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In normal epithelia, the epithelial cell adhesion molecule (EpCAM) expression is relatively low and only present at the basolateral cell surface. In contrast, EpCAM is aberrantly overexpressed in various human carcinomas. Therefore, EpCAM is considered to be a highly promising target for antibody-based cancer immunotherapy. Here, we present a new and fully human cytolytic fusion protein (CFP), designated “anti–EpCAM(scFv)-MAP,” that is comprised of an EpCAM-specific antibody fragment (scFv) genetically fused to the microtubule-associated protein tau (MAP). Anti–EpCAM(scFv)-MAP shows potent EpCAM-restricted proapoptotic activity toward rapidly proliferating carcinoma cells. In vitro assays confirmed that treatment with anti–EpCAM(scFv)-MAP resulted in the colocalization and stabilization of microtubules, suggesting that this could be the potential mode of action. Dose-finding experiments indicated that anti–EpCAM(scFv)-MAP is well tolerated in mice. Using noninvasive far-red in vivo imaging in a tumor xenograft mouse model, we further demonstrated that anti–EpCAM(scFv)-MAP inhibited tumor growth in vivo. In conclusion, our data suggest that anti–EpCAM(scFv)-MAP may be of therapeutic value for the targeted elimination of EpCAM+ carcinomas. Mol Cancer Ther; 13(9); 2194–202. ©2014 AACR.

Supplementary Figure 1. Cell-binding activity of anti-EpCAM(scFv)-MAP to further target cell lines.

Medulloblastoma (MB) is the most frequent malignant brain tumour in children with a poor outcome. Divided into four molecular subgroups, MB of the Sonic hedgehog (SHH) subgroup accounts for approximately 25% of the cases and is driven by mutations within components of the SHH pathway, such as its receptors PTCH1 or SMO. A fraction of these cases additionally harbour PIK3CA mutations, the relevance of which is so far unknown. To unravel the role of Pik3ca mutations alone or in combination with a constitutively activated SHH signalling pathway, transgenic mice were used. These mice show mutated variants within Smo, Ptch1 or Pik3ca genes in cerebellar granule neuron precursors, which represent the cellular origin of SHH MB. Our results show that Pik3ca mutations alone are insufficient to cause developmental alterations or to initiate MB. However, they significantly accelerate the growth of Shh MB, induce tumour spread throughout the cerebrospinal fluid, and result in lower survival rates of mice with a double Pik3caH1047R/SmoM2 or Pik3caH1047R/Ptch1 mutation. Therefore, PIK3CA mutations in SHH MB may represent a therapeutic target for first and second line combination treatments.

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt-Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor accounting for ~20 % of childhood brain tumors. One third of all MB are characterized by constitutive activation of the Sonic Hedgehog (SHH)-signaling pathway. This tumor type shows overexpression of the epidermal growth-factor receptor (EGFR), which we detected in SHH-MB patient samples, transgenic SHH MB mouse models, and MB-cell lines. In contrast, non-neoplastic cells only express EGFR at low levels. Intensive radio-/chemotherapy often leaves the young patients with significant long-term burdens including problems in brain development and cognitive deficits. Thus, there is an urgent need to develop new targeted therapies that can prevent tumor recurrence without affecting healthy cells. We selected EGFR as a potential therapy target using EGFR-specific antibody fragments (scFvs) as part of immunoconjugates, namely bispecific T-cell engagers (BiTEs) and immunotoxins (ITs). Both, the EGFR-specific BiTEs and the ITs showed specific binding and cytotoxic activity in MB cells. Effector- and target-cell specificity was demonstrated via flow cytometry for the BiTEs. BiTEs and ITs selectively killed MB-tumor cells and showed pro-apoptotic effects without unspecific effects. Furthermore, preliminary results from an innovative hiPSC-based in vitro-BBB-model suggest, that the ITs are able to cross the BBB. Finally, by having a functional cloning- and expression system for the BiTEs and ITs available, target-scFvs can be easily exchanged by novel antigens or peptides to obtain additional targeted immunotherapies. Together, these results pave the way for preclinical in vivo experiments and future clinical trials in patients with SHH MB.

