U.S. patent application number 15/395233 was filed with the patent office on 2017-06-29 for fluorescent fusion polypeptides and methods of use.
The applicant listed for this patent is STC.UNM. Invention is credited to Ravi Venkata Durvasula, Angray Singh Kang, Anatoliy Markiv.
Application Number | 20170183663 15/395233 |
Document ID | / |
Family ID | 44858410 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170183663 |
Kind Code |
A1 |
Markiv; Anatoliy ; et
al. |
June 29, 2017 |
FLUORESCENT FUSION POLYPEPTIDES AND METHODS OF USE
Abstract
Embodiments of the present invention provide for the facile
generation of a stable recombinant fusion polypeptides with
intrinsic fluorescent properties. The recombinant antibodies may be
suitable for qualitative and/or quantitative immunofluorescence
analysis. Generally, the fluorescent polypeptides include a
fluorescent domain comprising a C-terminus and an N-terminus; a
first antibody domain covalently linked to the C-terminus; and a
second antibody domain covalently linked to the N-terminus.
Inventors: |
Markiv; Anatoliy;
(Middlesex, GB) ; Durvasula; Ravi Venkata;
(Albuquerque, NM) ; Kang; Angray Singh; (Leyton,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STC.UNM |
Albuquerque |
NM |
US |
|
|
Family ID: |
44858410 |
Appl. No.: |
15/395233 |
Filed: |
December 30, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14496605 |
Sep 25, 2014 |
9566353 |
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15395233 |
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13096695 |
Apr 28, 2011 |
8877898 |
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14496605 |
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61343627 |
Apr 30, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0045 20130101;
C07K 16/32 20130101; C12N 15/62 20130101; C07K 2319/00 20130101;
C12Q 1/04 20130101; A61K 49/0058 20130101; C07K 2317/62 20130101;
C07K 2317/622 20130101; C07K 2319/60 20130101; C12N 15/63 20130101;
G01N 33/53 20130101; C07K 16/30 20130101; A61N 5/062 20130101; C07K
16/18 20130101; G01N 33/582 20130101; C07K 14/001 20130101 |
International
Class: |
C12N 15/62 20060101
C12N015/62; C12N 15/63 20060101 C12N015/63 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under R01
AI066045 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-18. (canceled)
19. A polynucleotide that encodes the polypeptide comprising: a
fluorescent domain comprising a C-terminus and an N-terminus; a
first antibody domain covalently linked to the C-terminus; and a
second antibody domain covalently linked to the N-terminus.
20. A cell comprising the polynucleotide of claim 19.
21-22. (canceled)
23. The cell of claim 20 wherein the cell comprises a microbe.
24. The cell of claim 20 wherein the cell comprises a tumor
cell.
25. A method comprising introducing into a cell a polynucleotide
that encodes a polypeptide that comprises: a fluorescent domain
comprising a C-terminus and an N-terminus; a first antibody domain
covalently linked to the C-terminus; and a second antibody domain
covalently linked to the N-terminus.
26. The method of claim 25 wherein the cell comprises a tumor
cell.
27-51. (canceled)
52. A method of making a fusion polypeptide, the method comprising:
creating an expression vector that comprises a polynucleotide
operably linked to a promoter, wherein the polynucleotide encodes
an fusion polypeptide comprising: a fluorescent domain comprising a
C-terminus and an N-terminus; a first antibody domain covalently
linked to the C-terminus; and a second antibody domain covalently
linked to the N-terminus; introducing the expression vector into a
host cell; and growing the host cell comprising the expression
vector in conditions effective for the host cell to express the
fusion polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/343,627, filed Apr. 30, 2010, which is
hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0003] This application contains a Sequence Listing electronically
submitted via EFS-Web to the United States Patent and Trademark
Office as an ASCII text file entitled
"31000670103_SequenceListing_ST25.txt" having a size of 19
kilobytes and created on Jan. 30, 2017. The information contained
in the Sequence Listing is incorporated by reference herein.
BACKGROUND
[0004] Nanotechnology and synthetic biology are converging areas
with the potential to create novel drugs and diagnostic molecules,
and to inspire a plethora of innovative applications exemplified by
the advances made in the area of antibody engineering since the
late 1980s. Recently efforts have been directed towards creating
antibody fragments with novel effector functions as fusions with
fluorescent proteins. These have used scFv fused directly to the
fluorescent protein either at the C-terminus or the N-terminus,
resulting in additive properties of the scFv and the fluorophore.
Alternative efforts have produced a green fluorescent protein (GFP)
scaffold that retains fluorophore activity and is capable of
accommodating two proximal, randomized binding loops.
SUMMARY OF THE INVENTION
[0005] In one aspect, the invention provides a polypeptide that
generally includes a fluorescent domain that possesses a C-terminus
and an N-terminus, a first antibody domain covalently linked to the
C-terminus, and a second antibody domain covalently linked to the
N-terminus.
[0006] In some embodiments, at least one covalent link comprises a
linker comprising no more than 10 amino acids.
[0007] In some embodiments, at least one of the fluorescent domain,
the first antibody domain, or the second antibody comprises an
affinity tag.
[0008] In some embodiments, the first antibody domain and the
second antibody domain specifically bind to a single target
molecule, while in other embodiments, the first antibody domain and
the second antibody domain specifically bind to different target
molecules.
[0009] In some embodiments, the first antibody domain comprises a
variable light chain (V.sub.L) comprising an N-terminus. In some
embodiments, the second antibody domain comprises a variable heavy
chain (V.sub.H) comprising a C-terminus. In some embodiments, the
polypeptide can include both a first antibody domain comprises a
variable light chain (V.sub.L) comprising an N-terminus and a
second antibody domain comprises a variable heavy chain (V.sub.H)
comprising a C-terminus. In certain of these embodiments, the
N-terminus of first antibody domain and C-terminus of the second
antibody domain are separated by a distance of no less than 30
.ANG. and no more than 40 .ANG..
[0010] In some embodiments, the fluorescent domain comprises at
least a portion of a monomeric fluorescent protein sufficient to
emit a fluorescent signal.
[0011] In some embodiments, the fluorescent domain comprises at
least a portion of a dimeric fluorescent protein sufficient to emit
a fluorescent signal.
[0012] In some embodiments, the fluorescent domain comprises an
amino acid sequence comprising at least 90% amino acid sequence
similarity to at least one of: amino acids 122-338 of SEQ ID NO:2,
amino acids 3-219 of SEQ ID NO:4, amino acids 3-235 of SEQ ID ON:6,
or amino acids 3-235 of SEQ ID ON:8.
[0013] In some embodiments, the fluorescent domain comprises an
amino acid sequence comprising at least 90% amino acid sequence
identity to at least one of: amino acids 122-338 of SEQ ID NO:2,
amino acids 3-219 of SEQ ID NO:4, amino acids 3-235 of SEQ ID ON:6,
or amino acids 3-235 of SEQ ID ON:8.
[0014] Moreover, in certain embodiments, the polypeptide can
include features of any combination of two or more embodiments
summarized above.
[0015] In another aspect, the invention provides a composition that
generally includes two or more different fluorescent fusion
polypeptides summarized above.
[0016] In some embodiments, such a composition can include a first
fluorescent fusion protein that includes a first fluorescent domain
and a second fluorescent fusion protein that includes a second
fluorescent domain. In certain of these embodiments, the first
fluorescent domain can emit a first fluorescent signal and the
second fluorescent domain can emit a second fluorescent signal. In
some of these embodiments, the first fluorescent signal comprises a
first emission peak, the second fluorescent signal comprises a
second emission peak, and the first emission peak is different than
the second emission peak.
[0017] In some embodiments, a first fluorescent polypeptide
specifically binds to a different target molecule than a second
fluorescent fusion polypeptide.
[0018] In another aspect, the invention provides a polynucleotide
that encodes any one of the polypeptides summarized above.
[0019] In another aspect, the invention provides a cell that
includes such a polynucleotide.
[0020] In some embodiments, the cell can further include a second
polynucleotide that encodes a second polypeptide as summarized
above. In some of these embodiments, the fluorescent domain of one
polypeptide can emit a first fluorescent signal comprising a first
emission peak, the fluorescent domain of the second polypeptide can
emit a second fluorescent signal comprising a second emission peak,
and the first emission peak can be different than the second
emission peak.
[0021] In another aspect, the invention provides a cell that
includes any one of the polypeptides summarized above. In some of
these embodiments, the cell may be a microbe, while in other
embodiments, the cell may be an animal cell such as, for example, a
tumor cell.
[0022] In another aspect, the invention provides a cell that
includes the any one of the compositions summarized above. In some
of these embodiments, the cell may be a microbe, while in other
embodiments, the cell may be an animal cell such as, for example, a
tumor cell.
