[Treatment of Opioid-dependent Inmates - Attitudes and Treatment Practice of medical Staff in Bavarian Prisons]. / Behandlung opioidabhängiger Inhaftierter ­ Einstellungen und Behandlungspraxis des medizinischen Personals in bayerischen Justizvollzugsanstalten.

BACKGROUND: The present study examines the treatment practice and attitudes of medical staff towards opioid-dependent inmates in Bavarian prisons. METHOD: We interviewed medical staff (n=20) from 18 Bavarian prisons about substitution practice and attitudes by semi-structured interviews. RESULTS: With regard to the treatment routines and the attitudes of the medical staff, we found mixed results. From the perspective of the medical staff, the treatment decision depends on the patients' wishes, the severity and duration of the dependence, the length of sentence and organizational factors. Problems were discussed in particular with regard to the care situation inside and outside the prisons and difficulties in transition management. CONCLUSIONS: Substitution therapy is considered a standard treatment method in prisons today. However, our respondents highlighted some disadvantages (e. g. passing on the substitution drug). Although the treatment goal of complete abstinence was generally viewed positively by a part of the medical staff, it was not considered very realistic. From the point of view of the respondents, special attention should be paid to the continuity of the chosen treatment strategy in the context of discharge management.

[Thrombotic Microangiopathies]. / Thrombotische Mikroangiopathien.

Thrombotic microangiopathies are almost devastating diseases leading to death at high frequency if untreated. They consist of at least five distinct entities, TTP, HUS, aHUS, TMA due to drug interference, systemic disease or post therapy TMA. Around 10 years ago there was only one established therapeutic approach as plasmapheresis. Meanwhile, there are new drugs been licensed or within licensing process. Patients with atypical HUS can be treated successfully by the Complement inhibitor Eculizumab. In addition, there is a new inhibitor of von-Willebrand-Polymerisation available. Caplacizumab provides a significantly better remission and decrease in TMA-related death. For therapy associated TMA as VOD/SOS Defibrotide could be established within a phase III study to significantly improve outcomes. In order to select these new medical approaches individual diagnostic parameters need to be established in order to rapidly distinguish between the TMA entities and start targeted therapy.

Antibody-dependent cellular cytotoxicity in patients on chronic hemodialysis.

BACKGROUND: Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is considered to be a relevant mechanism of action of Ab-based tumor therapies. However, knowledge about ADCC capacity of dialysis patients (DP) is limited. The aim of our study was to investigate if ADCC capacity of effector cells obtained from DP differed from those of healthy individuals (HI). METHODS: First, we performed ADCC assays with isolated polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC), mediated by the epidermal growth factor receptor Ab cetuximab or panitumumab. As cetuximab is of human IgG1 and panitumumab of human IgG2 isotype, both Abs differ in their affinity to Fcγ receptors and effector cell recruitment. RESULTS: Using PMN as effectors, ADCC levels via panitumumab proved to be higher than via cetuximab, but did not differ between DP and HI. In contrast, IgG2-mediated ADCC with PBMC from DP was significantly enhanced compared to HI. IgG2 Abs predominantly bind to FcγRIIa. Within the PBMC, monocytes are the only cytotoxic cells physiologically expressing this receptor. ADCC experiments with isolated monocytes confirmed them to be the pivotal cells for the observed effect. Analysis of monocytes' Fc receptor expression demonstrated no difference between DP and HI, but monocytes of DP proved to be numerically increased and appeared preactivated. CONCLUSION: Our studies implicate that ADCC capacity is not impaired in DP and that it might particularly be reasonable to apply human IgG2 Abs as therapeutics for these patients.

IgA EGFR antibodies mediate tumour killing in vivo.

Currently all approved anti-cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti-tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti-tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10-EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short-term syngeneic peritoneal model using EGFR-transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.

Effect of a tail piece cysteine deletion on biochemical and functional properties of an epidermal growth factor receptor-directed IgA2m(1) antibody.

