Displaying Anti-Neutrophil Antibodies at the Host-Skin Graft Interface using IgG-Binding Injectable Fibrils
Wen Liu1, Yiwei Wang1, Ngoc Pham1, Yong Fan3,4, Ellen S. Gawalt2,5, Nick Giannoukakis3,4, and Wilson S. Meng1,5
1
Graduate School of Pharmaceutical Sciences, and 2Department of Chemistry and Biochemistry, Duquesne University, PA
15282; 3Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, PA 15212; 4Department of Biological
Sciences, Carnegie Mellon University, Pittsburgh, PA 15224; 5McGowan Institute for Regenerative Medicine, University of
Pittsburgh, PA 15219
Statement of Purpose: The purpose of this work is to
characterize the functions of coacervates formed by two
peptides, namely EAK16-II and the novel construct
wZ4c_EAK2, both containing the AEAEAKAK
amphiphilic sequence. Z34C is a 4.18 kDa disulfidelinked two-helix domain that binds to IgG at an affinity
close to that of the parent B domain of Staphylococcal
protein A (Mw~42 kDa) but predicted to be less
immunogenic. We have engineered tryptophan-inserted
wZ34c_EAK2 into a plasmid vector and expressed the
bifunctional protein in E.coli. Upon intermixing the
peptides coassemble through ionic complementarity
between the common AEAEAKAK in both. We
hypothesized that the coacervates formed by the
intermixing can be used to display IgG antibodies at the
interface of skin graft and host cutaneous tissues. The
film was tested for interdicting neutrophil infiltration into
the graft as a modality to preserve tissue viability and
reduce inflammation.
Methods: A pEX-N-His plasmid encoding wZ34c was
used to express the protein in E.coli (BL21). The protein
was purified, dialyzed and underwent endotoxin removal
step. The predicted molecular weight of the expressed
fusion protein was confirmed using MALDI-TOF mass
spectrometry. Coassembly of wZ34c_EAK into EAK16II membranes was studied using electrophoretic (SDSPAGE), microscopic (fluorescence) and spectroscopic
(FTIR) methods. The capacity of the composite to
capture antibodies in vitro was studied using a mouse IgG
labeled with the fluorescent dye Phycoerythrin. Retention
of antibodies in vivo was monitored in wild-type
C57BL/6 mice using IgG conjugated with a near infrared
dye (λem=800 nm). Images of live animals were captured
using the Pearl Impulse system. The biological impact
was characterized in a mouse skin allograft model.
Results: Described herein is a second-generation design
of IgG-binding EAK coacervates [1-6] in which the novel
bifunctional peptide wZ34c_EAK2 is used. We have in
vitro evidence showing IgG binds significantly higher in
coacervates formed by wZ34c_EAK2 and EAK16-II
compared to EAK6-II alone. Interdicting neutrophils at
the host-graft interface is a novel concept from which
potential anti-rejection therapies might be developed. We
have evidences showing that neutrophils are prominent in
skin allografts and antibodies can be stably concentrated
at the host-graft interface (Fig. 1). The coacervate-IgG
complex was found to remain stable underneath skin graft
in vivo for at least 48 h. In addition, high activity of
elastase was detected in implanted allografts.
Neutrophilic elastase was illuminated using NE680,
Figure 1: (Left) Generation of IgG-binding fibrils by
intermixing wZ34c_EAK2 and EAK16-II. (Right) In
vivo imaging of dye-labeled IgG injected with the
system of materials remained stable at the host-graft
interface for at least 48 h.
indicating robust infiltration of neutrophils in the
allogeneic transplantation. Higher elastase activity
detected in skin graft recovered (k=1.8e-4) from a
transplanted mouse compared to fresh ear skin (k=0.9e-4)
using the chromogenic substrate MeOSuc-AAPV-AMC.
The system of materials was applied to interdict
neutrophils. The coacervates were armed anti-neutrophil
(Ly6G and CD11b) antibodies on wZ34c. We have
evidence showing that the extent of neutrophil infiltration
was reduced in wound beds applied with the wZ34c_EAK
film armed with Ly6G and CD11b antibodies. Lower
fluorescent intensity was found in the treated compared to
control, with all mice injected (i.v.) with cFLFLF-PEG76Cy7, a dye-conjugated ligand of the formyl peptide
receptors expressed on neutrophils.
Conclusions: The data indicate that intermixing
wZ34c_EAK with molar excess of EAK results in an IgG
binding coacervates remained stable at the host-graft
interface. The therapeutic potential was tested in
impeding neutrophil trafficking.
References:
[1] Saunders MJ. Bioconjug Chem (2013); 15:803-10
[2] Zheng Y. Biomaterials (2011); 32:249-57
[3] Wen Y. Mol Pharm (2013); 10:1035-44
[4] Wen Y. Biomaterials (2014); 35:5196-205
[5] Wen Y. Acta Biomater (2014); 10:4759-67
[6] Liu W. J Control Rel, (2016); 230: 1-12