[0023] In another aspect, the invention provides a method that
generally includes introducing into a cell any one of the
polynucleotides summarized above. In some of these embodiments, the
cell may be a microbe, while in other embodiments, the cell may be
an animal cell such as, for example, a tumor cell.
[0024] In another aspect, the invention provides a method that
generally includes releasing a microbe into an environment, wherein
the microbe comprises any one of the polynucleotides summarized
above and at least one additional heterologous polynucleotide, and
detecting the microbe by detecting the fluorescent signal. In some
embodiments, the method can include detecting the fluorescent
signal at a plurality of time points.
[0025] In another aspect, the invention provides a method that
generally includes releasing a microbe into an environment
inhabited by a pathogen, wherein the microbe include a
polynucleotide summarized above that encodes a polypeptide that
interferes with transmission of the pathogen, and permitting the
cell to express and export the polypeptide encoded by the
polynucleotide summarized above so that the polypeptide interferes
with transmission of the pathogen. In some of these embodiments,
the exported polypeptides binds to the pathogen. In other of these
embodiments, the exported polypeptide binds to a compound produced
by the pathogen. In some embodiments, the microbe is a symbiont of
a host organism, which is further a host organism to the pathogen.
In certain embodiments, the method can further include detecting a
fluorescent signal produced by the fluorescent domain of the
polypeptide encoded by the polynucleotide summarized above. In some
embodiments, the method can include detecting the fluorescent
signal at a plurality of time points.
[0026] In another aspect, the invention provides a method that
generally includes detecting a fluorescent signal produced by cell,
wherein the cell comprises a polynucleotide as summarized above
that encodes a polypeptide comprising a fluorescent domain. In some
embodiments, the method further includes detecting the fluorescent
signal before, during, or after surgery to remove tumor cells
emitting the fluorescent signal. In some embodiments, the method
includes detecting the fluorescent signal at a plurality of time
points. In some embodiments, the method includes exposing the cell
to white light under conditions effective for the fluorescent
signal to generate an amount of a reactive oxygen species effective
to kill the cell.
[0027] In another aspect the invention provides a method that
generally includes administering to a subject any one of the
polypeptides summarized above, wherein at least one antibody domain
of the polypeptide specifically binds to a marker of inflammation,
and detecting a fluorescent signal produced by the fluorescent
domain of the polypeptide. In some embodiments, the method includes
detecting the fluorescent signal at a plurality of time points. In
some embodiments, detecting the fluorescent signal may be performed
non-invasively.
[0028] In another aspect, the invention provides a method that
generally includes providing a sample that comprises the analyte,
contacting the sample with any one or more of the polypeptides
summarized above, wherein at least one antibody domain specifically
binds to the analyte, removing unbound polypeptides, and detecting
a fluorescent signal produced by the polypeptide specifically bound
to the analyte, thereby detecting presence of the analyte in the
sample. In some embodiments, the method can further include
immobilizing at least a portion of the sample on a substrate. In
some embodiments, the method can further include contacting at
least a portion of the sample with a second polypeptide as
summarized above, wherein at least one antibody domain of the
second polypeptide specifically binds to the second analyte
removing unbound second polypeptide, and detecting fluorescent
signal produced by the second polypeptide specifically bound to the
second analyte, thereby detecting presence of the second analyte in
the sample. In some embodiments, the method can further include
quantifying the fluorescent signal.
[0029] In another aspect, the invention provides a kit that
generally includes a first container comprising a first polypeptide
as summarized above comprising a first fluorescent domain that
produces a fluorescent signal comprising a first emission peak, and
at least one antibody domain that specifically binds to a first
analyte and a second container comprising a second polypeptide as
summarized above comprising a second fluorescent domain that
produces a fluorescent signal comprising a second emission peak,
and at least one antibody domain that specifically binds to a
second analyte.
[0030] In another aspect, the invention provides a method of making
a fusion polypeptide. Generally, the method includes creating an
expression vector that comprises a polynucleotide operably linked
to a promoter, wherein the polynucleotide encodes an fusion
polypeptide comprising: a fluorescent domain comprising a
C-terminus and an N-terminus, a first antibody domain covalently
linked to the C-terminus, and a second antibody domain covalently
linked to the N-terminus; introducing the expression vector into a
host cell; and growing the host cell comprising the expression
vector in conditions effective for the host cell to express the
fusion polypeptide.
[0031] The above summary of the present invention is not intended
to describe each disclosed embodiment or every implementation of
the present invention. The description that follows more
particularly exemplifies illustrative embodiments. In several
places throughout the application, guidance is provided through
lists of examples, which examples can be used in various
combinations. In each instance, the recited list serves only as a
representative group and should not be interpreted as an exclusive
list.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1: Ribbon representation of a REDantibody molecule 3D
model. The V.sub.H chain is shown in green, RFP domain in red, the
V.sub.L chain in yellow. His-tag is shown in blue and linkers
between V.sub.H-RFP and RFP-V.sub.L are in cyan with the distances
indicated. Antigen binding site is indicated as a black circle on
antibody structure. All distances are shown as a grey lines and are
indicated in the angstroms (.ANG.).
[0033] FIG. 2: Generation of expression plasmid (A) and E. coli
expression cassette (B) of REDantibody. (A) The single chain Fv
encoding fragment flanked by NcoI and NotI sites with an inframe
BamHI site between the V.sub.H and V.sub.L was inserted into pBAK1
vector to generate pBAK1B72.3, pBAK1CA19.9 and pBAK14D5. The mRFP
encoding fragment flanked by BamHI sites was inserted into the
pBAK1B72.3, CA19.9 and 4D5 to generate pBAK172.3RFP, pBAK1CA19.9RFP
and pBAK14D5RFP. (B) The expression cassette has a T7 promoter to
initiate transcription using T7 RNA polymerase, lac operator is for
the control of transcription using IPTG. RBS is ribosome binding
site where translation begins, pelB sequence to guide newly
synthesized molecule into the E. coli periplasm where this sequence
is cleaved and disulphide bonds are formed. V.sub.H is variable
heavy chain of antibody, Link is an additional 5 amino acids to
flank either side of the mRFP1 gene, mRFP is a gene of red
fluorescent protein and V.sub.L is variable light domain of
antibody. This is followed by His-tag sequence for immobilized
metal affinity purification and stop codon. T7 terminator is to
terminate transcription by T7 RNA polymerase.
[0034] FIG. 3: Expression and purification (A) CA19.9 scFv and (B)
CA19.9 REDantibody. The purified scFvCA19.9 (A) and REDantibody
CA19.9 (B) were resolved by 12% SDS-PAGE and stained with Coomassie
Brilliant Blue R250. Lane 1: molecular weight markers, 70, 40, 30
and 20 kDa; lane 2: Ni-NTA column elution 1; lane 3: Ni-NTA column
elution 2; lane 4: Ni-NTA column elution 3; lane 5: Ni-NTA column
elution 4.
[0035] FIG. 4: Gel filtration on Sephadex G200 column. Panel
A--standard proteins (peak1--200 kDa, peak 2--66 kDa, peak 3--29
kDa, peak 4--12.4 kDa.); Panel B--mRFP1 and panel
C--REDantibodyCA19.9.
[0036] FIG. 5: REDantibody expression and purification. (A) E. coli
cells expressing REDantibody (A1) and scFv (A2) resuspended in
periplasmic buffer, (B) periplasmic fraction of extracted
REDantibody (B1) and scFv antibody (B2) and (C) is REDantibody
fractions 1-5 eluted from the Ni-NTA column and labeled as C1-C5
respectively.
[0037] FIG. 6: Immunofluorescent microscopic analysis of T. cruzi
epimastigotes stained with REDantibody CA19.9 (A), REDantibody
B72.3 (B) and negative control REDantibody 4D5 (C). T. cruzi
epimastigote is stained with REDantibody CA19.9 and viewed by
microscopy reveals a maximum intensity fluorescent image (D). The
surface staining combined with confocal microscopy reveals a slight
invagination on the parasite surface (E). A cut-away of the image
(E) reveals the staining is limited to the surface of the T. cruzi
epimastigote (F).
[0038] FIG. 7 shows the PCR primers used in certain exemplary
embodiment.