The interaction of CNS tumors with infiltrating lymphocytes plays an important role in their initiation and progression and might be related to therapeutic responses. Gene expression-based methods have been successfully used to characterize the tumor microenvironment. However, methylation data are now increasingly used for molecular diagnostics and there are currently only few methods to infer information about the microenvironment from this data type. Using an approach based on differential methylation and principal component analysis, we developed DIMEimmune (Differential Methylation Analysis for Immune Cell Estimation) to estimate CD4+ and CD8+ T cell abundance as well as tumor-infiltrating lymphocytes (TILs) scores from bulk methylation data. Well-established approaches based on gene expression data and immunohistochemistry-based lymphocyte counts were used as benchmarks. The comparison of DIMEimmune to the previously published MethylCIBERSORT and MeTIL algorithms showed an improved correlation with both gene expression-based and immunohistological results across different brain tumor types. Further, we applied our method to large datasets of glioma, medulloblastoma, atypical teratoid/rhabdoid tumors (ATRTs) and ependymoma. High-grade gliomas showed higher scores of tumor-infiltrating lymphocytes than lower-grade gliomas. There were overall only few tumor-infiltrating lymphocytes in medulloblastoma subgroups. ATRTs were highly infiltrated by lymphocytes, most prominently in the MYC subgroup. DIMEimmune-based estimates of TILs were a significant prognostic factor in the overall cohort of gliomas and medulloblastomas, but not within methylation-based diagnostic subgroups. To conclude, DIMEimmune allows for robust estimates of TIL abundance and might contribute to establishing them as a prognostic or predictive factor in future studies of CNS tumors.

PLOS ONE 15(12), e0243286 (2020). doi:10.1371/journal.pone.0243286, Published by PLOS, San Francisco, California, US

CREB (cyclic AMP response element binding protein) binding protein (CBP, CREBBP) is a ubiquitously expressed transcription coactivator with intrinsic histone acetyltransferase (KAT) activity. Germline mutations within theCBPgene are known to cause Rubinstein-Taybi syndrome (RSTS), a developmental disorder characterized by intellectual disability, specific facial features and physical anomalies. Here, we investigate mechanisms of CBP function during brain development in order to elucidate morphological and functional mechanisms underlying the development of RSTS. Due to the embryonic lethality of conventional CBP knockout mice, we employed a tissue specific knockout mouse model (hGFAP-cre::CBPFl/Fl, mutant mouse) to achieve a homozygous deletion of CBP in neural precursor cells of the central nervous system.Our findings suggest that CBP plays a central role in brain size regulation, correct neural cell differentiation and neural precursor cell migration. We provide evidence that CBP is both important for stem cell viability within the ventricular germinal zone during embryonic development and for unhindered establishment of adult neurogenesis. Prominent histological findings in adult animals include a significantly smaller hippocampus with fewer neural stem cells. In the subventricular zone, we observe large cell aggregations at the beginning of the rostral migratory stream due to a migration deficit caused by impaired attraction from the CBP-deficient olfactory bulb. The cerebral cortex of mutant mice is characterized by a shorter dendrite length, a diminished spine number, and a relatively decreased number of mature spines as well as a reduced number of synapses.In conclusion, we provide evidence that CBP is important for neurogenesis, shaping neuronal morphology, neural connectivity and that it is involved in neuronal cell migration. These findings may help to understand the molecular basis of intellectual disability in RSTS patients and may be employed to establish treatment options to improve patients’ quality of life.

Brahma-related gene 1 (Brg1) is one of the two mutually exclusive catalytic subunits of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. Several roles of Brg1 have been described including acting as a tumor suppressor but also functioning in neural stem cell (NSC) maintenance, neural crest development, or differentiation of oligodendrocytes and Schwann cells. Here, we generated human glial fibrillary acidic protein (hGFAP)-cre::Brg1fl/fl mice to analyze the function of Brg1 in multipotential NSCs during late stages of neural development. hGFAP-cre::Brg1fl/fl mice died approximately 2 weeks after birth. Macroscopic examination revealed a severe hydrocephalus and a decreased brain weight caused by the loss of Brg1. The cerebellum of hGFAP-cre::Brg1fl/fl mice displayed disorganized cortical layers as well as a massive hypoplasia due to a dramatically reduced number of granule neurons. The cerebrum presented with less proliferative and more apoptotic precursor cells in the subventricular zone (SVZ). Furthermore, the cerebral cortex stood out with significantly thinned upper layers and with impressive dendrite pathology. Finally, the hippocampus was severely underdeveloped with only a sparse number of detectable neurons. We conclude that NSCs depend on Brg1 to give rise to major essential brain structures including the cerebellum, the cerebral cortex, and the hippocampus.

Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives.

Human cytolytic fusion proteins (hCFPs) offer a promising immunotherapeutic approach for the treatment of solid tumors, avoiding the immunogenicity and undesirable side-effects caused by immunotoxins derived from plants or bacteria. The well-characterized human serine protease granzyme B has already been used as a therapeutic pro-apoptotic effector domain. We therefore developed a novel recombinant hCFP (GbR201K-scFv1711) consisting of an epidermal growth factor receptor-specific human antibody fragment and a granzyme B point mutant (R201K) that is insensitive to serpin B9 (PI9), a natural inhibitor of wild-type granzyme B that is often expressed in solid tumors. We found that GbR201K-scFv1711 selectively bound to epidermoid cancer and rhabdomyosarcoma cells and was rapidly internalized by them. Nanomolar concentrations of GbR201K-scFv1711 achieved the specific killing of epidermoid cancer cells by inducing apoptosis, and similar effects were observed in rhabdomyosarcoma cells when GbR201K-scFv1711 was combined with the endosomolytic substance chloroquine. The novel hCFP was stable in serum and bound to human rhabdomyosarcoma tissue ex vivo. These data confirm that GbR201K-scFv1711 is a promising therapeutic candidate suitable for further clinical investigation.

Brahma-related gene 1 (Brg1) is one of the two mutually exclusive catalytic subunits of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. Several roles of Brg1 have been described including acting as a tumor suppressor but also functioning in neural stem cell (NSC) maintenance, neural crest development, or differentiation of oligodendrocytes and Schwann cells. Here, we generated human glial fibrillary acidic protein (hGFAP)-cre::Brg1fl/fl mice to analyze the function of Brg1 in multipotential NSCs during late stages of neural development. hGFAP-cre::Brg1fl/fl mice died approximately 2 weeks after birth. Macroscopic examination revealed a severe hydrocephalus and a decreased brain weight caused by the loss of Brg1. The cerebellum of hGFAP-cre::Brg1fl/fl mice displayed disorganized cortical layers as well as a massive hypoplasia due to a dramatically reduced number of granule neurons. The cerebrum presented with less proliferative and more apoptotic precursor cells in the subventricular zone (SVZ). Furthermore, the cerebral cortex stood out with significantly thinned upper layers and with impressive dendrite pathology. Finally, the hippocampus was severely underdeveloped with only a sparse number of detectable neurons. We conclude that NSCs depend on Brg1 to give rise to major essential brain structures including the cerebellum, the cerebral cortex, and the hippocampus.

The TCF4 gene encodes for the basic helix–loop–helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.

The treatment of rhabdomyosarcoma (RMS) is challenging, and the prognosis remains especially poor for high-grade RMS with metastasis. The conventional treatment of RMS is based on multi-agent chemotherapy combined with resection and radiotherapy, which are often marked by low success rate. Alternative therapeutic options include the combination of standard treatments with immunotherapy. We generated a microtubule-associated protein (MAP)-based fully human cytolytic fusion protein (hCFP) targeting the fetal acetylcholine receptor, which is expressed on RMS cells. We were able to express and purify functional scFv35-MAP from Escherichia coli cells. Moreover, we found that scFv35-MAP is rapidly internalized by target cells after binding its receptor, and exhibits specific cytotoxicity toward FL-OH1 and RD cells in vitro. We also confirmed that scFv35-MAP induces apoptosis in FL-OH1 and RD cells. The in vivo potential of scFv35-MAP will need to be considered in further studies.

