Download (RBC) coated with IgG anti-D bind transforming growth factor-beta

Biochemical Society Transactions (2002) Volume 30, Part 3
87
A single-domain antibody fragment that stabilises the native
state of two amyloidogenic lysozyme variants and hence
prevents aggre ation
M. Dumoulin , A . Last2, D. Canet2, L. Wyns3,
f
S. Muylderrnans3, A. Matagne4, C. Redfield2, C.V. Robinson1
and C.M. Dobsonl
‘Department of Chemistry, University of Cambridge, Lensfield
Road, Cambridge CB2 1 E W , UK; 20xford Center for Molecular
Sciences, University of Oxford, South Park Road, Oxford O X 1 347;
U.K; 3Department of Ultrastructure, Vrije Universiteit Brussel,
Paardenstraat 65, B-1640 St. Genesius Rode, Belgium; lLaboratoire
d’Enzymologie, Institut de Chimie B6, Universite de Liege, B-4000
Liege, Belgium.
Amyloid diseases are characterized by the change of a normally
soluble protein into an abnormal insoluble form. A number of
studies suggest that a common property of the amyloidogenic
proteins is their ability, due to their reduced stability, to undergo
conformational changes into partially unfolded states. These
amyloidogenic intermediates arc likely to be the precursors
which permit self-ordered assembly into amyloid structures.
The conformational dynamics of two amyloidogenic variants of
human lysozyme [I] were probed in the presence of a single-domain
antibody fragment ( V H H [2]) derived from a camelid source.
Hydrogen exchange experiments, combined with N M R and mass
spectrometry measurements indicate that the specific binding of the
V H H significantly stabilizes both lysozyme variants and prevents
their conversion into aggregates.
[ l ] Pepys, M.B., et al. (1993) Nature 362, 553-557.
[2] Muyldermans ct al. (2001) Trends Biochem. Sci. 26,230-235.
88 Red blood cells (RBC) coated with IgG anti-D bind
transforming growth factor-beta (TGF-beta)
B.M. Kumpel
International Blood Group Reference Laboratory, Southmead Road,
Bristol BSlO SND
Clinical and experimental observations (trials of monoclonal anti-D)
indicate long-term suppression of primary anti-D responses by
passive IgG anti-D (RhD prophylaxis). T G F - p may bind to
anti-D o n RBC and increase the IgG-mediated suppression of
antigen-specific B cells (Kumpel & Elson, Trends Immunol.
2001;22:26). To investigate this hypothesis, D+ RBC were washed,
incubated with human IgG anti-D (polyclonal anti-D, IgGl or IgG3
monoclonal anti-D)or other preparations not containing anti-D
(saline, intravenous IgG, fresh plasma) and then incubated with
fresh human plasma. Samples of RBC were tested with anti-IgG
and shown to be opsonised by anti-D but they had not bound IgG
with the control reagents. The remaining RBC were then washed,
solubilised and human TGF-pl concentrations in the lysates
determined by ELISA (DuoSet, R&D Systems). Mean ? SEM
values were 408 2 25 pg/ml and 256 ? 25 pg/ml for the anti-D
coated and uncoated RBC, respectively (p=0.0124, unpaired t test).
The anti-D sensitised RBC bound approximately 3600 molecules
of TGF-PI per RBC more than the control RBC. Thus inhibition
of anti-D specific B cells through co-crosslinking the B cell receptor
and the IgG Fc receptor (FcyRIIb) by RhD antigen and RBCbound passive anti-D may be enhanced by T G F - b o n the RBC.
A93
89
Targeted cytokine delivery to neuroblastoma
P. K. Dehal M. J. Embleton J. T. Kemshead R. E. Hawkins
Medical Oncology, Paterson Institute for Cancer Research,
Wilmslow Road, Manchester M20 4BX U.K.
The aim of this study was t o construct a fusion protein from the
cytokine GMCSF and a single chain Fv fragment (scFv D29) and
t o investigate its potential to activate cells of the immune system
against neuroblastoma cells expressing neural cell adhesion molecule
(NCAM). Mammalian cell expression of the scFv D29-GMCSF
fusion protein was compared using a number of vectors, including
retroviral and adenoviral vectors. The resultant fusion protein,
expressed by HeLa cells, was found by ELISA t o bind immobilised
recombinant NCAM, and FACS analysis revealed binding to the
human neuroblastoma cell line SKNBE and a murine neuroblastoma
cell line engineered to express the G P I form of human N C A M
(N2A-rKNIE). The fusion protein was also found to stimulate
the proliferation of the FDC-PI haemopoietic cell line, which is
dependent on GMCSF (or IL-3) for continued growth. In vitroclonogenic assays indicated that the scFv-GMCSF could selectively
induce growth inhibition of SKNBE cells by murine lymphoid cells.
Parallel studies investigating the potential of D29-Fc fusion protein
showed that it could mediate A D C C of SKNBE cells by murine
mononuclear cells.
90
Highly stable single-domain antibody fragments
from camelids
M. D ~ m o u l i nK.
~ ~Conrath2,
~,
E Meersrnan3,
K. Heremans3, S. Muyldermans2, L. Wyns2 and A. Matagnel
Laboratoire d’Enzymologie, Centre d’lnginerie des Protiines,
Institut de chimie B6, UniversitP de Liege, B-4000 Liege, Belgium;
2Departrnent of Ultrastructure VI U.B, 8-1640 St. Genesius
Rode, Be1 ium; 3Department of Chemistry, K. U.L. B-3001 Leuven,
Belgium; $Present address: University of Cambridge, U.K.
Camels, dromedaries and llamas generate functional antibodies
devoid of light chains. Following immunization, recombinant
antigen binders can be isolated, and produced as soluble monomers
in E . coli [l]. A number of V H H fragments have been generated
against selected proteins o r haptens. Despite the absence of a light
chain, these V H H s display high affinities for their antigens, with
values of the dissociation constant (KD) in the nanomolar range,
i.e. very similar to the affinity of most conventional antibodies.
The equilibrium folding properties of six V H H s have been studied
by a variety of techniques, including high pressure unfolding
monitored by Fourier transform infrared spectroscopy, fluorescence,
circular dichroism and surface plasmon resonance spectroscopy.
With all binders, chemical-induced unfolding is fully reversible and
proceeds via a simple two-state mechanism. All the fragments
display remarkably high conformational stabilities (A(H20) = 30-60
kJ mol-l). Hence, the reduced size, improved solubility and higher
stability of the camelid heavy-chain antibody fragments arc of
special interest for biotechnological and medical applications.
[l] Muyldermans, S . (2001) Rev. Mol. Biotech. 74, 277-302.
0 2002 Biochemical Society