Tumor cell lysis and synergistically enhanced antibody-dependent cell-mediated cytotoxicity by NKG2D engagement with a bispecific immunoligand targeting the HER2 antigen.

Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC.

Suppression of IgE-mediated anaphylaxis and food allergy with monovalent anti-FcεRIα mAbs.

BACKGROUND: Mast cell and basophil activation by antigen cross-linking of FcεRI-bound IgE is central to allergy pathogenesis. We previously demonstrated global suppression of this process by rapid desensitization with anti-FcεRIα mAbs. OBJECTIVES: We sought to determine whether use of monovalent (mv) anti-FcεRIα mAbs increases desensitization safety without loss of efficacy. METHODS: mv anti-human (hu) FcεRIα mAbs were produced with mouse-derived immunoglobulin variable regions and huIgG1 or huIgG4 C regions and were used to suppress murine IgE-mediated anaphylaxis and food allergy. mAbs were administered as a single dose or as serially increasing doses to mice that express hu instead of mouse FcεRIα; mice that additionally have an allergy-promoting IL-4Rα mutation; and hu cord blood-reconstituted immunodeficient, hu cytokine-secreting, mice that have large numbers of activated hu mast cells. Anaphylaxis susceptibility was sometimes increased by treatment with IL-4 or a ß-adrenergic receptor antagonist. RESULTS: mv anti-hu FcεRIα mAbs are considerably less able than divalent mAbs are to induce anaphylaxis and deplete mast cell and basophil IgE, but mv mAbs still strongly suppress IgE-mediated disease. The mv mAbs can be safely administered as a single large dose to mice with typical susceptibility to anaphylaxis, while a rapid desensitization approach safely suppresses disease in mice with increased susceptibility. Our huIgG4 variant of mv anti-huFcεRIα mAb is safer than our huIgG1 variant is, apparently because reduced interactions with FcεRs decrease ability to indirectly cross-link FcεRI. CONCLUSIONS: mv anti-FcεRIα mAbs more safely suppress IgE-mediated anaphylaxis and food allergy than divalent variants of the same mAbs do. These mv mAbs may be useful for suppression of huIgE-mediated disease.

Fc-engineering significantly improves the recruitment of immune effector cells by anti-ICAM-1 antibody MSH-TP15 for myeloma therapy.

Despite several therapeutic advances, patients with multiple myeloma (MM) require additional treatment options since no curative therapy exists yet. In search of a novel therapeutic antibody, we previously applied phage display with myeloma cell screening and developed TP15, a scFv targeting intercellular adhesion molecule 1 (ICAM-1/CD54). To more precisely evaluate the antibody's modes of action, fully human IgG1 antibody variants were generated bearing wild-type (MSH-TP15) or mutated Fc to either enhance (MSH-TP15 Fc-eng.) or prevent (MSH-TP15 Fc k.o.) Fc gamma receptor binding. Especially MSH-TP15 Fc-eng. induced potent antibody-dependent cell-mediated cytotoxicity (ADCC) against malignant plasma cells by efficiently recruiting NK cells and engaged macrophages for antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Binding studies with truncated ICAM-1 demonstrated MSH-TP15 binding to ICAM-1 domain 1-2. Importantly, MSH-TP15 and MSH-TP15 Fc-eng. both prevented myeloma cell engraftment and significantly prolonged survival of mice in an intraperitoneal xenograft model. In the subcutaneous model MSH-TP15 Fc-eng. was superior to MSH-TP15, whereas MSH-TP15 Fc k.o. was not effective in both models - reflecting the importance of Fc-dependent mechanisms of action also in vivo. The efficient recruitment of immune cells and the potent anti-tumor activity of the Fc-engineered MSH-TP15 antibody hold significant potential for myeloma immunotherapy.

Meprin metalloproteases: Molecular regulation and function in inflammation and fibrosis.

The zinc-endopeptidases meprin α and meprin ß are extracellular proteases involved in connective tissue homeostasis, intestinal barrier function and immunological processes. Meprins are unique among other extracellular proteases with regard to cleavage specificity and structure. Meprin α and meprin ß have a strong preference for negatively charged amino acids around the scissile bond, reflected by cleavage sites identified in procollagen I, the amyloid precursor protein (APP) and the interleukin-6 receptor (IL-6R). In this review we report on recent findings that summarize the complex molecular regulation of meprins, particular folding, activation and shedding. Dysregulation of meprin α and meprin ß is often associated with pathological conditions such as neurodegeneration, inflammatory bowel disease and fibrosis. Based on mouse models and patient data we suggest meprins as possible key regulators in the onset and progression of fibrotic disorders, leading to severe diseases such as pulmonary hypertension. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin ß and promotes transendothelial cell migration.

