The in vitro and in vivo anti-tumour activity of N-AcMEL-(Fab')2 conjugates.

To increase the accessibility of drug-antibody complexes to tumours and to decrease non-specific binding via Fc receptors N-acetyl-melphalan (N-AcMEL) was conjugated to F(ab')2 fragments. These fragments were synthesised by pepsin degradation of IgG MoAb. Up to 20 molecules of N-AcMEL could be successfully coupled to each F(ab')2 fragment (compared with 25 molecules/intact IgG) with retention of both drug and antibody activity. The N-AcMEL-F(ab')2 conjugates demonstrated specific cytotoxicity in vitro however despite the absence of non specific Fc receptor binding and greater permeability when using F(ab')2 fragments, the N-AcMEL-F(ab')2 and N-AcMEL-IgG conjugates had similar anti-tumour activity in vivo. Conjugates made with whole IgG and F(ab')2 were equally effective in eradicating subcutaneous solid tumours in mice when injected intravenously. The lower immunogenicity of F(ab')2 fragments compared with whole IgG and the similar cytotoxicity of their conjugates, suggests that the F(ab')2 conjugate has greater clinical utility

Tanggung Jawab Pemerintah Desa Dalam Bidang Pendidikan Di Desa Lutur Kecamatan Aru Selatan Kabupaten Kepulauan Aru

The study aims to determine the responsibility of the village government in the field of education in Lutur Village. Normative legal research on village government or what is commonly referred to as the village head is supported by village officials as one of the elements of village government administration. The village head is a village official who has the authority, obligations and responsibilities to manage his village. Details of the position, duties and function of these community leaders based on domestic regulation number 84 0f 2015 as stipulated in article 6 the village administration who leads the administration of the village. The formulation of the problem to answer this question above is first whether the village government is responsible in the filed of education, seond what are the legal consequences does not carry out its responsibilities. The method used is normative law based on research conducted, the village government is responsible for carrying out development in the filed of education and is legally following

MUC1 immunotherapy against a metastatic mammary adenocarcinoma model: Importance of IFN-gamma

Abstract Immunotherapy using mucin 1 (MUC1) linked to oxidised mannan (MFP) was investigated in an aggressive MUC1+ metastatic tumour, DA3-MUC1 because, unlike many MUC1+ tumour models, DA3-MUC1 is not spontaneously rejected in mice making it an alternative model for immunotherapy studies. Further, DA3-MUC1 cells are resistant to lysis by anti-MUC1 cytotoxic T cells (CTLs). The inability of DA3-MUC1 tumours to be rejected in naïve mice as well as vaccination to MUC1 was attributed to a deficiency of expression of MHC class I molecules on the tumour cell surface. In vitro and in vivo analysis of subcutaneous tumours and lung metastases demonstrated that DA3-MUC1 tumour cells have a low expression (&lt; 6%) of MHC class I which can be upregulated (&gt; 90%) following culturing with IFN-γ. Results from flow cytometry analysis and immunoperoxidase staining indicated that the in vitro up-regulation of MHC class I could be maintained for up to seven days in vivo, without affecting the expression levels of MUC1 antigen. Interestingly, MUC1-specific CTL that lyse DA3-MUC1 targets in vitro were induced in MFP immunised mice but failed to protect mice from a DA3-MUC1 tumour challenge. These results highlight the importance of MHC class I molecules in the induction of anti-tumour immunity and the MFP immune response.</jats:p

Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells

Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations

Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1

The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUC1 expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUC1. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUC1 synthetic peptides is low (approximately 100 n for the IgG and 10 μ for the Fab), with particularly low but similar dissociation rate constants (0.031–0.095 s−1). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly (the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody

Rise and Fall of an Anti-MUC1 Specific Antibody

So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology