A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells

BACKGROUND: Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy with bispecific diabodies could be a promising novel treatment option for prostate cancer. METHODS: A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv V_HCD3-V_LPSMA and V_HPSMA-V_LCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model. RESULTS: By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor xenografts with the diabody and PBL efficiently inhibited tumor growth. CONCLUSIONS: The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease

Single-Batch Production of Recombinant Human Polyclonal Antibodies

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the SympressTM I technology. The SympressTM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the SympressTM I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the SympressTM II technology. Here we describe proof-of-principle data demonstrating the feasibility of the SympressTM II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices

[EN] A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against (RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl-1,1,2,2-tetrafluoroethyl ether (tetraconazole), 2-(4-triazolyl)benzimidazole (thiabendazole), and (RS)-1-(beta-allyloxy-2,4-dichlorophenylethyl)imidazole (imazalil) were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices.This work was funded by Ministerio de Educacion y Ciencia (MEC, Spain, Project AGL2002-03266). E. P. was the recipient of a doctoral fellowship from Conselleria d'Educacio (Generalitat Valenciana, Spain).Moreno Tamarit, MJ.; Plana Andani, E.; Manclus Ciscar, JJ.; Montoya Baides, Á. (2014). Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices. Food Analytical Methods. 7(2):481-489. https://doi.org/10.1007/s12161-013-9655-zS48148972Abad A, Manclús JJ, Moreno M, Montoya A (2001) J AOAC Int 84:1–6Alcocer MJC, Doyen C, Lee HA, Morgan MRA (2000) J Agric Food Chem 48:4053–4059Brichta J, Vesela H, Franek M (2003) Vet Med 48:237–247Brichta J, Hnilova M, Viskovic T (2005) Vet Med 50:231–252Charlton K, Harris WJ, Potter AJ (2001) Biosens Bioelec 16:639–646EU Pesticide Database (2013) Pesticide EU-MRLs. http://ec.europa.eu/sanco_pesticides/public/index.cfm . Accessed Jan 2013Ferrer C, Martínez-Bueno MJ, Lozano A, Fernández-Alba AR (2011) Talanta 83:1552–1561Garret SD, Appleford DJA, Wyatt GM, Lee HA, Morgan MRA (1997) J Agric Food Chem 45:4183–4189Graham BM, Porter AJ, Harris WJ (1995) J Chem Technol Biotech 63:279–289Hiemstra M, de Kok A (2007) J Chromatog A 1154:3–25Kipriyanov SM, Moldenhauer G, Little M (1997) J Immunol Meth 200:69–77Kramer K, Hock B (2007) Recombinant antibodies for agrochemicals: Evolutionary optimization. In: Kennedy IR, Solomon KR, Gee SJ, Crossan AN, Wang S, Sánchez-Bayo F (eds) Rational environmental management of agrochemicals: Risk assessment, monitoring, and remedial action. ACS Symposium Series, vol. 966, pp 155−170Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A (1997) J Immunol Meth 201:35–55Leong SSJ, Chen WN (2008) Chem Engin Sci 63:1401–1414Li T, Zhang Q, Liu Y, Chen D, Hu B, Blake DA, Liu F (2006) J Agric Food Chem 54:9085–9091Manclús JJ, Moreno M, Plana E, Montoya A (2008) J Agric Food Chem 56:8790–8800Markus V, Janne L, Urpo L (2011) Trends Anal Chem 30:219–226Mersmann M, Schmidt A, Tesar M, Schöneberg A, Welschof M, Kipriyanov S, Terness P, Little M, Pfizenmaier K, Moosmayer D (1998) J Immunol Meth 220:51–58Moreno M, Plana E, Montoya A, Caputo P, Manclús JJ (2007) Food Addit Contam 24:704–712Morozova VS, Levashova AI, Eremin SA (2005) J Anal Chem 60:202–217Nishi K, Imajuku Y, Nakata M, Ohde K, Miyake S, Morimune K, Kawata M, Ohkawa H (2003) J Pest Sci 28:301–309Nishi K, Ishiuchi M, Morimune K, Ohkawa H (2005) J Agric Food Chem 53:5096–5104Scholthof KB, Whang G, Karu AE (1997) J Agric Food Chem 45:1509–1517Sheedy C, MacKenzie CR, Hall JC (2007) Biotech Adv 25:25333–25352Tout NL, Yau KYF, Trevors JT, Lee H, Hall JC (2001) J Agric Food Chem 49:3628–3637Webb SR, Lee H, Hall JC (1997) J Agric Food Chem 45:535–541Yau KYF, Tout NL, Trevors JT, Lee H, Hall JC (1998) J Agric Food Chem 46:4457–4463Yoshioka N, Akiyama Y, Matsuoka T, Mitsuhashi T (2010) Food Control 21:212–21

On some generalizations of the properties of the multidimensional generalized Erdélyi-Kober operators and their applications

In this paper we investigate the composition of a multidimensional generalized Erdélyi-Kober operator with differential operators of high order. In particular, with powers of the differential Bessel operator. Applications of proved properties to solving the Cauchy problem for a multidimensional polycaloric equation with a Bessel operator are show

Antibody Engineering Using Phage Display with a Coiled-Coil Heterodimeric Fv Antibody Fragment

A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to VH and VL for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of VH frameworks and VH-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering

Transmutation operators as a solvability concept of abstract singular equations

One of the methods of studying differential equations is the transmutation operators method. Detailed study of the theory of transmutation operators with applications may be found in the literature. Application of transmutation operators establishes many important results for different classes of differential equations including singular equations with Bessel operato

Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage

Background: Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display. Methodology/Principal Findings: Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient. Conclusions/Significance: Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

A novel human recombinant single-chain antibody targeting CD166/ALCAM inhibits cancer cell invasion in vitro and in vivo tumour growth

Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control