The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin ß and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin ß with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin ß would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin ß induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin ß in Mep1b-/- mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin ß could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin ß inhibitor-treated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin ß and promotes transendothelial cell migration.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

An Fc Double-Engineered CD20 Antibody with Enhanced Ability to Trigger Complement-Dependent Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity.

BACKGROUND: Engineering of the antibody's fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies. METHODS AND RESULTS: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody. CONCLUSION: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.

Targeting Importin-α7 as a Therapeutic Approach against Pandemic Influenza Viruses.

Viral drug resistance is believed to be less likely to occur if compounds are directed against cellular rather than viral proteins. In this study, we analyzed the feasibility of a crucial viral replication factor, namely, importin-α7, as a cellular drug target to combat pandemic influenza viruses. Surprisingly, only five viral lung-to-lung passages were required to achieve 100% lethality in importin-α7⁻/⁻ mice that otherwise are resistant. Viral escape from importin-α7 requirement was mediated by five mutations in the viral ribonucleoprotein complex and the surface glycoproteins. Moreover, the importin-α7⁻/⁻ mouse-adapted strain became even more virulent for wild-type mice than the parental strain. These studies show that targeting host proteins may still result in viral escape by alternative pathways, eventually giving rise to even more virulent virus strains. Thus, therapeutic intervention strategies should consider a multitarget approach to reduce viral drug resistance. IMPORTANCE Here, we investigated the long-standing hypothesis based on in vitro studies that viral drug resistance occurrence is less likely if compounds are directed against cellular rather than viral proteins. Here, we challenged this hypothesis by analyzing, in an in vivo animal model, the feasibility of targeting the cellular factor importin-α7, which is crucial for human influenza virus replication and pathogenesis, as an efficient antiviral strategy against pandemic influenza viruses. In summary, our studies suggest that resistance against cellular factors is possible in vivo, and the emergence of even more virulent viral escape variants calls for particular caution. Thus, therapeutic intervention strategies should consider a multitarget approach using compounds against viral as well as cellular factors to reduce the risk of viral drug resistance and potentially increased virulence.

PB2 mutations D701N and S714R promote adaptation of an influenza H5N1 virus to a mammalian host.

UNLABELLED: Mutation D701N in the PB2 protein is known to play a prominent role in the adaptation of avian influenza A viruses to mammalian hosts. In contrast, little is known about the nearby mutations S714I and S714R, which have been observed in some avian influenza viruses highly pathogenic for mammals. We have generated recombinant H5N1 viruses with PB2 displaying the avian signature 701D or the mammalian signature 701N and serine, isoleucine, and arginine at position 714 and compared them for polymerase activity and virus growth in avian and mammalian cells, as well as for pathogenicity in mice. Mutation D701N led to an increase in polymerase activity and replication efficiency in mammalian cells and in mouse pathogenicity, and this increase was significantly enhanced when mutation D701N was combined with mutation S714R. Stimulation by mutation S714I was less distinct. These observations indicate that PB2 mutation S714R, in combination with the mammalian signature at position 701, has the potential to promote the adaptation of an H5N1 virus to a mammalian host. IMPORTANCE: Influenza A/H5N1 viruses are avian pathogens that have pandemic potential, since they are spread over large parts of Asia, Africa, and Europe and are occasionally transmitted to humans. It is therefore of high scientific interest to understand the mechanisms that determine the host specificity and pathogenicity of these viruses. It is well known that the PB2 subunit of the viral polymerase is an important host range determinant and that PB2 mutation D701N plays an important role in virus adaptation to mammalian cells. In the present study, we show that mutation S714R is also involved in adaptation and that it cooperates with D701N in exposing a nuclear localization signal that mediates importin-α binding and entry of PB2 into the nucleus, where virus replication and transcription take place.

Differential host determinants contribute to the pathogenesis of 2009 pandemic H1N1 and human H5N1 influenza A viruses in experimental mouse models.

