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Abstracts / Molecular Imm

sing a monoclonal antibody that differentiates between these twosotypes we were able to separate and purify the product of theFH/CFHR3 gene. Functional analysis of this showed a profound

mpairment of both co-factor and decay accelerating activity inell-based assays. In conclusion we have in a large aHUS family dis-overed a novel CFH/CFHR3 hybrid gene in all affected individuals.he protein product of this is a novel 24 SCR complement proteinhich has profoundly impaired regulatory function.

oi:10.1016/j.molimm.2010.05.262

1

omplement deficiency ameliorates cigarette smoke inducedung injury

arah Casey, Fei Qiao, Andrew Elvington, Stephen Tomlinson, Carltkinson

Medical University of South Carolina, Division of Biomedical Sciences,harleston, SC, United States

Cigarette smoke inhalation is the single largest risk factoror the development of emphysema, and is characterized byroteinase/anti-proteinase imbalances that result in inflammatoryell infiltration and lung alveolar tissue destruction. Studies per-ormed more than a decade ago demonstrated that componentsontained within cigarette smoke have the capacity to activatehe alternative pathway of complement in vitro by modificationf C3. Therefore we sought to investigate whether deficiency ofither C3 of fB provided protection in a murine model of acuteigarette smoke induced lung injury. We exposed wild type, C3−/−nd fB−/− mice to 3 days of whole body cigarette smoke exposure,here mice received 2 exposures/per day of 4 cigarettes/exposure.

ixteen hours after the final exposure, lungs and lavage fluidere collected for analysis. In wild type animals, smoke expo-

ure resulted in complement deposition in lungs, increased lavageoncentrations of C3, C3a, C5, and C5a, and increased gene tran-cription for C3, C4, C5 and fB. Smoke exposure of wild type animalsas also associated with a significant increase in neutrophils andacrophages and the pro-inflammatory cytokines KC, IL-6, MIP-

, TNF alpha, and MCP-1. Deficiency in either C3 or fB resulted insignificant reduction in inflammatory cell infiltrates in both the

avage and lung tissues, and a significant reduction in all cytokinesnalyzed with the exception of MCP-1. Given these findings weerformed chronic smoke exposure experiments with wild typend fB−/− mice. Mice were exposed to 4 cigarettes/day for 6onths. Analysis of lung structure demonstrated that fB deficiency

esulted in preservation of lung morphology evidenced by reducedean linear intercept values, as compared to wild type. Further,

B deficiency was also associated with reduced inflammatory cell

nfiltrates. Taken together these data indicate a key role for thelternative pathway of complement in the inflammatory reactionollowing acute and chronic cigarette smoke exposure.

oi:10.1016/j.molimm.2010.05.263

gy 47 (2010) 2198–2294 2287

82

Development of human bispecific antibodies against CD20/CD55or CD20/CD59 for the treatment of Burkitt lymphoma

P. Macor a, E. Secco a, N. Mezzaroba a, L. De Maso a, P. Durigutto a,T. Gaiotto a, C. Garrovo b, F. Biffi b, S. Zorzet a, D. Sblattero c, F.Tedesco a

a Department of Life Sciences, University of Trieste, Trieste, Italyb Optical Imaging Laboratory, CBM, Area Science Park, Trieste, Italyc Department of Medical Sciences, University of Eastern Piedmont,Novara, Italy

We have shown that human neutralizing anti-CD55 or anti-CD59 miniantibodies (MBs) increase the effect of Rituximab in thetreatment of a Hu/SCID model of lymphoma (Macor et al., 2007,Cancer Res.). To avoid binding of these MBs to circulating and tissuecells and to target selectively lymphoma cells we have developedbispecific antibodies (BsAbs) containing anti-CD20 (MB20) com-bined with MBs to CD55 (MB20/55) or to CD59 (MB20/59). A singlevector containing the sequences of 2 MBs on a single open read-ing frame was constructed and heterodimerization of the MBs wasobtained by a “knob-into-hole” mutagenesis of the CH3 domain.The 2A self-processing sequence from the foot-and-mouth diseasevirus was introduced into the vector to allow an equal level of MBssynthesis. The effect of the BsAbs was tested in vitro in a stan-dard complement-dependent cytotoxicity assay on BJAB, a Burkittlymphoma cell line. The results obtained showed that the killingactivity of MB20/55 and MB20/59 was 3–4 times higher than thatof MB20. The in vivo pharmacokinetic of BsAbs was evaluated bytime-domain optical imaging technology in a Hu-SCID model ofBurkitt lymphoma. The distribution profile of the BsAbs injectedi.p. mimics that MB20. In particular the signal: (i) was recorded inthe tumor mass developing in the peritoneum, as well as in the liver,spleen and bone marrow, all involved in tumor cell dissemination;(ii) peaked in tumor mass at 3–4 days; (iii) and was still seen 10days after antibody injection. To evaluate the therapeutic effect ofBsAbs, tumor bearing mice received 2 i.p. injections of the antibod-ies 4 and 11 days after tumor cell engraftment. The treated animalsexhibited a marked reduction in tumor mass size associated withincreased survival. Based on these results, we propose this strategyto target tumor cells with neutralizing antibodies to CD55 and CD59to enhance complement-dependent cell killing. This approach canbe adapted to all complement-fixing anti-tumor antibodies.

doi:10.1016/j.molimm.2010.05.264

83

Recruitment of human endothelial cells expressing alpha-galepitopes into tumor vessels as a novel strategy to promote com-plement dependent tumor regression

P. Macor a, N. Mezzaroba a, F. Bossi a, F. Biffi b, S. Zorzet a, C.Garrovo b, F. Tedesco a

a Department of Life Sciences, University of Trieste, Trieste, Italyb Optical Imaging Laboratory, CBM, Area Science Park, Trieste, Italy

Tumor vessels represent one of the potential targets of can-cer therapy and anti-angiogenic drugs are currently used to starvetumor cells. Our approach was to exploit the effect of complement(C) in inducing functional alteration of the tumor vessels in orderto enhance the therapeutic effect of anti-tumor antibodies. To this

end, human endothelial cells (ECs) were engineered to expressalpha-gal epitopes as target of circulating natural human anti-bodies. Adult dermal microvasculature or umbilical vein ECs weretransfected with murine alpha 1,3-galactosyl-transferase cloned in