Antibodies of human IgA isotype are critical components of the mucosal immune system, but little is known about their immunotherapeutic potential. Compared with IgG antibodies, IgA molecules carry a C-terminal tail piece extension of 18 amino acids with a free cysteine at position 471. This cysteine is required for the formation of dimeric IgA antibodies, but may impair molecular characteristics of monomeric IgA antibodies as therapeutic reagents. Thus, we generated and characterized a d471-mutated antibody against the epidermal growth factor receptor (EGFR) and compared it to its respective IgA2m(1) wild type antibody. Both wild type and mutated IgA antibodies demonstrated similar EGFR binding and were similarly efficient in inhibiting EGF binding and in blocking EGF-mediated cell proliferation. Recruitment of Fc-mediated effector functions like antibody-dependent cell-mediated cytotoxicity by monocytes, macrophages or PMN was similar, but the d471-mutated IgA exhibited different biochemical properties compared with wild type antibody. As expected, mutated IgA did not form stable dimers in the presence of human joining (J)-chain, but we also observed reduced levels of dimeric aggregates in the absence of J-chain. Furthermore, glycoprofiling revealed different glycosylation patterns for both antibodies, including considerably less mannosylation of d471-mutated antibodies. Overall, our results demonstrate that the deletion of the C-terminal cysteine of IgA2 did not affect the investigated effector functions compared with wild type antibody, but it improved biochemical properties of an IgA2m(1) antibody against EGFR, and may thereby assist in exploring the immunotherapeutic potential of recombinant IgA antibodies.

Characterization of a mutated IgA2 antibody of the m(1) allotype against the epidermal growth factor receptor for the recruitment of monocytes and macrophages.

IgA antibodies constitute an important part of the mucosal immune system, but their immunotherapeutic potential remains rather unexplored, in part due to biotechnological issues. For example, the IgA2m(1) allotype carries an unusual heavy and light chain pairing, which may confer production and stability concerns. Here, we report the generation and the biochemical and functional characterization of a P221R-mutated IgA2m(1) antibody against the epidermal growth factor receptor (EGFR). Compared with wild type, the mutated antibody demonstrated heavy chains covalently linked to light chains in monomeric as well as in joining (J)-chain containing dimeric IgA. Functional studies with wild type and mutated IgA2m(1) revealed similar binding to EGFR and direct effector functions such as EGFR down-modulation and growth inhibition. Furthermore, both IgA molecules triggered similar levels of indirect tumor cell killing such as antibody-dependent cell-mediated cytotoxicity (ADCC) by isolated monocytes, activated polymorphonuclear cells, and human whole blood. Interestingly, the dimeric IgA antibodies demonstrated higher efficiency in direct as well as in indirect effector mechanisms compared with their respective monomeric forms. Both wild type and mutated antibody triggered effective FcαRI-mediated tumor cell killing by macrophages already at low effector to target cell ratios. Interestingly, also polarized macrophages mediated significant IgA2-mediated ADCC. M2 macrophages, which have been described as promoting tumor growth and progression, may convert to ADCC-mediating effector cells in the presence of EGFR-directed antibodies. In conclusion, these results provide further insight into the immunotherapeutic potential of recombinant IgA antibodies for tumor immunotherapy and suggest macrophages as an additional effector cell population.

Oncogenic KRAS impairs EGFR antibodies' efficiency by C/EBPß-dependent suppression of EGFR expression.

Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS(G12V)) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS(G12V) on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS(G12V) impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS(G12V) also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS(G12V) downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBPß-LIP. Thus, siRNA-mediated knockdown of C/EBPß led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS(G12V) signaling induced C/EBPß-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents.

Combined Fc-protein- and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances CD16a binding but does not further enhance NK-cell mediated ADCC.

Protein- or glyco-engineering of antibody molecules can be used to enhance Fc-mediated effector functions. ScFv-Fc fusion proteins (scFv-Fc) represent interesting antibody derivatives due to their relatively simple design and increased tissue penetration. Here, the impact of protein- and glyco-engineering on ADCC potency of a panel of human IgG1-based scFv-Fc was tested. Three matched sets of scFv-Fc variants targeting CD7, CD20 or HLA class II and optimized for CD16a binding by mutagenesis, lack of core-fucose, or their combination, were generated and functionally tested in comparison to the corresponding wild type scFv-Fc. Antigen binding activity was not compromised by altered glycosylation or Fc mutagenesis, whereas Fc binding to CD16a was significantly enhanced in the order: non-core fucosylated/Fc-mutated double-engineered≫Fc-mutated≥non-core-fucosylated>wild-type IgG1-Fc. All engineered variants triggered potent ADCC with up to 100-fold reduced EC50 values compared to non-engineered variants. Interestingly, double-engineered variants were similarly effective in triggering ADCC compared to single-engineered variants irrespective of their 1 log greater CD16a binding affinity. Thus, these data demonstrate that protein- and glyco-engineering enhances NK-cell mediated ADCC of scFv-Fc similarly and show that enhancing CD16a affinity beyond a certain threshold does not result in a further increase of NK-cell mediated ADCC.

Complement-mediated tumor-specific cell lysis by antibody combinations targeting epidermal growth factor receptor (EGFR) and its variant III (EGFRvIII).