[0039] FIG. 8A-B shows plasmid maps and 3D model of genetically
encoded fluorescent antibody. (A) Molecular model (ribbon
representation) of the HERCEPTIN V.sub.H and V.sub.L (Genentech,
Inc., South San Francisco, Calif.) antigen-binding fragment (Fv)
complexed with human HER2 (PDB 1N8Z) with mRFP1 linker based on the
DsRed (PDB 1G7K). A portion of HER2 is shown in orange, V.sub.H
chain of 4D5-8 in green, mRFP1 in red and V.sub.L chain of 4D5-8
antibody in navy blue. (B) Plasmid maps of pBAK1 vector, p4D5-8Bam
and p4D5-8mRFP1 used to clone and express red fluorescent antibody.
The expression cassette has a T7 promoter region, a lac operator, a
ribosome-binding site followed by a cloning region with NdeI, NcoI
and NotI restriction sites for the insertion of the DNA sequences.
At the end, there is a T7 termination sequence to restrict
translation to the expression of RNA for the recombinant protein.
The vector also has the kanamycin resistance gene, ColE1 pBR322
origin of replication and lacI repressor gene. The 4D5-8mRFP1
construct starts with pelB leader sequence to direct the protein to
the periplasmic space of E. coli where it is cleaved off. The
mature protein begins with V.sub.H chain, followed by a 5-amino
acid linker, mRFP1 sequence, second 5-amino acid linker, V.sub.L
chain and terminates with an octa-His-tag.
[0040] FIG. 9 shows purification and characterization of the
4D5-8mRFP1 recombinant protein. (a) SDS-PAGE and Coomassie stained
gel of 4D5-8mRFP1: molecular markers (Lane 1), uninduced sample
(Lane 2), IPTG induced sample (Lane 3), soluble fraction (Lane 4),
flow-through Ni.sup.2+-nitrilotriacetic acid column (Lane 5), wash
fraction (Lane 6), elution fractions using 500 mM concentration
imidazole (Lanes 7-10). (b) Western blotting of SDS-PAGE gel of
4D5-8mRFP1 (as in panel a) analyzed using HRP-labelled anti-His-tag
antibody. (c) Gel filtration chromatography of standards proteins
(peaks 1-4) and Ni.sup.2+-nitrilotriacetic acid purified 4D5-8mRFP1
protein using Sephadex G 200 gel bead column. (d) SDS-PAGE and
Coomassie stained gel of gel filtration chromatography 4D5-8mRFP1
(pooled fractions 32-37): molecular markers (Lane 1), mRFP1 (Lane
2) 4D5-8mRFP1 (Lane 3).
[0041] FIG. 10 shows binding properties of purified 4D5-8mRFP1. (a)
Detection of 4D5-8mRFP1 binding to
p185.sup.HER-2-ECD-overexpressing SKBr-3 cells by flow cytometry.
Cells incubated with 4D5-8mRFP1 are shown as filled peak and cells
used as controls (solid line) were treated with MACS buffer alone.
All experiments were normalized to 20,000 cells, excluding
doublets. (b) Flow cytometry analysis of p185.sup.HER-2-ECD non
expressing MDA-MD-231 cells incubated with 4D5-8mRFP1. (c)
Fluorescence images of SKBr-3 cells incubated with 4D5-8mRFP1 and
DNA (nucleus) binding dye GelGreen.TM. and (d) Bright-field images
of the same SKBr-3 cells incubated with 4D5-8mRFP1.
[0042] FIG. 11 shows the nucleic acid (SEQ ID NO:1) and amino acid
sequence (SEQ ID NO:2) for the V.sub.H, V.sub.L, and fluorophore of
B72.3RFP. The tertiary structure is illustrated in FIG. 1. Gray
shaded amino acids correspond to the V.sub.H domain; bold text
amino acids correspond to the fluorescent domain (mRFP domain); and
underlined amino acids correspond to the V.sub.L domain.
[0043] FIG. 12 shows the nucleic acid (SEQ ID NO:3) and amino acid
sequence (SEQ ID NO:4) of mRFP1. The fluorescent domain is
reflected in amino acids 3-219 of SEQ ID NO:4.
[0044] FIG. 13 shows the nucleic acid (SEQ ID NO:5) and amino acid
sequence (SEQ ID NO:6) of mCitrine. The fluorescent domain is
reflected in amino acids 3-235 of SEQ ID NO:6.
[0045] FIG. 14 shows the nucleic acid (SEQ ID NO:7) and amino acid
sequence (SEQ ID NO:8) of mCerulean. The fluorescent domain is
reflected in amino acids 3-235 of SEQ ID NO:8.
[0046] FIG. 15: (A) Excitation and emission peaks of REDantibody
4D5-8. (B) Detection of REDantibody 4D5-8 based on concentration
dependent emission at 607 nm.
[0047] FIG. 16A-E shows plasmid maps of vectors used to express
4D5-8 antibodies. Fluorescent proteins were inserted in-between the
V.sub.H/V.sub.L regions of 4D5-8. A) The plasmid map for parent
plasmid pA4D5-81. B) The plasmid map and expression cassette for
construct pA4D5-8mRFP. C) The plasmid map for construct
pA4D5-81-mCit. D) The plasmid map for construct pA4D5-81-mCer. E)
The plasmid map for construct pA4D5-81-BFP.
[0048] FIG. 17A-D shows excitation and emission spectra of 4D5-8
fluorescent fusion antibodies 4D5-8_mRFP (A), 4D5-8_mCit (B),
4D5-8_Cerul (C), and 4D5-8_BFP (D).
DETAILED DESCRIPTION OF EXAMPLARY EMBODIMENTS
[0049] The present invention involves novel fusion proteins
constructs, polynucleotides that encode the constructs, and
methods, including therapeutic, diagnostic, and detection methods,
employing such constructs.
[0050] We describe herein fusion proteins that include a
fluorescent protein domain linked to one or more antibody domains.
In one exemplary embodiment, illustrated in FIG. 1, we used
monomeric red fluorescent protein (mRFP) derived from Discosoma to
link the V.sub.H and V.sub.L domain pairing of the recombinant
anti-carbohydrate antibodies B72.3, CA19.9, and 4D5
anti-p185HER2.
[0051] Many antibody structures have been determined by X-ray
crystallography. These analyses have revealed that the native
distance between the C-terminus on the variable heavy chain and the
N-terminus on the variable light chain is often approximately 35
.ANG.. To generate conventional single chain fragment of the
variable region (scFv) antibodies, a 20-30 amino acid linker may be
introduced between these two sites and thus provide a flexible
region of approximately 35 .ANG.. This spacing between the V.sub.H
and V.sub.L can influence the functionality of the scFv because the
non-covalent interactions between V.sub.H/V.sub.L interfaces are
involved in antigen recognition. Nevertheless, the V.sub.H/V.sub.L
pairing exist in equilibrium with the unpaired state, often
resulting in aggregation of the variable chains and, therefore,
reduced antigen recognition and decreased stability relative to the
Fab fragment or whole immunoglobulin.
[0052] We have engineered a fusion polypeptide that uses a
.beta.-barrel fluorescent domain to bridge the V.sub.H and the
V.sub.L and enhance stability of the antibody domains by
maintaining the correct spatial geometry between the antibody
domains. Moreover, the fluorescent domain anchors the N-terminus
and the C-terminus on the same plane with similar spatial dimension
to the Fv in the context of a Fab fragment so that appropriate
V.sub.H/V.sub.L interface interactions may be achieved resulting in
functional binding sites.
[0053] Generally, prior antibody-fluorophore conjugates have been
established as valuable tools in basic and applied research,
despite having limitations of photo-bleaching, batch variation, Fc
receptor binding, aggregation, and partial loss of binding. Some of
these issues can be addressed by using recombinant single-chain
fragment variable antibody fused directly to genetically encoded
fluorescent reporters. However, conventional single-chain fragment
variable domains linked by long flexible linkers are themselves
prone to disassociation and aggregation.
[0054] Here we report the design, assembly, bacterial production,
and purification of a modular model that may be used generally to
produce novel antibody-fluorescent protein fusion molecules. In one
particular embodiment, the insertion of fluorescent protein mRFP1
in-between the V.sub.H/V.sub.L regions of anti-p185HER2-ECD
antibody 4D5-8 resulted in optimal V.sub.H/V.sub.L interface
interactions to create 4D5-8 REDantibody. The bacterially expressed
monomeric molecule used in flow cytometry and cell staining studies
with SKBr-3 cells retained the fluorophore properties and antibody
specificity functions. The molecular model may be generalized
beyond the use of mRFP1 and the 4D5-REDantibody and may, instead,
use the fluorescent domain of any suitable fluorescent protein, as
described in more detail below. Thus, references to REDantibody are
merely an exemplary and should not be construed as limiting.
[0055] Thus, in one aspect, the invention provides a fusion
polypeptide that includes a fluorescent domain comprising a
C-terminus and an N-terminus, a first antibody domain covalently
linked to the C-terminus, and a second antibody domain covalently
linked to the N-terminus.
