-cetuximab suggested that it is suitable as a diagnostic tool and maybe a potent radioimmunotherapy agent in EGFR-positive cancers.

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

Development of a novel thermal-sensitive multifunctional liposome with antibody conjugation to target EGFR-expressing tumors

A novel EGFR-targeting, thermal-sensitive multifunctional liposome (TSML) was developed based on manganese-doped magnetism-engineered iron oxide nanoparticles (MnMEIOs) and gold nanorods (AuNRs) for efficient photothermal therapy and magnetic resonance (MR) imaging. An Erbitux-conjugated TSML (Erb-TSML) was encapsulated with doxorubicin and gold nanorods conjugated with manganese-doped magnetism-engineered iron oxide nanoparticles, for theranostic applications of EGFR-positive tumors. The Erb-TSML selectively targeted EGFR-positive tumors and promoted tumor destruction by laser activation. Using confocal microscopy, MR and optical imaging, we demonstrated that Erb-TSML specifically bound to A431 tumor cells. No signs of major morphological damages to the normal tissues were observed in mice treated with Erb-TSML and laser, indicating this theranostic platform protected heart against doxorubicin-induced toxicity to normal tissues. These results indicate that the Erb-TSML may be a promising diagnostic and therapeutic platform for EGFR-overexpressing tumors.

1510TiP - The GATTO study: A phase I of the anti-EGFR tomuzotuximab (TO) in combination with the anti-MUC1 gatipotuzumab (GAT) in patients with EGFR positive solid tumors

1510TiP - The GATTO study: A phase I of the anti-EGFR tomuzotuximab (TO) in combination with the anti-MUC1 gatipotuzumab (GAT) in patients with EGFR positive solid tumors

Abstract 641: Cell free DNA in the supernatant of pleural effusion can detect driver and resistance mutations and can guide TKI treatment decisions

Abstract Introduction: Molecular profiling of tumors has become the mainstay of diagnostics for metastasized solid malignancies and guides personalized treatment, especially in non-small cell lung cancer (NSCLC). In current practice it is often challenging to obtain sufficient tumor material for reliable molecular analysis. Cell free (cf) DNA in blood or other bio-sources could present an alternative approach to obtain genetic information from the tumor. In a retrospective cohort we analyzed the added value of cfDNA analysis in pleural effusions for molecular profiling. Methods: We retrospectively analyzed both the supernatant and the cell pellet of 44 pleural effusions sampled from 39 patients with KRAS (23) or EGFR (16) positive tumors for the original driver gene mutation as well as for EGFR T790M resistance mutations. Patients were diagnosed with either NSCLC (32), colon carcinoma (4), appendiceal carcinoma (2) or adenocarcinoma of unknown primary (1). Samples collected in the context of routine clinical care were stored in the NKI-AVL biobank. We used Bio-Rad QX200 droplet digital PCR for analysis. Results: The original driver gene mutation could be detected in 37 of the 44 pleural effusions by analysis of both supernatant (35/44 positive) and cell pellet (29/44 positive). In 7 out of 20 pleural effusions from patients with EGFR mutation positive tumors, a T790M mutation was detected. All 7 supernatants were positive as were 5 of the 7 cell pellets. The EGFR T790M mutation was confirmed in all supernatants (4/4) and in 3 of the 4 cell pellets sampled from patients with T790M positive tumors (4). Conclusions: Cell free DNA in pleural effusion proved to be a valuable bio-source and can be used to detect driver gene mutations as well as resistance mechanisms like EGFR T790M in pleural effusion. Citation Format: Karlijn Hummelink, Mirte Muller, Dorothe Linders, Vincent van der Noort, Petra Nederlof, Sjaak Burgers, Gerrit Meijer, Michel van den Heuvel, Daan van den Broek, Kim Monkhorst. Cell free DNA in the supernatant of pleural effusion can detect driver and resistance mutations and can guide TKI treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 641.

PO-298 Pegylated recombinant human hyaluronidase PH20 (PEGPH20) increases tumour uptake and efficacy of cetuximab in a human pancreatic cancer xenograft model

IntroductionHyaluronan (HA) accumulation in the ECM of many solid tumours correlates with tumour progression and poor prognosis. In preclinical models, enzymatic degradation of ECM HA with PEGPH20 is associated with remodelling of the tumour stroma, reduction of tumour interstitial fluid pressure, and expansion of tumour blood vessels, resulting in facilitated delivery of anti-cancer agents. Cetuximab (CET), a chimeric monoclonal antibody, targets human EGFR preventing tyrosine kinase-mediated phosphorylation and subsequent signal transduction. Studies were performed to quantitatively assess tumour CET content, plasma PK, and efficacy of CET±PEGPH20 in human xenograft tumour bearing mice.Material and methodsNude mice bearing EGFR-positive peritibial human pancreatic BxPC-3 tumours overexpressing human HA synthase 3 (HAS3) were used to evaluate the effects of PEGPH20 in combination with CET. To evaluate PK, mice were dosed IV with 37.5 µg/kg PEGPH20 and IP with 0.03 mg/kg CET. Plasma samples were collected and analysed for CET concentration by ECL immunoassay. To evaluate tumour uptake, CET was radiolabeled with 89Zr and administered IV±PEGPH20, and PET scans were acquired over 24 hours. To evaluate anti-tumour efficacy, 37.5 µg/kg PEGPH20 was delivered IV, 0.03 mg/kg CET was administered IP biweekly, and tumour growth was measured over time.Results and discussionsSignificantly higher 89Zr uptake into BxPC-3/HAS3 tumours was observed by PET imaging in PEGPH20-treated mice, compared to vehicle alone. PEGPH20 administration immediately prior to 89Zr-CET resulted in significantly increased tumour maximum standardised uptake value (SUV) compared to CET alone (9.4 vs 6.5 meanSUVmax; p=0.039). Similarly, PEGPH20 administration 24 hour prior to 89Zr-CET showed increased tumour SUV that was nearly significant compared to CET alone (8.8 vs 6.5 meanSUVmax; p=0.051). PK analysis indicated decreased plasma CET exposure when combined with PEGPH20. Whether PEGPH20 was administered immediately prior to CET or administered 24 hour prior to CET, plasma CET levels at 24 hour after CET dosing were approximately half the concentration of that obtained when CET was administered alone. Administration of PEGPH20 alone inhibited tumour growth by 46% compared to vehicle. The addition of PEGPH20 increased CET tumour growth inhibition (TGI) from 67% to 82% (p<0.05).ConclusionTumour imaging and PK studies in nude mice demonstrated increased tumour uptake and reduced systemic exposure of CET, which corresponded with enhanced TGI when CET was combined with PEGPH20.

Abstract 2786: Pharmacokinetics and in vitro/in vivo characterization of high-affinity bispecific EGFR/CD16A NK cell engagers for the treatment of EGFR-expressing tumors

Abstract The epidermal growth factor receptor (EGFR) is a validated target for the treatment of several solid tumor types. Current EGFR-targeting monoclonal antibodies (mAbs) and tyrosine kinase inhibitors function mainly through blocking of signal-transduction. Moreover, treatment with these agents is dependent on the receptor's mutational status which may cause treatment resistance in a large number of patients. In addition, EGFR-targeting therapies have been associated with side effects considered to impact prescription rates, in particular in the U.S. A natural killer (NK) cell-based EGFR-targeting approach has the potential to widen the therapeutic window and overcome intrinsic and acquired resistance. Therefore, it offers a promising and well differentiated therapeutic option. We describe the engineering of different bispecific NK cell engagers designed to redirect NK cell-mediated killing to EGFR-positive tumors. Different bispecific EGFR/CD16A antibody constructs targeting human and cynomolgus EGFR and CD16A were designed using novel human Fv antibody binding domains from a toolbox of formats. The Fv domains were evaluated in terms of their pharmacokinetic (PK) properties. Furthermore, the antibody constructs were characterized in antigen and cell binding assays, as well as in in vitro cytotoxicity assays against tumor cell lines expressing Raswt or mutated Ras. PK parameters were determined in CD1 mice and in vivo efficacy of selected antibodies was investigated in an A-431 tumor model in humanized mice. We generated antibody constructs with varying half-lives, including antibodies with a PK profile which allows for dosing comparable to IgG-based antibodies, while aiming at an improved safety profile compared to other EGFR-targeting therapies. Different high affinity tetravalent, bispecific antibodies binding to CD16A and the extracellular domain of EGFR were characterized. While binding to CD16A of classical mAbs is impaired by serum IgG, no substantial changes in binding affinity of our constructs to NK cells were observed. The antibodies showed superior potency and efficacy compared to mAbs or Fc-enhanced antibodies when tested in cytotoxicity assays. In vivo efficacy of selected antibodies was demonstrated in an A-431 tumor model in humanized mice. We have successfully designed and characterized several novel, highly potent and differentiated tetravalent bispecific antibody constructs to redirect NK cell-mediated cell killing to EGFR-positive tumor cells. These drug candidates are suitable for the treatment of EGFR-expressing malignancies with the potential to overcome resistance to other EGFR-targeting agents and offering a potentially improved safety profile. These candidates are currently in development as monotherapy and are also explored in combination with immune activating agents. Citation Format: Michael Kluge, Michael Tesar, Uwe Reusch, Stefan Knackmuss, Torsten Haneke, Kristina Ellwanger, Ivica Fucek, Thomas Mueller, Ute Schniegler-Mattox, Martin Treder. Pharmacokinetics and in vitro/in vivo characterization of high-affinity bispecific EGFR/CD16A NK cell engagers for the treatment of EGFR-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2786.