Influenza viruses are responsible for high morbidities in humans and may, eventually, cause pandemics. Herein, we compared the pathogenesis and host innate immune responses of a seasonal H1N1, two 2009 pandemic H1N1, and a human H5N1 influenza virus in experimental BALB/c and C57BL/6J mouse models. We found that both 2009 pandemic H1N1 isolates studied (A/Hamburg/05/09 and A/Hamburg/NY1580/09) were low pathogenic in BALB/c mice [log mouse lethal dose 50 (MLD(50)) >6 plaque-forming units (PFU)] but displayed remarkable differences in virulence in C57BL/6J mice. A/Hamburg/NY1580/09 was more virulent (logMLD(50) = 3.5 PFU) than A/Hamburg/05/09 (logMLD(50) = 5.2 PFU) in C57BL/6J mice. In contrast, the H5N1 influenza virus was more virulent in BALB/c mice (logMLD(50) = 0.3 PFU) than in C57BL/6J mice (logMLD(50) = 1.8 PFU). Seasonal H1N1 influenza revealed marginal pathogenicity in BALB/c or C57BL/6J mice (logMLD(50) >6 PFU). Enhanced susceptibility of C57BL/6J mice to pandemic H1N1 correlated with a depressed cytokine response. In contrast, enhanced H5N1 virulence in BALB/c mice correlated with an elevated proinflammatory cytokine response. These findings highlight that host determinants responsible for the pathogenesis of 2009 pandemic H1N1 influenza viruses are different from those contributing to H5N1 pathogenesis. Our results show, for the first time to our knowledge, that the C57BL/6J mouse strain is more appropriate for the evaluation and identification of intrinsic pathogenicity markers of 2009 pandemic H1N1 influenza viruses that are "masked" in BALB/c mice.

Mesenchymal stem cells as all-round supporters in a normal and neoplastic microenvironment.

Mesenchymal stem cells (MSC) represent a heterogeneous population exhibiting stem cell-like properties which are distributed almost ubiquitously among perivascular niches of various human tissues and organs. Organismal requirements such as tissue damage determine interdisciplinary functions of resident MSC including self-renewal, migration and differentiation, whereby MSC support local tissue repair, angiogenesis and concomitant immunomodulation. However, growth of tumor cells and invasion also causes local tissue damage and injury which subsequently activates repair mechanisms and consequently, attracts MSC. Thereby, MSC exhibit a tissue-specific functional biodiversity which is mediated by direct cell-to-cell communication via adhesion molecule signaling and by a tightly regulated exchange of a multifactorial panel of cytokines, exosomes, and micro RNAs. Such interactions determine either tumor-promoting or tumor-inhibitory support by MSC. Moreover, fusion with necrotic/apoptotic tumor cell bodies contributes to re-program MSC into an aberrant phenotype also suggesting that tumor tissue in general represents different types of neoplastic cell populations including tumor-associated stem cell-like cells. The present work summarizes some functional characteristics and biodiversity of MSC and highlights certain controversial interactions with normal and tumorigenic cell populations, including associated modulations within the MSC microenvironment.

Involvement of CD11b integrin in the alteration of metabolic factors after phorbol ester stimulation of human myeloid leukemia cells.

Previous work has demonstrated that phorbol ester (TPA)-induced adherence of human U937 myeloid leukemia cells can be blocked upon down-modulation of the ß2-integrin CD11b after stable transfection of U937 cells with a pMTH1 vector-containing the CD11b gene in antisense orientation (asCD11b-U937) [Otte et al., (2011)]. In the present study, alterations in metabolism-associated factors, particularly intra- and extracellular proteases were investigated. A measurement of telomerase activity in the leukemic cells revealed continuously decreasing telomere adducts within 72 h of TPA treatment in pMTH1-U937 cells. In contrast, telomerase activity sustained in asCD11b-U937 upon TPA-induced differentiation. Flow cytometric analysis confirmed unchanged CD11b levels in TPA-induced asCD11b-U937 in contrast to elevated levels in pMTH1-U937 whereby the expression of other ß2-integrins including CD11a, CD11c and CD18 was increased in both populations after TPA treatment. Moreover, adherent pMTH1-U937 demonstrated the expression of monocytic differentiation markers including F4-80 and CD14 and an increased MIP-1α production which remained at low or undetectable in TPA-induced asCD11b-U937. These effects indicated an altered response of the different cell populations to the TPA-induced differentiation process. Indeed, Western blot analysis revealed differences in the expression levels of intracellular metabolic factors including MnSOD and p97/VCP and after measurement of 20 S proteasomal proteolytic activity. In addition, increased levels of extracellular metabolic factors including the matrix metalloproteinases MMP-1, MMP-7 and MMP-9 were observed in pMTH1-U937 cells in contrast to unaltered levels in asCD11b-U937 cells.