[0056] In some embodiments, the polypeptide can include a linker
that provides the covalent linkage between the fluorescent domain
and one of the antibody domains. In such embodiments, the
polypeptide can include two linkers so that a linker provides the
link between the fluorescent domain and each of the antibody
domains. The linker may be of any suitable length. In some
embodiments, the linker assists in providing the stability of the
molecule by contributing to spacing between the C-terminus of one
antibody domain and the N-terminus of the second antibody domain
that reflects the spacing natively found in a Fab fragment or full
IgG. In such embodiments, the length of the linker may be no more
than 10 amino acids such as, for example, no more than nine amino
acids, no more than eight amino acids, no more than seven amino
acids, no more than six amino acids, no more than five amino acids,
no more than four amino acids, no more then three amino acids, or
no more than two amino acids. When two linkers are present, the
length of one linker may be identical or different than the length
of the other linker. In one embodiment, the linker can include five
amino acids such as, for example, Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:21).
[0057] As used herein, an antibody domain is that portion of the
fusion polypeptide that exhibits affinity interaction with a ligand
that is to some degree specific. As used herein, "specific" and
variations thereof refer to having a differential or a non-general
affinity, to any degree, for a particular target. In some
embodiments, an antibody domain can include any suitable portion of
an immunoglobulin. As immunoglobulin structure and function are
well characterized, those of skill in the art are well equipped to
determine the amount and the particular portions of an
immunoglobulin necessary to provide desired target recognition. In
some embodiments, an antibody domain can include, for example, a
V.sub.L or a V.sub.H chain. In some of these embodiments, one
antibody domain can include a V.sub.L chain while the second
antibody domain can include a V.sub.H chain. In particular
embodiments, a V.sub.L chain can be covalently linked to the
C-terminus of the fluorescent domain and a V.sub.H chain can be
covalently linked to the N-terminus of the fluorescent domain.
[0058] In some embodiments, the terminus of the antibody domains
that are covalently linked to the fluorescent domain--either
directly or via a linker, if present--may be separated by a
predetermined distance. The minimum predetermined distance may be
at least 30 .ANG. such as, for example, at least 31 .ANG., at least
32 .ANG., at least 33 .ANG., at least 34 .ANG., at least 35 .ANG.,
at least 36 .ANG., at least 37 .ANG., at least 38 .ANG., or at
least 39 .ANG.. The maximum predetermined distance may be no more
than 40 .ANG. such as, for example, no more than 39 .ANG., no more
than 38 .ANG., no more than 37 .ANG., no more than 36 .ANG., no
more than 35 .ANG., no more than 34 .ANG., no more than 33 .ANG.,
no more than 32 .ANG., or no more than 31 .ANG.. The predetermined
distance may be within a range defined by any minimum and any
maximum distance described herein. Also, in certain embodiments,
the predetermined distance may be any of the minimum or maximum
endpoints described herein. Thus, the predetermined distance may be
30 .ANG., 31 .ANG., 32 .ANG., 33 .ANG., 34 .ANG., 35 .ANG., 36
.ANG., 37 .ANG., 38 .ANG., 39 .ANG., or 40 .ANG..
[0059] In some embodiments, the antibody domains may specifically
bind to the same or to different target molecules. Thus,
embodiments in which both antibody domains specifically bind to the
same target molecule can be characterized as mono-specific. In
contrast, a fluorescent fusion polypeptide that includes antibody
domains that specifically bind to different target molecules may be
characterized as bi-specific polypeptides.
[0060] The antibody domains may be, or be derived from, any
suitable antibody. For example, we report herein producing a
fluorescent fusion polypeptide that includes antibody domains
derived from antibodies that specifically bind to human breast
cancer markers (FIG. 8 through FIG. 10) as well as a fluorescent
fusion polypeptide that includes antibody domains derived from
antibodies that specifically bind to T. cruzi markers (FIG. 2
through FIG. 6). Other exemplary antibody domains can include, for
example, CA125 anti-MUC16, H23 anti-MUC1 and 1C3 anti-Plasmodium
falciparum chitinase. The modular nature of the fluorescent fusion
polypeptides described herein permit great design flexibility and
the possibility of exploiting the known binding affinity and
specificity of any known antibody.
[0061] The fluorescent domain includes at least a portion of any
suitable fluorescent polypeptide sufficient to emit a fluorescent
signal. The structure and function of fluorescent polypeptides are
well characterized. Thus, a person of ordinary skill in the art can
readily determine the portion of a fluorescent polypeptide that is
required to maintain fluorescent functionality.
[0062] In some embodiments, the fluorescent domain can optionally
provide structural integrity to the molecule in addition to
providing a source for the fluorescent signal. Thus, in some
embodiments, the fluorescent domain can include a portion of the
fluorescent polypeptide sufficient to emit a fluorescent signal and
to provide desired steric stability.
[0063] In some embodiments, the fluorescent fusion polypeptide can
include at least a portion of a monomeric fluorescent protein
sufficient to emit a fluorescent signal. Many monomeric fluorescent
polypeptides possess a .beta.-barrel structure that is capable of
maintaining a desired predetermined distance between the C-terminus
of one antibody domain and the N-terminus of the second antibody
domain.
[0064] Portions of certain dimeric fluorescent polypeptides also
may suitable for use as the fluorescent domain. Dimeric fluorescent
polypeptides may naturally orient so that they can provide steric
stability as described above while avoiding steric interference
with the antigen-binding site formed by the antibody domains.
[0065] Thus, in some embodiments, the fluorescent domain can
include at least a portion of mRFP, mCitrine, mCerulean, GFP(wt),
EBFP, Sapphire, T-Sapphire, ECFP, mCFP, CyPet, Midori-Ishi Cyan,
mTFP1, EGFP, AcGFP, TurboGFP, Emerald, Azami Green, EYFP, Topaz,
Venus, yPet, PhiYFP, mBanana, Kusabira Orange, mOrange, dTomato,
DsRed-Monomer, mTangerine, mStrawberry, Jred, mCherry, HcRed1,
mRasberry, or mPlum. Thus, while reference is occasionally made
herein to REDantibody, the use of this particular construct is
merely an exemplary embodiment.
[0066] In some embodiments, the fluorescent domain can include an
amino acid sequence that bears a specified level of amino acid
sequence similarity to a reference polypeptide. The reference
polypeptide may be, or be a fluorescently active portion of, a
fluorescent polypeptide.
[0067] Amino acid similarity of two polypeptides can be determined
by aligning the residues of the two polypeptides (for example, a
candidate polypeptide and the fluorescent domain of, for example,
any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8)
to optimize the number of identical amino acids along the lengths
of their sequences; gaps in either or both sequences are permitted
in making the alignment in order to optimize the number of
identical amino acids, although the amino acids in each sequence
must nonetheless remain in their proper order. A candidate
polypeptide is the polypeptide being compared to the reference
polypeptide. In some embodiments, the reference polypeptide may be,
e.g., the fluorescent domain of any one of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, or SEQ ID NO:8 such as, for example, amino acids
122-338 of SEQ ID NO:2, amino acids 3-219 of SEQ ID NO:4, amino
acids 3-235 of SEQ ID NO:6, or amino acids 3-235 of SEQ ID
NO:8.
[0068] A pair-wise comparison analysis of amino acid sequences can
be carried out using the BESTFIT algorithm in the GCG package
(version 10.2, Madison Wis.). Alternatively, polypeptides may be
compared using the Blastp program of the BLAST 2 search algorithm,
as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250
(1999)), and available on the National Center for Biotechnology
Information (NCBI) website. The default values for all BLAST 2
search parameters may be used, including matrix=BLOSUM62; open gap
penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10,
wordsize=3, and filter on.
[0069] In the comparison of two amino acid sequences, structural
similarity may be referred to by percent "identity" or may be
referred to by percent "similarity." "Identity" refers to the
presence of identical amino acids. "Similarity" refers to the
presence of not only identical amino acids but also the presence of
conservative substitutions. A conservative substitution for an
amino acid in a polypeptide of the invention may be selected from
other members of the class to which the amino acid belongs. For
example, it is well-known in the art of protein biochemistry that
an amino acid belonging to a grouping of amino acids having a
particular size or characteristic (such as charge, hydrophobicity
and hydrophilicity) can be substituted for another amino acid
without altering the activity of a protein, particularly in regions
of the protein that are not directly associated with biological
activity. For example, nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and tyrosine. Polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine and
glutamine. The positively charged (basic) amino acids include
arginine, lysine and histidine. The negatively charged (acidic)
amino acids include aspartic acid and glutamic acid. Conservative
substitutions include, for example, Lys for Arg and vice versa to
maintain a positive charge; Glu for Asp and vice versa to maintain
a negative charge; Ser for Thr so that a free --OH is maintained;
and Gln for Asn to maintain a free --NH2. Likewise, biologically
active analogs of a polypeptide containing deletions or additions
of one or more contiguous or noncontiguous amino acids that do not
eliminate a functional activity of the polypeptide are also
contemplated.