The GATTO study: A phase I of the anti-MUC1 Gatipotuzumab (GAT) in combination with the anti-EGFR Tomuzotuximab (TO) in patients with EGFR positive solid tumors.

TPS2596Background: TO (CetuGEX) is a second-generation anti-EGFR antibody that specifically binds to EGFR and acts as a competitive antagonist at the ligand binding site. GAT (PankoMab-GEX) is a no...

IgG-single-chain TRAIL fusion proteins for tumour therapy

Single-chain formats of TNF-related apoptosis inducing ligand (scTRAIL) can serve as effector components of tumour-associated antigen-targeted as well as non-targeted fusion proteins, being characterized by high tumour cell-specific induction of apoptosis through death receptor activation. We studied the suitability of immunoglobulin G as a scaffold for oligovalent and bispecific TRAIL fusion proteins. Thus, we developed novel targeted hexa- and dodecavalent IgG-scTRAIL molecules by fusing scTRAIL to the C-terminus of either light (LC-scTRAIL) or heavy immunoglobulin chain (HC-scTRAIL), or to both ends (LC/HC-scTRAIL) of the anti-EGFR IgG antibody hu225. The binding specificity to EGFR and death receptors was retained in all IgG-scTRAIL formats and translated into high antigen-specific bioactivity on EGFR-positive Colo205, HCT116 and WM1366 tumour cell lines, with or without sensitization to apoptosis by bortezomib. In vivo, therapeutic potential was assessed for one of the targeted variants, HC-scTRAIL, compared to the non-targeted Fc-scTRAIL. Both molecules showed a significant reduction of tumour volume and synergism with a Smac mimetic in a Colo205 xenograft tumour model. The IgG-scTRAIL format allows directing a defined, highly bioactive form of TRAIL to a wide variety of tumour antigens, enabling customized solutions for a patient-specific targeted cancer therapy with a reduced risk of side effects.

Development of radioimmunoconjugate for diagnosis and management of head-and-neck subclinical cancer and colorectal carcinoma

ABSTRACT Scientific innovations in diagnostic methods are important drivers of cancer control and prevention. Noninvasive imaging of the epidermal growth factor receptor (EGFR) in head-and-neck squamous, cell carcinoma and colorectal cancer could be valuable to select patients for EGFR-targeted therapy, as well as to monitor the efficacy and occurrence of resistance to immunotherapy. In order to develop the first Brazilian radioimmunoconjugate for diagnosis, Cetuximab has been conjugated to p-SCN-Bn-DTPA chelator and radiolabeled with Indium-111. The conjugation methodology was optimized using different mAb:DTPA molar ratios, time was then reduced for immunoconjugate preparation, besides the protein recovery’ percentage increased after purification (m = 83.8 ± 0.91 %). The stability of Cetuximab-DTPA at - 20 oC was evaluated for six months, and its integrity was greater than 90% (m =93.9 ± 1.5%, N = 24). The radioimmunoconjugate with specific activity of 185 MBq/mg showed radiochemical purity above 95% (m=96.8 ± 1.31 %, N = 15). We conclude that the radioimmunoconjugate 111In-DTPA-cetuximab is stable and may be applied to the diagnosis of EGFR-positive tumors.

Characterization of ABBV-221, a Tumor-Selective EGFR-Targeting Antibody Drug Conjugate.