Monoclonal antibodies (mAb) against variant III of epidermal growth factor receptor (EGFRvIII) hold promise for improving tumor selectivity of EGFR-targeted therapy. Here, we compared Fc-mediated effector functions of three mAb against EGFRvIII (MR1-1, ch806, 13.1.2) with those of zalutumumab, a high affinity EGFR mAb in advanced clinical trials. MR1-1 and ch806 demonstrated preferential and 13.1.2 exclusive binding to EGFRvIII, in contrast to zalutumumab, which bound both wild-type and EGFRvIII. All four human IgG1κ mAb mediated antibody-dependent cellular cytotoxicity (ADCC) of EGFRvIII-expressing cells with mononuclear cells and isolated monocytes, while only zalutumumab in addition triggered ADCC by polymorphonuclear cells. Interestingly, combinations of zalutumumab and EGFRvIII mAb specifically mediated complement-dependent cytotoxicity (CDC) of EGFRvIII-transfected but not wild-type cells. Moreover, EGFRvIII-specific CDC was significantly enhanced when zalutumumab was combined with a Fc-engineered variant of MR1-1 (K326A/E333A). These observations confirm the immunotherapeutic potential of antibody combinations against EGFR, and demonstrate that tumor selectivity can be improved by combining therapeutic EGFR mAb with an antibody against EGFRvIII.

Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing.

Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human κ and anti-His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs.

Impact of human IgA antibodies on complement-dependent cytotoxicity mediated by combinations of EGF-R-directed antibodies.

Dual combinations of non-crossblocking epidermal growth factor receptor (EGF-R)-directed monoclonal antibodies were demonstrated to effectively induce complement-dependent cytotoxicity (CDC) of tumor cells, whereas individual antibodies were ineffective. Here the modulating effects of different antibody isotypes on CDC were studied by adding them as a third antibody. Two different combinations of non-crossblocking EGF-R antibodies of human IgG1 isotype, 018/003 and 425/005, were investigated against the A431 and A1207 cell lines. As a third antibody, human IgG1, IgA1, and IgA2 isotype variants of the therapeutic EGF-R antibody 225 were employed that bind to an EGF-R epitope distinct from the other EGF-R antibodies. In this model, the human IgG1 antibody proved to further enhance CDC, whereas both IgA antibodies significantly blocked CDC. The IgG1 and IgA variants increased target opsonization at similar levels, but the isotypes differed in their effects on C1q fixation. Addition of IgG1 significantly enhanced complement factor binding on the target surface, whereas both IgA antibodies reduced complement binding. Control experiments revealed this blocking effect to be not specific to IgA antibodies, but to antibody constructs incapable of activating the complement system. Interestingly, the effects caused by the IgA2 isotype were consistently stronger than those by IgA1, which may be caused by stronger steric hindrance due to its reduced hinge flexibility. These results demonstrate that monoclonal IgA antibodies inhibit IgG-mediated complement activation in vitro and suggest that the appearance of IgA antibodies within a polyclonal immune response might inhibit complement activation in vivo.

Fc-engineered EGF-R antibodies mediate improved antibody-dependent cellular cytotoxicity (ADCC) against KRAS-mutated tumor cells.

Oncogenic mutations of the KRAS gene have emerged as a common mechanism of resistance against epidermal growth factor receptor (EGF-R)-directed tumor therapy. Mutated KRAS leads to ligand-independent activation of signaling pathways downstream of EGF-R. Thereby, direct effector mechanisms of EGF-R antibodies, such as blockade of ligand binding and inhibition of signaling, are bypassed. Thus, a humanized variant of the approved EGF-R antibody Cetuximab inhibited growth of wild-type KRAS-expressing A431 cells, but did not inhibit KRAS-mutated A549 tumor cells. We then investigated whether killing of tumor cells harboring mutated KRAS can be improved by enhancing antibody-dependent cellular cytotoxicity (ADCC). Protein- and glyco-engineering of antibodies' Fc region are established technologies to enhance ADCC by increasing antibodies' affinity to activating Fcgamma receptors. Thus, EGF-R antibody variants with increased affinity for the natural killer (NK) cell-expressed FcgammaRIIIa (CD16) were generated and analyzed. These variants triggered significantly enhanced mononuclear cell (MNC)-mediated killing of KRAS-mutated tumor cells compared to wild-type antibodies. Additionally, cells transfected with mutated KRAS were killed as effectively by ADCC as vector-transfected control cells. Together, these data demonstrate that KRAS mutations are not sufficient to render tumor cells resistant to ADCC. Consequently Fc-engineered EGF-R antibodies may prove effective against KRAS-mutated tumors, which are not susceptible to signaling inhibition by EGF-R antibodies.