[0070] The fluorescent domain can include a polypeptide with at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 81%, at least 82%, at least
83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% amino acid sequence similarity
to the reference amino acid sequence.
[0071] In certain embodiments, the fluorescent domain can include a
polypeptide with at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% amino acid
sequence identity to the reference amino acid sequence.
[0072] In some embodiments, at least one of the fluorescent domain,
the first antibody domain, or the second antibody can include an
affinity tag. Affinity tags are routinely used to assist with the
isolation and/or collection of recombinant polypeptides. Affinity
tags, their use, and the methods of isolating polypeptides equipped
with an affinity tag are well known to those of skill in the art.
Exemplary affinity tags include, for example, a Histidine-rich tag
(His-tag). A recombinant protein containing a His-tag can be
purified and detected easily because the string of histidine
residues binds to several types of immobilized metal ions such as,
for example, nickel, cobalt or copper, under specific buffer
conditions. In addition, anti-His-tag antibodies are commercially
available for use in assay methods involving His-tagged proteins.
In either case, the tag provides a means of specifically purifying
or detecting the recombinant protein without a protein-specific
antibody or probe. It is also possible to use alternative
conventional tags including, for example, tags that include three
or more amino acids, which bind to known corresponding affinity
acceptors.
[0073] Another feature of the fluorescent fusion polypeptide
involves the isoelectric point (pI). Table 2 summarizes the
predicted pI values for exemplary antibody single chains with the
predicted pI values for corresponding antibody-RFP constructs.
Generally, incorporating the antibody chain into a fluorescent
fusion polypeptide lowers the pI.
TABLE-US-00001 TABLE 2 Molecular Weight Predicted pI B72.3 26.5 kDa
7.32 B72.3-RFP 51.5 kDa 6.37 CA19.9 26.4 kDa 8.30 CA19.9-RFP 51.4
kDa 6.54 H23 26.2 kDa 9.03 H23-RFP 51.2 kDa 7.37 4D5-8 27.0 kDa
8.32 4D5-8-RFP 45.1 kDa 6.19 CA125 26.3 kDa 7.86 CA125-RFP 51.3 kDa
6.43
[0074] A compound can precipitate from solution when in an
environment close to its pI. At physiological pH (approximately pH
7.4), the pI values of many single chain antibodies are close
enough to the environmental pI that they are at risk for
precipitating out of solution. Incorporating the antibody chain
into a fluorescent fusion polypeptide can alter the pI so that the
antibody chain is less likely to precipitate at physiological pH,
thereby broadening the effective utility of the antibody chain. The
V.sub.H and V.sub.L in the context of a whole antibody has a pI
that is compatible with physiological pH. Once the V.sub.H and
V.sub.L are engineered as single chain Fv, the pI can shift
considerably resulting in a pI close to the physiological pH, this
would result in the protein having a net zero charge and lead to
aggregation and precipitation. We observed 4D5-8 scFv with a
(Gly.sub.4Ser).sub.3 linker with a predicted pI of 8.32 to
precipitate at pH 7.4. Whereas the corresponding REDantibody 4D5-8
with a predicted pI of 6.19 was soluble at pH 7.4.
[0075] One further feature of the fluorescent fusion polypeptides
described herein results from the polypeptides being able to be
generated in situ. Because the polypeptides are genetically
encoded, once a polynucleotide encoding the fluorescent fusion
polypeptide is introduced into the cell, the cell will produce the
polypeptide. Thus, intracellular target molecules can be detected
without disrupting the cell to allow antibody access to these
target molecules.
[0076] In some embodiments, the fluorescent domain can include a
photoactivated fluorescent polypeptide. The mRFP1 (SEQ ID NO:4) is
one such fluorescent polypeptide. Photoactivated fluorescent
polypeptides can be regenerated by changing the light source. This
can allow one to obtain repeated signal measurements from a single
fluorescent fusion protein by regenerating the molecules ability to
emit the fluorescent signal.
[0077] In another aspect, the invention provides compositions that
include two or more fluorescent fusion polypeptide described
herein. The fluorescent domain, first antibody domain, and second
antibody domain of the second fluorescent fusion polypeptide can,
independent of the other domains, be similar or different than the
corresponding domain of any other fluorescent fusion polypeptide in
the composition. Thus, a composition may include a mixture of
fluorescent fusion polypeptides that may have similar or identical
antibody domains so that the two different fluorescent fusion
polypeptides bind to the same target molecules, but possess
different fluorescent domains so that the two different fluorescent
fusion polypeptides emit different fluorescent signals. Conversely,
in other embodiments, a composition can include fluorescent fusion
polypeptides that have different antibody domains so that the two
fluorescent fusion polypeptides bind to different target molecules,
but have the same fluorescent domain so that they produce the same
fluorescent signal. Finally, the composition can include two
entirely distinct fluorescent polypeptides so that the two
fluorescent fusion polypeptides in the composition bind to
different target molecules and emit different fluorescent
signals.
[0078] Different fluorescent signals may be characterized in any
suitable manner. For example, fluorescent signals may be
characterized by the color of the signal emitted by the fluorescent
domain. When more precision is desired, the fluorescent signal may
be characterized in terms of the wavelength of one or more
characteristic emission peaks.
[0079] In another aspect, the invention provides polynucleotides
that encode any of the fluorescent fusion polypeptides described
herein, and the complements of such polynucleotide sequences. Also
included in the present invention are polynucleotides that
hybridize, under standard hybridization conditions, to a
polynucleotide that encodes any of the fluorescent fusion
polypeptides described herein, and the complements of such
polynucleotide sequences. Also included in the present invention
are polynucleotides having a sequence identity of at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99% to a nucleotide sequence that encodes any of
the fluorescent fusion polypeptides described herein.
[0080] As used herein, "sequence identity" refers to the identity
between two polynucleotide sequences. Sequence identity is
generally determined by aligning the residues of the two
polynucleotides to optimize the number of identical nucleotides
along the lengths of their sequences; gaps in either or both
sequences are permitted in making the alignment in order to
optimize the number of shared nucleotides, although the nucleotides
in each sequence must nonetheless remain in their proper order. A
candidate sequence is the sequence being compared to a known
sequence. For example, two polynucleotide sequences can be compared
using the Blastn program of the BLAST 2 search algorithm, as
described by Tatiana et al., FEMS Microbiol Lett., 1999; 174:
247-250, and available on the world wide web at
ncbi.nlm.nih.gov/BLAST/. The default values for all BLAST 2 search
parameters may be used, including reward for match=1, penalty for
mismatch=-2, open gap penalty=5, extension gap penalty=2, gap
x_dropoff=50, expect=10, wordsize=11, and filter on.
[0081] In another aspect, the invention provides a method that
includes introducing a polynucleotide as just described into a
cell. In some embodiments, the cell can be a prokaryote or a
eukaryote. Thus, the cell may be a single-celled organism such as,
for example, a bacterium or a yeast cell. In other embodiments, the
cell may be a cell from a multicellular organism. Exemplary cells
from a multicellular organism include, for example, tumor cells.
The method includes introducing the polynucleotide into the cell
using methods routine to those of skill in the art and appropriate
for introducing foreign nucleic acid molecules into the host cell.
For example, routine heat shock transformation methods may be used
to introduce the polynucleotide into, for example, a bacterial
cell. For mammalian cells, the polynucleotide may be introduced
into the cells using, for example, nanoparticle delivery.
[0082] Thus, in yet another aspect, the invention provides a cell
that includes a fluorescent fusion protein as described herein, a
composition as described herein that includes one or more
fluorescent fusion proteins, or a polynucleotide that encodes a
fluorescent fusion protein as described herein. The cells may be in
vitro--as may be the case with a tumor cell obtained through a
biopsy being analyzed--or in vivo--as may be the case in methods,
discussed in more detail below, in which the fluorescent fusion
polypeptide is used to detect a particular cell population in
situ.
[0083] In some embodiments, the method can further include
detecting a fluorescent signal produced by the fluorescent domain
of the fluorescent fusion polypeptide encoded by the polynucleotide
and expressed by the host cell. Depending upon the cell and the
nature of the antibody domains of the fluorescent fusion
polypeptide encoded by the polynucleotide, methods that include
detecting the fluorescent signal can have varying applications. For
example, the polynucleotide may be introduced into a microbe that
is further genetically modified so that, after the genetically
modified microbe is released into an environment, the movement or
spread of the microbe in the environment may be monitored. As such,
the method can include detecting the fluorescent signal over a
plurality of time points so that time course data may be obtained.