Depatuxizumab mafodotin (depatux-m, ABT-414) is a tumor-selective antibody drug conjugate (ADC) comprised of the anti-EGFR antibody ABT-806 and the monomethyl auristatin F (MMAF) warhead. Depatux-m has demonstrated promising clinical activity in glioblastoma multiforme (GBM) patients and is currently being evaluated in clinical trials in first-line and recurrent GBM disease settings. Depatux-m responses have been restricted to patients with amplified EGFR, highlighting the need for therapies with activity against tumors with nonamplified EGFR overexpression. In addition, depatux-m dosing has been limited by corneal side effects common to MMAF conjugates. We hypothesized that a monomethyl auristatin E (MMAE) ADC utilizing an EGFR-targeting antibody with increased affinity may have broader utility against tumors with more modest EGFR overexpression while mitigating the risk of corneal side effects. We describe here preclinical characterization of ABBV-221, an EGFR-targeting ADC comprised of an affinity-matured ABT-806 conjugated to MMAE. ABBV-221 binds to a similar EGFR epitope as depatux-m and retains tumor selectivity with increased binding to EGFR-positive tumor cells and greater in vitro potency. ABBV-221 displays increased tumor uptake and antitumor activity against wild-type EGFR-positive xenografts with a greatly reduced incidence of corneal side effects relative to depatux-m. ABBV-221 has similar activity as depatux-m against an EGFR-amplified GBM patient derived xenograft (PDX) model and is highly effective alone and in combination with standard-of-care temozolomide in an EGFRvIII-positive GBM xenograft model. Based on these results, ABBV-221 has advanced to a phase I clinical trial in patients with advanced solid tumors associated with elevated levels of EGFR. Mol Cancer Ther; 17(4); 795-805. ©2018 AACR.

89Zr-nimotuzumab for immunoPET imaging of epidermal growth factor receptor I.

RationaleEpidermal growth factor receptor (EGFR) upregulation is associated with enhanced proliferation and drug resistance in a number of cancers. Nimotuzumab is a humanized monoclonal antibody with high affinity for EGFR. The objective of this study was to determine if 89Zr-DFO-nimotuzumab could be suitable for human use as a PET probe for quantifying EGFR in vivo.MethodsTo evaluate the pharmacokinetics, biodistribution, microPET imaging, radiation dosimetry, and normal tissue toxicity in tumor and non-tumor bearing mice of 89Zr-desferoxamine-nimotuzumab (89Zr-DFO-nimotuzumab) of a product prepared under GMP conditions. Nimotuzumab was conjugated to DFO and radiolabeled with 89Zr. 89Zr-DFO-nimotuzumab was characterized by in vitro gel-electrophoresis, biolayer interferometry (BLI) and flow cytometry. 89Zr-DFO-nimotuzumab was evaluated in vivo by microPET and ex vivo by biodistribution in healthy and EGFR-positive tumor bearing mice.ResultsFlow cytometry with A431 cells showed no significant difference in the dissociation constant of nimotuzumab (13 ± 2 nM) compared with DFO-nimotuzumab (17 ± 4 nM). PET imaging in mice xenografts showed persistently high tumor uptake with the highest uptake obtained in DLD-1 xenograft (18.3 %IA/cc) at 168 hp.i. The projected human effective dose was low and was 0.184 mSv/MBq (0.679 rem/mCi) in females and 0.205 mSv/MBq (0.757 rem/mCi) in males. There was no apparent normal tissue toxicity as shown by cell blood counts and blood biochemistry analyses at 168-fold and 25-fold excess of the projected human radioactive and mass dose of the agent.Conclusion89Zr-DFO-nimotuzumab had low organ absorbed dose and effective dose that makes it suitable for potential human use.

Anti-EGFR lipid micellar nanoparticles co-encapsulating quantum dots and paclitaxel for tumor-targeted theranosis.

Cancer theranosis is an emerging field of personalized medicine which enables individual anti-cancer treatment by monitoring the therapeutic responses of cancer patients. Based on a consideration of the nano-bio interactions related to the blood circulation of systemically administered nanoparticles in humans, as well as extravasation and active targeting, lipid micellar nanoparticles were co-loaded with paclitaxel (PTX) and quantum dots (QDs) to generate a theranostic delivery vehicle. To provide with a tumor-targeting capability, either an antibody or an aptamer against the epidermal growth factor receptor (EGFR) was conjugated to the micelle surface. The QD-containing micelles (QDMs), antibody-coupled QDMs (immuno-QDMs), and aptamer-coupled QDMs (aptamo-QDMs) were able to effectively circulate in blood for at least 8 h when administered intravenously into mice bearing EGFR-positive LS174T tumor xenografts. In vivo fluorescence imaging and a bio-distribution study showed that both the immuno-QDMs and aptamo-QDMs were largely localized in the tumor tissue. The tumor targeting capability enhanced the therapeutic efficacy of PTX for the target cancer cells. Both the immuno-PTX-QDMs and the aptamo-PTX-QDMs caused a stronger inhibition of LS174T tumor growth in mice, compared to the non-targeted PTX-QDMs. These results suggest that the anti-EGFR immuno-PTX-QDMs and anti-EGFR aptamo-PTX-QDMs could be utilized as a tumor-targeted theranostic delivery system for cancer treatment in the clinic.