Abolished adherence alters signaling pathways in phorbol ester-induced human U937 cells.

Phorbol ester (TPA) treatment of human U937 myeloid leukemia cells is associated with increasing adherence and monocyte-like maturation whereby the role of ß2 integrin-mediated attachment for subsequent growth properties and the differentiation program remains unclear. Here, stably-transfected U937 cells with a pMTH1 vector containing the ß2 integrin gene of CD11b in antisense orientation (asCD11b-U937) demonstrated a significantly reduced proliferative capacity in contrast to control vector transfectants (pMTH1-U937) or wild-type U937 cells. Phorbol ester exposure induced adherence and growth arrest in more than 90% of pMTH1-U937 and wild-type U937 cells after 72 h. In contrast, TPA-treated asCD11b-U937 failed to attach and the proliferation continued in more than 30% of the cells. Moreover, increased apoptosis appeared in asCD11b-U937 after TPA induction in contrast to pMTH1-U937 cells. In addition, non-specific inhibition of adherence on an agarose surface demonstrated internucleosomal DNA fragmentation in both, pMTH1-U937 and asCD11b-U937 after TPA treatment indicating a functional relationship between abolished adherence, regulation of proliferation and induction of apoptosis. Western blot analysis revealed differences in the expression levels and altered phosphorylation patterns of Pyk-2, pp60src and p42/p44 MAP kinases between pMTH1-U937 and asCD11b-U937 following TPA exposure which was also substantiated by Pyk-2 immunoprecipitation. These findings suggested that induced adherence predominantly mediated by a functional CD11b/CD18 integrin in U937 cells is involved in the activation of downstream signaling kinases and contributes to cell cycle regulation and apoptosis during monocytic maturation.

Childhood-onset Craniopharyngioma.

Craniopharyngiomas are rare embryonic malformational tumors of the sellar/parasellar region, classified by the World Health Organization (WHO) as tumors with low-grade malignancy (WHO I). The childhood adamantinomatous subtype of craniopharyngioma is usually cystic with calcified areas. At the time of diagnosis, hypothalamic/pituitary deficits, visual disturbances, and increased intracranial pressure are major symptoms. The treatment of choice in case of favorable tumor location (without hypothalamic involvement) is complete resection. It is important to ensure that optical and hypothalamic functionality are preserved. In case of unfavorable tumor location, that is with hypothalamic involvement, a hypothalamus-sparing surgical strategy with subsequent local irradiation of residual tumor is recommended. In the further course of the disease, recurrences and progression often occur. Nevertheless, overall survival rates are high at 92%. Severe impairment of quality of life and comorbidities such as metabolic syndrome, hypothalamic obesity, and neurological consequences can be observed in patients with disease- and/or treatment-related lesions of hypothalamic structures. Childhood-onset craniopharyngioma frequently manifests as a chronic disease so that patients require lifelong, continuous care by experienced multidisciplinary teams to manage clinical and quality of life consequences. For this review, a search for original articles and reviews published between 1986 and 2020 was performed in Pubmed, Science Citation Index Expanded, EMBASE, and Scopus. The search terms used were "craniopharyngioma, hypothalamus, pituitary obesity, irradiation, neurosurgery.

Cerebral Infarction in Childhood-Onset Craniopharyngioma Patients: Results of KRANIOPHARYNGEOM 2007.

BACKGROUND: Cerebral infarction (CI) is a known vascular complication following treatment of suprasellar tumors. Risk factors for CI, incidence rate, and long-term prognosis are unknown for patients with childhood-onset craniopharyngioma (CP). METHODS: MRI of 244 CP patients, recruited between 2007 and 2019 in KRANIOPHARYNGEOM 2007, were reviewed for CI. Risk factors for CI and outcome after CI were analyzed. RESULTS: Twenty-eight of 244 patients (11%) presented with CI based on reference assessment of MRI. One CI occurred before initial surgery and one case of CI occurred after release of intracystic pressure by a cyst catheter. 26 of 28 CI were detected after surgical tumor resection at a median postoperative interval of one day (range: 0.5-53 days). Vascular lesions during surgical procedures were documented in 7 cases with CI. No relevant differences with regard to surgical approaches were found. In all 12 irradiated patients, CI occurred before irradiation. Multivariable analyses showed that hydrocephalus and gross-total resection at the time of primary diagnosis/surgery both were risk factors for CI. After CI, quality of life (PEDQOL) and functional capacity (FMH) were impaired. CONCLUSIONS: CI occurs in 11% of surgically-treated CP cases. Degree of resection and increased intracranial pressure are risk factors, which should be considered in the planning of surgical procedures for prevention of CI.