As another example, the cell may be a tumor cell. The introduction
of a polynucleotide that encodes a fluorescent fusion polypeptide
may permit detection of tumor cells over time to, for example,
monitor the metastatic activity of the tumor cells. Alternatively,
the polynucleotide may be introduced into tumor cells in advance of
surgery so that a surgeon is better able to distinguish diseased
and healthy tissues. This can help improve the likelihood that all
tumor cells are surgically removed as well as decrease the extent
to which healthy cells or tissues are removed. As yet another
example, the method can include non-invasive imaging; the
fluorescent fusion polypeptide may be detectable non-invasively
through the skin. For example, the fluorescent fusion polypeptide
may be designed to detect markers of inflammation and a
polynucleotide encoding that polypeptide administered to a subject.
If the fluorescent domain emits a signal in the near-IR portion of
the spectrum, the signal may be visible through the skin.
[0084] In some embodiments, the fluorescent fusion polypeptide may
provide therapeutic activity. Fluorescence can induce the
conversion of molecular hydrogen (H.sub.2) and molecular oxygen
(O.sub.2) to hydrogen peroxide:
H.sub.2+O.sub.2.fwdarw.H.sub.2O.sub.2. With sufficient excitation
(e.g., from white light), fluorescence from the fluorescent domain
can produce enough H.sub.2O.sub.2 to kill a cell harboring the
fluorescent fusion polypeptide. This feature could be exploited in
conjunction with, for example, illuminating tumor cells. Expression
of the fluorescent fusion polypeptide may not only help identify
diseased cells, but may also provide some level of
cytotoxicity.
[0085] In another aspect, one or more of the fluorescent fusion
polypeptides may be used in any conventional detection method in
place of an antibody with corresponding antigen recognition
activity. Indeed, use of a fluorescent fusion polypeptide described
herein may eliminate the final labeling step of, for example, a
conventional sandwich assay.
[0086] In yet another aspect the invention provides a kit that
includes, in separate containers, a first fluorescent fusion
polypeptide and a second fluorescent fusion polypeptide. In other
embodiments, the kits can include, in separate containers, a first
polynucleotide that encodes a first fluorescent fusion polypeptide,
and a second polynucleotide that encodes a second fluorescent
fusion polypeptide.
[0087] In another aspect, the invention provides a method of using
the fluorescent fusion polypeptide as a nanoprobe. Although
immunodetection, the mainstay of clinical diagnostic laboratories
has changed formats considerably over the past 50 years, the basic
principles have remained fundamentally the same: i.e.,
antibody-antigen interaction determination, either qualitative
(yes/no), if yes, where (imaging), or quantitative (how much), and
various combinations of these metrics. The scope for the
application of immunoassays is wide, from over-the-counter
pregnancy tests, HIV testing, substance of abuse monitoring,
detecting mycotoxins in food and feed, measuring markers of cardiac
health to screening biopsies for tumor-associated markers. The
ability to readily manipulate antibody gene sequences using simple
bacterial expression systems allowed various strategies to be
developed for accessing and modifying antibodies. One application
of such modified antibodies involves creating recombinant antibody
fragments as fusions with marker molecules. Currently, it is
possible to tag antibodies with different colored fluorescent
chemical tags and to use these reagents in multi-analyte detection.
However, antibodies tagged in this manner can vary from batch to
batch in the amount of fluorescent chemical that is conjugated to
the antibody. That is, the amount of fluorescent chemical tagged to
the antibody is not absolute, some may have more than others and in
some instances the chemical coupling can interfere with the binding
properties of the antibody. Hence, use of these tagged antibodies
yields an `averaged` result, which is acceptable for a qualitative
assay, but is limited in quantitative assays.
[0088] In contrast, the fluorescent fusion polypeptides described
herein include a single fluorophore so that every labeled antibody
carries a single fluorescent molecule to tag antigens with a
particular color. These fluorescent fusion polypeptides make
possible new approaches based on the integration of multiple
readouts in a range of quantitative and/or qualitative settings,
from cell surfaces to antigens in microarrays, etc.
[0089] We have engineered monomeric red fluorescent protein mRFP
from a coral Discosoma (Campbell, R. E., et al., 2002, Proc Natl
Acad Sci USA 99, 7877-7882.) and inserted it between the V.sub.H
and V.sub.L domains of a range of recombinant anti-cancer
antibodies such as, for example, B72.3, CA19.9, and 4D5-8 replacing
the flexible peptide linker. This is, however, a general approach
that may be used to produce fluorescent fusion polypeptides that
include a portion of V.sub.H and V.sub.L domains of any antibody of
interest. Indeed, we have applied this approach successfully to six
different scFvs. Moreover, the mRFP can be readily interchanged
with a range of other fluorescent proteins that have almost
identical external tertiary structure (Tsien, R. Y., 2009, Angew
Chem Int Ed Engl 48, 5612-5626), thus opening up the possibility of
creating palettes of stable recombinant monoclonal antibodies with
defined spectral properties for use in, for example, protein
arrays, live cell imaging, and/or immunocytochemical imaging.
[0090] As noted above, in some applications, the fluorescent fusion
polypeptides may be used for in vivo imaging, using the fluorescent
fusion polypeptides to label, for example, diseased cells and/or
tissues to aid surgery. The fluorescent fusion polypeptides may
provide a navigational map to assist surgeons in identifying both
diseased and healthy tissue on a molecular level and, therefore,
assist in increasing the accuracy and precision of the surgical
procedure to remove diseased tissue while limiting the extent to
which healthy tissue is collaterally removed during surgery.
[0091] In the case of cancer, surgery with molecular fluorescence
imaging using activatable cell-penetrating peptides decreased the
residual cancer and improved survival in a mouse xenograft model
(Nguyen, Q. T., et al., Proc Natl Acad Sci USA 107, 4317-4322).
Thus, fluorescence imaging may provide a dual opportunity to
exploit the fluorescent fusion polypeptides described herein. The
fluorescent fusion polypeptides may be useful for detecting the
presence of tumors using in vitro detection methods on cells or
tissues obtained from a biopsy sample, then used in vivo for
guiding the subsequent surgical removal or ablation of diseased
cells and/or tissues.
[0092] In yet another aspect, fluorescent antibodies may be used in
connection with pest management and/or disease control. Evolving
methods for control of vector-borne diseases rely on modification
of insects rather than elimination of insects by, for example,
application of pesticides. These new strategies involve either
direct transformation of an insect genome via mobile DNA elements
(transgenesis) or expression of gene products in the host insect
via transformed symbiotic microbes (paratransgenesis). Transgenesis
and/or paratransgenesis may supplement conventional insect control
methods and, therefore, decrease the extent to which conventional
methods must be used--i.e., transgenesis and paratransgenesis may
not necessarily completely supplant traditional insecticide
campaigns but can decrease reliance on insecticides and, therefore,
the negative environmental effects of insecticide use.
[0093] Paratransgenesis is a "Trojan Horse" approach to control of
disease transmission. It employs the interactions between
disease-transmitting vectors, bacterial symbionts of the vectors,
and transmitted pathogens. Symbiotic bacteria are isolated and
genetically transformed in vitro to export molecules that interfere
with pathogen transmission. The genetically altered symbionts are
then introduced into the host vector where expression of engineered
molecules affects the host's ability to transmit the pathogen,
i.e., its vector competence. This approach attempts to decrease
pathogen transmission without adverse effects on the vectors
themselves. Furthermore, it can employ, as a gene delivery
mechanism, bacterial flora native to the host vector. There are
several requirements for such an approach to work: (1) A population
of symbiotic bacteria must exist within a given
disease-transmitting vector, (2) Symbiotic bacteria should be
specific to a given vector, (3) Bacterial symbionts should be
amenable to culture and genetic manipulation, (4) Genetically
altered symbionts should remain stable, (5) Fitness of the
genetically altered symbionts to re-infect host vectors should not
be compromised. Furthermore, their normal symbiotic functions
should not be altered, (6) Transgene products released from the
genetically altered symbionts should interact with the target
pathogen(s) and (7) A method must exist for dispersal of the
genetically altered symbionts amongst naturally occurring
populations of vectors with minimal non-target spread of foreign
genes to environmental bacteria and other arthropods.