Antibody-based immunotherapy of leukemia requires the targeting of specific antigens on the surface of blasts. The Fc gamma receptor (CD64) has been investigated in detail, and CD64-targeting immunotherapy has shown promising efficacy in the targeted ablation of acute myeloid leukemia (AML), acute myelomonocytic leukemia (AMML) and chronic myeloid leukemia cells (CML). Here we investigate for the first time the potential of FcαRI (CD89) as a new target antigen expressed by different myeloid leukemic cell populations. For specific targeting and killing, we generated a recombinant fusion protein comprising an anti-human CD89 single-chain Fragment variable and the well-characterized truncated version of the potent Pseudomonas aeruginosa exotoxin A (ETA'). Our novel therapeutic approach achieved in vitro EC50 values in range 0.2-3 nM depending on the applied stimuli, that is, interferon gamma or tumor necrosis factor alpha. We also observed a dose-dependent apoptosis-mediated cytotoxicity, which resulted in the elimination of up to 90% of the target cells within 72 hr. These findings were also confirmed ex vivo using leukemic primary cells from peripheral blood samples of three previously untreated patients. We conclude that CD89-specific targeting of leukemia cell lines can be achieved in vitro and that the efficient elimination of leukemic primary cells supports the potential of CD89-ETA' as a potent, novel immunotherapeutic agent.

In the original publication, the second name of the twentieth author was incorrect. It should read as 'Miguel Sáinz-Jaspeado'. The original publication of the article has been updated to reflect the change. This correction was authored by Ulrich Schüller on behalf of all authors of the original publication.

Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n = 66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed four subgroups among institutionally diagnosed ONB. The largest group (n = 42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n = 7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis, these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10, and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of tumors. Expression of cytokeratin, chromogranin A, the mutational status of IDH2 as well as DNA methylation patterns may greatly aid in the precise classification of ONB.

Treatment of cancer using standard chemotherapy still offers a poor prognosis combined with severe side effects. Novel antibody-based therapies have been shown to overcome low efficiency and lack of selectivity by targeting cancer-associated antigens, such as aminopeptidase CD13. We isolated a high-affinity CD13-specific single-chain fragment variable (scFv13) from a phage display library of V-genes from mice immunized with soluble antigen. An immunotoxin comprising the scFv13 and a truncated version of the exotoxin A of Pseudomonas aeruginosa (ETA′, scFv13–ETA′) and a bispecific scFv targeting CD13 and CD16 simultaneously (bsscFv[13xds16]) was generated and investigated for their therapeutic potential. Both fusion proteins bound specifically to target cells with high affinity. Furthermore, scFv13–ETA′ inhibited the proliferation of human cancer cell lines efficiently at low concentrations (IC50 values of 408 pM–7 nM) and induced apoptosis (40–85% of target cells). The bsscFv triggered dose-dependent antibody-dependent cell-mediated cytotoxicity, resulting in the lysis of up to 23.9% A2058 cells, 18.0% MDA-MB-468 cells and 19.1% HL-60 cells. The provided data demonstrate potent therapeutic activity of the scFv13–ETA′ and the bsscFv[13xds16]. The CD13-specific scFv is therefore suitable for the direct and specific delivery of both cytotoxic agents and effector cells to cancer-derived cells, making it ideal for further therapeutic evaluation.

In normal epithelia, the epithelial cell adhesion molecule (EpCAM) expression is relatively low and only present at the basolateral cell surface. In contrast, EpCAM is aberrantly overexpressed in various human carcinomas. Therefore, EpCAM is considered to be a highly promising target for antibody-based cancer immunotherapy. Here, we present a new and fully human cytolytic fusion protein (CFP), designated “anti–EpCAM(scFv)-MAP,” that is comprised of an EpCAM-specific antibody fragment (scFv) genetically fused to the microtubule-associated protein tau (MAP). Anti–EpCAM(scFv)-MAP shows potent EpCAM-restricted proapoptotic activity toward rapidly proliferating carcinoma cells. In vitro assays confirmed that treatment with anti–EpCAM(scFv)-MAP resulted in the colocalization and stabilization of microtubules, suggesting that this could be the potential mode of action. Dose-finding experiments indicated that anti–EpCAM(scFv)-MAP is well tolerated in mice. Using noninvasive far-red in vivo imaging in a tumor xenograft mouse model, we further demonstrated that anti–EpCAM(scFv)-MAP inhibited tumor growth in vivo. In conclusion, our data suggest that anti–EpCAM(scFv)-MAP may be of therapeutic value for the targeted elimination of EpCAM+ carcinomas. Mol Cancer Ther; 13(9); 2194–202. ©2014 AACR.