Syndecan-1 shedding by meprin ß impairs keratinocyte adhesion and differentiation in hyperkeratosis.

Dysregulation of proteolytic enzymes has huge impact on epidermal homeostasis, which can result in severe pathological conditions such as fibrosis or Netherton syndrome. The metalloprotease meprin ß was found to be upregulated in hyperproliferative skin diseases. AP-1 transcription factor complex has been reported to induce Mep1b expression. Since AP-1 and its subunit fos-related antigen 2 (fra-2) are associated with the onset and progression of psoriasis, we wanted to investigate if this could partially be attributed to increased meprin ß activity. Here, we demonstrate that fra-2 transgenic mice show increased meprin ß expression and proteolytic activity in the epidermis. To avoid influence by other fra-2 regulated genes, we additionally generated a mouse model that enabled tamoxifen-inducible expression of meprin ß under the Krt5-promotor to mimic the pathological condition. Interestingly, induced meprin ß expression in the epidermis resulted in hyperkeratosis, hair loss and mottled pigmentation of the skin. Employing N-terminomics revealed syndecan-1 as a substrate of meprin ß in skin. Shedding of syndecan-1 at the cell surface caused delayed calcium-induced differentiation and impaired adhesion of keratinocytes, which was blocked by the meprin ß inhibitor fetuin-B.

Fc Glyco- and Fc Protein-Engineering: Design of Antibody Variants with Improved ADCC and CDC Activity.

Monoclonal antibodies are established treatment options in cancer therapy. However, not all patients benefit from antibody therapy. Basic research and findings from clinical trials revealed that certain Fc-mediated effector mechanisms triggered by monoclonal antibodies are essential for efficient antitumor activity. Today, next-generation monoclonal antibodies can be designed displaying tailor-made improved effector functions. The introduction of Fc-engineering technologies offers the potential to fine-tune Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDC). Fc-engineered antibodies hopefully will overcome some limitations of current forms of antibody therapy.

SCCOHT tumors acquire chemoresistance and protection by interacting mesenchymal stroma/stem cells within the tumor microenvironment.

Chemotherapeutic drug testing of SCCOHT-1 and BIN-67 tumor cells revealed synergistic growth-inhibition of >95% in vitro with a combination of foretinib and FK228. Application of this drug combination in vivo in NODscid mice-induced SCCOHT-1GFP tumors was associated with ~6-fold reduction in tumor mass within 10 days, whereby synergistic effects of the two compounds remained undetectable compared to previous results with foretinib treatment alone. Histopathologic evaluation revealed a reduced vascularization and a lower amount of proliferating cells in the treated tumors. Surprisingly, a simultaneous significant accumulation of extracellular matrix structures with positive elastin-van Gieson staining was observed following foretinib/FK228 exposure. Expression analysis of treated animal tumors exhibited various changes including increased mouse transcript levels of elastin, laminin, and fibronectin. In parallel, markers for mesenchymal stroma/stem cells (MSC) including CD73 and CD90 were detectable in all mouse tumors suggesting a possible involvement of these cells in extracellular matrix restructure. Indeed, incubation of MSC with FK228 or foretinib/FK228 demonstrated morphologic alterations and enhanced expression of laminin and fibronectin. Moreover, a co-culture of MSC with lentiviral-labeled SCCOHT-1GFP cells contributed to protection of the tumor cells against FK228-mediated cytotoxicity. Furthermore, explant cultures of SCCOHT-1GFP-induced tumors acquired an increased resistance to FK228 and a combination of foretinib/FK228 in contrast to foretinib alone. Together, these data suggested that FK228-mediated extracellular matrix protein expression by MSC contributes to increased protection and enhanced resistance of SCCOHT tumors which could represent a more general mechanism of MSC during drug-induced alterations of a tumor microenvironment.