[0094] So, for example, Pierce's Disease is a deadly disease of
grapevines that causes significant economic loss to the wine
industry of California. It is caused by the bacterium Xylella
fastidiosa, which is spread by xylem-feeding sharpshooters. The
predominant vector of this disease in the U.S. is the Glassy Winged
Sharpshooter (GWSS), Homalodisca vitripennis. Xylella is
xylem-limited in host plants and is not thought to survive outside
the vector insect or host plant. GWSS is a xylem-feeder and carpets
of bacteria on the surface of the precibarial chamber of the
mouthparts of the insect have long been thought to be the principle
source of transmission of pathogenic Xylella strains into
susceptible plants. The anterior mouthparts of H. vitripennis are
colonized with a variety of other environmental bacteria some of
which appear to have a symbiotic association with the insect. Two
such organisms are Alcaligenes xylosoxidans dentrificans (AXD) and
Pantoea agglomerans, both environmental gram-negative bacteria
commonly found in the rhizosphere of grape plants. Both X.
fastidiosa and the two symbionts are located in physical proximity
in the cibarium of GWSS in an extracellular environment.
Fluorescent antibodies as described herein may be engineered into
AXD and/or P. agglomerans so that the fluorescent antibodies are
expressed, target the surface of Xylella, and prevent transmission
of this pathogen by H. vitripennis. The fluorescent antibodies may
provide two related but distinct functions: they can (1) function
as transmission-blocking molecules that disrupt Xylella
pathogenesis in grape plants, and (2) intrinsically fluoresce so
that one can monitor gene flow in the environment as a risk
assessment strategy.
[0095] To develop a panel of anti-X. fastidiosa surface proteins,
five scFv libraries were constructed using spleens from mice
previously immunized with whole, heat-inactivated X. fastidiosa
cells. These libraries were used to select and isolate scFv's
against the surface molecule MopB (4XfMopB1 & 4XfMopB3) of X.
fastidiosa. Antibody scFv's selected against surface components of
X. fastidiosa can be engineered into the fluorescent antibody
format described herein and evaluated for plasmid-based expression
in E. coli and P. agglomerans. Candidates that are expressed and
secreted in P. agglomerans can be used to establish stable bacteria
that can express the antibody using transposon insertion into the
P. agglomerans genome.
[0096] On the outer membrane of Gram-negative bacteria,
macromolecules such as lipopolysaccharides and proteins are
produced. MopB is the major outer membrane protein in X.
fastidiosa. MopB is a member of ompA protein family. Two constructs
of X. fastidiosa MopB (truncated and full length mature) have been
expressed and purified.
[0097] Furthermore, mouse immunoglobulin DNA libraries against
heat-killed whole X. fastidiosa have been constructed and used in
ribosome technology to select specific scFv antibodies against MopB
and these have been characterized to confirm binding to X.
fastidiosa. Using these libraries, one can isolate scFv antibodies
against other surface proteins that may play a critical role in X.
fastidiosa attachment or pathogenicity in the insect or plant.
Since the complete genome of X. fastidiosa is known, using a
bioinformatics approach can identify additional potential X.
fastidiosa surface proteins. Thus, one can clone these candidates
in a similar manner as MopB and use the recombinant proteins to
isolate alternative surface specific scFv's.
[0098] In other approaches, paratransgenesis using a fluorescent
polypeptide as described herein may be used to inhibit the effects
of pathogens affecting other agricultural species, maricultural
species, or livestock species. With the platform established as
just described, analogous corresponding paratransgenesis systems
may be designed. Such systems can permit inhibition of the pathogen
and/or detection of the engineered microbe.
[0099] In another aspect, microbes may be genetically-engineered to
produce and export fluorescent antibodies that interfere with
animal pathogens. For example, E. coli Nissle (EcN) may be used as
a bacterial delivery system to produce and export fluorescent
antibodies that interfere with pathogenesis of, for example,
Clostridium difficile. In this aspect, EcN are used for
paratransgenesis. Resident bacteria such as, for example, EcN are
isolated and genetically transformed in vitro to export molecules
that interfere with pathogen transmission. The genetically altered
bacteria are then introduced into the host where expression of
engineered molecules disrupts virulence of the pathogen such as,
for example, C. difficile. This approach results in a decrease of
deleterious effects of the pathogen by delivering local passive
immunity. Furthermore, it employs, as a gene delivery mechanism,
bacterial flora that can reside in the host. This paratransgenic
platform can be used to produce recombinant EcN that expresses
antibodies that specifically bind C. difficile Toxin A and/or Toxin
B.
Molecular Design
[0100] The X-ray crystallographic structure of Fab B72.3 (PDB:
1BBJ) reveals that the distance between the C-terminus Ser114 on
the variable heavy chain and the N-terminus Asp1 on the variable
light chain is approximately 34 .ANG.. A search of the Protein Data
Base (PDB) was carried out to identify structures with N- and
C-termini in the same spatial plane with spacing close to 20-30
.ANG.. Fortuitously, mRFP met these criteria with the distance
between Val7 and His221 of the termini in fast maturing red
fluorescent protein DsRed variant (PDB 2VAE) being approximately 26
.ANG.. Thus, direct fusion of the V.sub.H to the N-terminus and
V.sub.L to the C-terminus would have resulted in a linkage 8 .ANG.
shorter than the optimal spacing revealed by X-ray crystallographic
structure of B72.3. This was overcome by modeling the N- and
C-termini to include Gly.sub.4Ser linkers on each end of RFP (FIG.
1). The resulting model could accommodate the variable regions of
the antibody with a molecular geometry approaching that determined
from the X-ray studies.
Molecular Biology
[0101] The V.sub.H-RFP-V.sub.L-His-Tag (REDantibody) constructs
were assembled in a modified pET-26b vector (EMD Chemicals Inc.,
Gibbstown, N.J.) with a pelB leader sequence to direct secretion to
the periplasm of E. coli (FIG. 2A) or in a modified pET-32a vector
(EMD Chemicals Inc., Gibbstown, N.J.) without a pelB leader for
cytoplasmic expression. The synthetic genes of the V.sub.H-V.sub.L
scFv antibodies were designed to encode an in-frame BamHI site
between the V.sub.H and V.sub.L regions and the whole flanked by
NcoI and NotI sites for in-frame directional cloning. The mRFP gene
was inserted into each of these plasmids to make expression
plasmids pBAK1B72.3RFP and pBAK1CA19.9RFP as shown in FIG. 2A. The
pBAK14D5RFP was assembled in a similar manner after removing an
internal BamHI site. The expression cassettes starts from pelB
leader sequence followed by V.sub.H chain, RFP, V.sub.L chain and
an octa-His-tag at the C terminus of the resulting protein sequence
as shown in FIG. 2B.
Protein Expression & Purification
[0102] The recombinant scFv and the corresponding REDantibody
proteins were expressed in BL 21 (DE3) pRARE E. coli strain and
recovered from the periplasmic extract or in Rosetta gami B(DE3) E.
coli strain and recovered from the cytoplasmic extract and purified
via Ni-NTA affinity chromatography. Protein expression and
purification processes were monitored by SDS-PAGE. The protein
concentration of the recovered functional protein was determined
for each construct: mRFP (20 mg/L), REDantibody 4D5 (5 mg/L),
REDantibody CA19.9 (0.88 mg/L), and REDantibody 72.3 (0.9 mg/L).
The predicted molecular weights of the scFv and REDantibody
recombinant proteins were approximately 25 kDa and 51 kDa,
respectively, as shown in FIG. 3. The SDS-PAGE analysis of the
Ni-NTA affinity enriched recombinant antibodies also had other
protein bands corresponding to co-enriched E. coli host proteins.
The size exclusion column was calibrated using a standard range 200
kDa-12 kDa (Sigma-Aldrich, St. Louis, Mo.) prior to use (FIG. 4A).
The mRFP protein elution corresponded to monomer of 25 kDa (FIG.
4B). A single REDantibody peak eluted at fractions/time
corresponding to .about.51 kDa, (FIG. 4C). Moreover, the color of
REDantibody E. coli culture, periplasmic, and elution fractions
were pink (FIG. 5 A-C, respectively).
Fluorophor Measurements
[0103] The excitation and emission peaks of REDantibody 4D5-8 were
determined to be 584 nm and 607 nm as shown in FIG. 15A. The
concentration dependent emission at 607 nm permitted accurate
detection of 9.5 pmole of this REDantibody as shown in FIG.
15B.
BIAcore Measurements
[0104] The association and disassociation rate for REDantibody
4D5-8 binding to p185HER2-ECD was determined to be
2.19.times.10.sup.-4.+-.0.13.times.10.sup.-4 M.sup.-1s.sup.-1 and
4.43.times.10.sup.-5.+-.1.04.times.10.sup.-5 s.sup.-1, respectively
and the KD calculated to be 2.2.+-.0.8 nM.