A recombinant bispecific single-chain fragment variable specific for HLA class II and Fc alpha RI (CD89) recruits polymorphonuclear neutrophils for efficient lysis of malignant B lymphoid cells.

Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against Fc alpha RI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. Fc alpha RI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at K(D) values of 148 +/- 42 nM and 113 +/- 25 nM for the anti-Fc alpha RI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (Fc alphaRI x HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an Fc alpha RI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (Fc alpha RI x HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.

Human IgG2 antibodies against epidermal growth factor receptor effectively trigger antibody-dependent cellular cytotoxicity but, in contrast to IgG1, only by cells of myeloid lineage.

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcgammaRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcgammaRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcgammaRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R-directed immunotherapy.

Sensitivity and resistance to EGF-R inhibitors: approaches to enhance the efficacy of EGF-R antibodies.

The epidermal growth factor receptor (EGF-R) constitutes one of the most broadly targeted antigens in tumor therapy since it is commonly expressed on many epithelial cancers, as well as on glioblastomas. Both EGF-R-directed tyrosine kinase inhibitors and monoclonal antibodies have been approved, but clinical response rates are often limited. A more detailed understanding of the mechanisms underlying sensitivity or resistance against EGF-R inhibitors may assist in identifying patient populations who optimally benefit from currently available reagents. In addition, these insights may guide the development of more effective molecules. In this short review, we will summarize some of the current knowledge in this rapidly evolving field with particular emphasis on EGF-R-directed antibodies.

Effector mechanisms of therapeutic antibodies against ErbB receptors.

ErbB1 and ErbB2 constitute validated target antigens for tumor therapy-as documented by the approval of antibodies and tyrosine kinase inhibitors (TKIs) against both antigens. However, their complex biology in development and tumorigenesis poses significant challenges on the optimization of this targeted approach. Crystallographic studies have significantly improved concepts about structure/function relationships of these receptors, and may assist in improving the efficacy of ErbB-directed therapy over the following years. Here, we will review these recent advances and their implications for ErbB-directed therapies. Although we will focus on the mechanisms of action of ErbB therapeutic antibodies, we will also briefly discuss TKIs.

Complement-dependent tumor cell lysis triggered by combinations of epidermal growth factor receptor antibodies.

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) have advanced the treatment of colon and head and neck cancer, and show great promise for the development of treatments for other solid cancers. Antibodies against EGFR have been shown to act via inhibition of receptor signaling and induction of antibody-dependent cellular cytoxicity. However, complement-dependent cytotoxicity, which is considered one of the most powerful cell killing mechanisms of antibodies, seems inactive for such antibodies. Here, we show a remarkable synergy for EGFR antibodies. Combinations of antibodies against EGFR were identified, which resulted in potent complement activation via the classic pathway and effective lysis of tumor cells. Studies on a large panel of antibodies indicated that the observed synergy is a general mechanism, which can be activated by combining human IgG1 antibodies recognizing different, nonoverlapping epitopes. Our findings show an unexpected quality of therapeutic EGFR antibodies, which may be exploited to develop novel and more effective treatments for solid cancers.

Antibody fucosylation differentially impacts cytotoxicity mediated by NK and PMN effector cells.

Glycosylation of the antibody Fc fragment is essential for Fc receptor-mediated activity. Carbohydrate heterogeneity is known to modulate the activity of effector cells in the blood, in which fucosylation particularly affects NK cell-mediated killing. Here, we investigated how the glycosylation profile of 2F8, a human IgG(1) monoclonal antibody against epidermal growth factor receptor in clinical development, impacted effector function. Various 2F8 batches differing in fucosylation, galactosylation, and sialylation of the complex-type oligosaccharides in the Fc fragment were investigated. Our results confirmed that low fucose levels enhance mononuclear cell-mediated antibody-mediated cellular cytotoxicity (ADCC). In contrast, polymorphonuclear cells were found to preferentially kill via high-fucosylated antibody. Whole blood ADCC assays, containing both types of effector cells, revealed little differences in tumor cell killing between both batches. Significantly, however, high-fucose antibody induced superior ADCC in blood from granulocyte colony-stimulating factor-primed donors containing higher numbers of activated polymorphonuclear cells. In conclusion, our data demonstrated for the first time that lack of fucose does not generally increase the ADCC activity of therapeutic antibodies and that the impact of Fc glycosylation on ADCC is critically dependent on the recruited effector cell type.