7 research outputs found
Substance P Derivatives as Versatile Tools for Specific Delivery of Various Types of Biomolecular Cargo
The use of proteins or nucleic acids as therapeutic agents
has
been severely hampered by their intrinsic inability to cross the cell
membrane. Moreover, common techniques for driving the delivery of
macromolecules lack the ability to distinguish between healthy and
diseased tissue, precluding their clinical use. Recently, receptor-mediated
delivery (RMD) has emerged as a technology with the potential to circumvent
the obstacles associated with the delivery of drug targets by utilizing
the natural endocytosis of a ligand upon binding to its receptor.
Here, we describe the synthesis
of variants of substance P (SP), an eleven amino acid neuropeptide
ligand of the neurokinin type 1 receptor (NK1R), for the delivery
of various types of cargo. The variants of SP were synthesized with
an N-terminal maleimide moiety that allows conjugation to surface
thiols, resulting in a nonreducible thioether. Cargos lacking an available
thiol are conjugated to SP using commercially available cross-linkers.
In addition to the delivery of proteins, we expand the use of SP to
include nuclear delivery of DNA fragments that are actively expressed
in the target cells. We also show that SP can be used to deliver whole
bacteriophage particles as well as polystyrene beads up to 1 ÎĽm
in diameter. The results show the ability of SP to deliver cargo of
various sizes and chemical properties that retain their function within
the cell. Furthermore, the overexpression of the NK1R in many tumors
provides the potential for developing targeted delivery reagents that
are specific toward diseased tissue
Substance P Derivatives as Versatile Tools for Specific Delivery of Various Types of Biomolecular Cargo
The use of proteins or nucleic acids as therapeutic agents
has
been severely hampered by their intrinsic inability to cross the cell
membrane. Moreover, common techniques for driving the delivery of
macromolecules lack the ability to distinguish between healthy and
diseased tissue, precluding their clinical use. Recently, receptor-mediated
delivery (RMD) has emerged as a technology with the potential to circumvent
the obstacles associated with the delivery of drug targets by utilizing
the natural endocytosis of a ligand upon binding to its receptor.
Here, we describe the synthesis
of variants of substance P (SP), an eleven amino acid neuropeptide
ligand of the neurokinin type 1 receptor (NK1R), for the delivery
of various types of cargo. The variants of SP were synthesized with
an N-terminal maleimide moiety that allows conjugation to surface
thiols, resulting in a nonreducible thioether. Cargos lacking an available
thiol are conjugated to SP using commercially available cross-linkers.
In addition to the delivery of proteins, we expand the use of SP to
include nuclear delivery of DNA fragments that are actively expressed
in the target cells. We also show that SP can be used to deliver whole
bacteriophage particles as well as polystyrene beads up to 1 ÎĽm
in diameter. The results show the ability of SP to deliver cargo of
various sizes and chemical properties that retain their function within
the cell. Furthermore, the overexpression of the NK1R in many tumors
provides the potential for developing targeted delivery reagents that
are specific toward diseased tissue
Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins
<div><p>We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that <i>in vivo</i> biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 <i>in vivo</i> biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce <i>in vivo</i> biotinylated Fabs in <i>E</i>. <i>coli</i>. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.</p></div
Fab and IgG production.
<p>(A) Comparison of purification yields between different expression vectors using an anti-MBP Fab as an example. The large-scale purification method on the Ă„KTA Xpress including a heat denaturation step was used. (B) SDS-PAGE gel showing the anti-MBP Fab produced with various expression vectors and purified in triplicate. (C) IgG production yields with and without the dilution strategy.</p
Overview of targets and their success rates.
<p>Overview of the 265 antigen domains included in the study and what protein families they belong to. Family success rate was calculated as percentage of targets that were successful in selections and which also yielded at least one antibody that passed cell-based validation.</p><p>Overview of targets and their success rates.</p
Performance consistency among Fabs and IgGs generated against the same target.
<p>Multiple Fabs and IgGs against several targets were used to immunoprecipitate their corresponding FLAG-tagged antigens. Western blot was performed and the presence of the FLAG-tagged immunoprecipitated protein was detected with an antibody against the tag. A) CBX3. B) L3MBTl2, C) SFMBT2, D) TDRD3. FLAG-tagged GFP was used as control (data not shown). Fab batches are labeled with a trailer “-Bxxx” and IgG batches are labeled with a trailer “-IBxxx”. Fabs against CBX3 and SFMBT2 have been produced twice (CBX3 (B002, B004); SFMBT2 (B002, B004)) while Fabs against L3MBTL2 and TDRD3 have been produced only once (L3MBTL2 (B001); TDRD3 (B001)). Multiple IgGs have been produced with corresponding IB numbers. Fabs/IgGs derived from the same phagemid clone have similar efficiencies and show a high lot-to-lot consistency.</p
Comparison of antigen immobilization methods.
<p>(A) Three different affinity tags were tested for antigen immobilization in phage display; <i>in vivo</i> biotinylation through an Avi tag, SBP and GST tags. The diversity of Fabs derived from these differentially tagged antigens was then compared. (B) Immunoprecipitation with Fabs selected against either Avi-tagged antigen or GST-tagged antigen from a cell lysate expressing FLAG-tagged target protein. Immunoprecipitated antigen was detected with an M2 antibody against the FLAG tag. Fabs selected against Avi-tagged antigen generally show a higher recovery of the antigen.</p