PLoS one 12(7), e0180305 (2017). doi:10.1371/journal.pone.0180305, Published by PLoS, Lawrence, Kan.

Antibody-drug conjugates (ADCs) combine the potency of cytotoxic drugs with the specificity of monoclonal antibodies (mAbs). Most ADCs are currently generated by the nonspecific conjugation of drug-linker reagents to certain amino acid residues in mAbs, resulting in a heterogeneous product. To overcome this limitation and prepare ADCs with a defined stoichiometry, we use SNAP-tag technology as an alternative conjugation strategy. This allows the site-specific conjugation of O(6)-benzylguanine (BG)-modified small molecules to SNAP-tag fusion proteins. To demonstrate the suitability of this system for the preparation of novel recombinant ADCs, here we conjugated SNAP-tagged single chain antibody fragments (scFvs) to a BG-modified version of auristatin F (AURIF). We used two scFv-SNAP fusion proteins targeting members of the epidermal growth factor receptor (EGFR) family that are frequently overexpressed in breast cancer. The conjugation of BG-AURIF to EGFR-specific 425(scFv)-SNAP and HER2-specific αHER2(scFv)-SNAP resulted in two potent recombinant ADCs that specifically killed breast cancer cell lines by inducing apoptosis when applied at nanomolar concentrations. These data confirm that SNAP-tag technology is a promising tool for the generation of novel recombinant ADCs.

The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression. Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0 6-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model. The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 ± 6.3 (n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 ± 1.35 (n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 ± 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection. The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.

The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA′). The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA′ (from cetuximab) and scFv1711-ETA′ (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-tag imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies. Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed. These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA′-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

The treatment of rhabdomyosarcoma (RMS) remains challenging, with metastatic and alveolar RMS offering a particularly poor prognosis. Therefore, the identification and evaluation of novel antigens, which are suitable targets for immunotherapy, is one attractive possibility to improve the treatment of this disease. Here we show that chondroitin sulfate proteoglycan 4 (CSPG4) is expressed on RMS cell lines and RMS patient material. We evaluated the immunotoxin (IT) αMCSP-ETA', which specifically recognizes CSPG4 on the RMS cell lines RD, FL-OH1, TE-671 and Rh30. It is internalized rapidly, induces apoptosis and thus kills RMS cells selectively. We also demonstrate the specific binding of this IT to RMS primary tumor material from three different patients.

Although current cancer treatment strategies are highly aggressive, they are often not effective enough to destroy the collectivity of malignant cells. The residual tumor cells that survived the first-line treatment may continue to proliferate or even metastasize. Therefore, the development of novel more effective strategies to specifically eliminate also single cancer cells is urgently needed. In this respect, the development of antibody-based therapeutics, in particular example immunotoxins, has attracted broad interest. Since the internalization of immunotoxins is essential for their cytotoxic effectivity, it is of crucial importance to study their internalization behavior to assess the potential for their therapeutic use. In this study, we determined the internalization behavior of four different single-chain fragments variable (scFv) when binding to the corresponding target antigen as expressed on solid or non-solid tumor cell lines. The scFvs were recombinantly fused to the SNAP-tag, an engineered variant of the human repair enzyme O(6)-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine derivatives. Since a large number of highly sensitive organic fluorescent dyes are already available or can easily be derivatized to react with the self-labeling SNAP-tag, this system provides versatile applications for imaging of intraand extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface(®) Alexa Fluor(®) 488 or SNAP-surface(®) Alexa Fluor(®) 647 and binding as well as internalization was monitored by flow cytometry and confocal microscopy, respectively. Depending on the respective target antigen, we could distinguish between slow and rapid internalization behavior. Moreover, we detected increased internalization rate for bivalent scFv constructs. Our approach allows for rapid and early stage evaluation of the internalization characteristics of new antibodies designated for further therapeutic development.