Immunofluorescent Staining and Confocal Microscopy
[0105] Functional analysis of the REDantibody was based on
well-characterized properties of B72.3 and CA19.9 antibodies to
recognize sialyl-Tn and sialylated Lewis (Le).sup.a blood group
antigen, respectively, which are part of a panel of markers used in
cancer diagnostics. The same sialyl-Tn antigen has previously been
detected on the surface of the human pathogen Trypanosoma cruzi
using B72.3 monoclonal antibody.
[0106] The CA19.9 also binds to sialyl glycans on the parasite
surface. The 4D5 REDantibody was constructed for use as a negative
control since it recognizes a peptide epitope on p185HER2, but not
sialyl glycan. This was confirmed by the fluorescent staining of T.
cruzi epimastigotes using purified recombinant anti-glycan
REDantibody shown in FIG. 6A and FIG. 6B. The control REDantibody
4D5 did not label the parasites (FIG. 6C). The maximum intensity
fluorescence staining image with REDantibody CA19.9 (FIG. 6D)
revealed the appearance of a cleft area on the parasite surface
with reduced staining. Confocal three-dimensional imaging (FIG. 6E)
confirmed the presence of a depression corresponding to the cleft
observed earlier. The cut away image of the confocal image revealed
that the staining was restricted to the surface of the parasite
(FIG. 6F).
[0107] Immunofluorescent tagging of molecules, in particular
antibodies, is a widely used tool in clinical diagnostics and
research. Historically, fluorophores exemplified by FITC,
TRITC/CY-3, and TRITC/CY-5 have been chemically linked to
antibodies, resulting in some instances with reduced binding to
target antigen and/or a heterogeneous number of labels incorporated
into each antibody molecule. The heterogeneity of labels
incorporated into each antibody molecule limits the utility of
these molecules for quantitative analysis. Significant advances
have been made in conjugation chemistry to attempt to overcome some
of these difficulties. For example, approaches have been developed
that result in recombinant scFv that incorporate a free cysteine at
the C-terminus for specific conjugation chemistry. Others have gone
one step further and fused scFv directly to GFP. However
conventional scFv are prone to aggregation. It is not clear whether
fusing a scFv directly to GFP would impact the aggregation
properties of the scFv. More recently, GFP has been modified to
incorporate two loops that can form recognition motifs even though
the resulting molecule is not an antibody as such.
[0108] We sought to overcome two issues faced with creating
antibody fluorophores. Firstly, we incorporated a single
fluorophore per binding site. Secondly, we spatially orientated the
V.sub.H/V.sub.L interfaces for optimal pairing. Our earlier
attempts to construct similar bridged molecules using a related
.beta.-barrel structure of green fluorescent protein (GFP) resulted
in molecules that did not bind to the target antigen.
[0109] The vectors constructed encoding the REDantibodies are based
on the pET series as shown in FIG. 2. The use of the T7 promoter
and the BL21(DE3) host results in high level of expression of the
recombinant protein, the majority of which is retained within the
cell cytoplasm despite having a PelB leader sequence to direct the
polypeptide into the periplasm. The amount of REDantibody recovered
varied for each antibody construct. 4D5 had the highest recovery (5
mg/L) whereas the recovery of the corresponding anti-sialyl glycan
antibodies were both just below 1 mg/L. Both levels are comparable
to the recovery of scFv from E. coli. The recovery of anti-sialyl
glycan REDantibodies may have been reduced since the secreted,
correctly folded, and functional REDantibody in both cases bound to
sialic acid, which is present on the host bacterial surface.
[0110] Sufficient REDantibody was recovered from the periplasmic
extract to permit further affinity purification and functional
analysis. The SDS-PAGE of the affinity isolated CA19.9 scFv and the
corresponding REDantibody had proteins of 25 kDa and 51 kDa,
respectively, as shown in FIG. 3A and FIG. 3B, respectively. The
REDantibody also had a doublet band at 26 kDa, which could be due
to cleavage within the mRFP that occurs when the sample is boiled
in SDS as observed with DsRed.
[0111] Size exclusion chromatography on the affinity purified
proteins was used to determine whether the molecules were monomeric
or multimeric. Each of the mRFP and REDantibody eluted with a
single peak, corresponding to 25 kDa and 51 kDa, respectively, as
shown in FIG. 4B and FIG. 4C, respectively. These data indicate
that both proteins are monomeric.
[0112] Features of this REDantibody platform include the intrinsic
color of the bacteria producing the active molecule and the
intrinsic color of the purified proteins. Consequently,
sophisticated detection in the purification steps is not required.
The excitation wavelength of REDantibody is 584 nm and the emission
wavelength is 607 nm, which are identical to mRFP (Campbell et al.,
2002 Proc Natl Acad Sci USA 99:7877-82; Khrameeva et al., 2008
Biochemistry (Mosc) 73:1085-95) confirming that the fusing
immunoglobulin domains to both ends of a fluorescent protein does
not compromise the fluorescent properties. Moreover, it may be
possible to detect as little as 9.5 pmoles of REDantibody using a
standard fluorometer as shown in FIG. 15B. This may permit
quantitative analysis of target antigen. The kinetics of binding
for the anti-glycan REDantibodies were difficult to perform since a
defined antigen preparation was not readily available. Thus we
carried out the binding studies on the REDantibody 4D5-8. The
calculated K.sub.D value for REDantibody 4D5-8 of 2.19 nM was
within the values determined independently for the 4D5-8 scFv with
a V.sub.H-(Gly4 Ser)3 linker V.sub.L orientation of 194 pM (Worn
and Pluckthun, 1998 FEBS Lett 427:357-61), and 9.4 nM for HERCEPTIN
(Genentech, Inc., South San Francisco, Calif.) (Troise et al., 2008
FEBS J 275:4967-79), further confirming that the binding
characteristics are not compromised in this format.
[0113] These characteristics of the REDantibody permit its use with
other conventional dyes for FACs analysis, immunochemistry, and
confocal microscopy that utilize a range up to 543 nm. In this
study we used the REDantibody B72.3 and CA19.9 to visualize the
carbohydrate antigens sialylTn and sialylated Lewis (Le)a on the
surface of T. cruzi epimastigotes, respectively, using
immunofluorescence (FIG. 6A, FIG. 6B and FIG. 6D) and confocal
(FIG. 6E and FIG. 6F) microscopy. The binding of the REDantibody
CA19.9 is restricted to the surface of the parasite (FIG. 6B). The
negative control REDantibody 4D5-8 did not label the parasites when
examined using immunofluorescence microscopy, implying the
specificity of binding observed using the B72.3 and CA19.9
antibodies was introduced by the antibody binding domains (FIG.
6C).
[0114] One feature of the REDantibodies described herein include
robust stability; the slides prepared for this study could be
viewed for at least a week without noticeable loss of signal,
indicating robust stability of the REDantibody. Another feature is
that the monomeric red fluorescent protein (mRFP1) derived from
DsRed is stable within a wide range of pH 5.0-12.0 and in 6M urea.
Thus, the antibody/antigen interaction may be disrupted by
adjusting the pH and/or adding urea, and the released fluorophore
may be quantified in solution. Yet another feature is that the
REDantibody molecule is photo activated. Thus, the REDantibodies
described herein permit detecting a target with increased
sensitivity through the use of, for example, time-resolved dual
laser pulsed analysis. Yet another feature is that the REDantibody
can generate reactive oxygen species (e.g., H.sub.2O.sub.2) upon
exposure to white light. This may potentially provide a wide range
of therapeutic applications such as, for example, photo-ablation of
target cells. Additionally, with a far red emission spectra, these
types of molecule may have utility in in vivo whole body
imaging.
[0115] Assembling the REDantibody using the fluorophore as a bridge
introduces certain features. Firstly, with an integrated
fluorophore, a single reagent has been created, which reduces time
and cost and increases reproducibility of the binding assay.
Secondly, the antibody binding site and fluorophore are
stoichiometric. Thus, the signal generated is directly proportional
to the amount of antibody bound (i.e., quantitative). Finally,
expression and purification steps can be monitored without the need
for the expensive equipment since the bacteria and the recombinant
proteins are visible with a distinct red color.
[0116] The platform may be applied using other suitable fluorescent
protein domains. Thus, the fluorescent domain can include at least
a portion of mRFP, mCitrine, mCerulean, GFP(wt), EBFP, Sapphire,
T-Sapphire, ECFP, mCFP, CyPet, Midori-Ishi Cyan, mTFP1, EGFP,
AcGFP, TurboGFP, Emerald, Azami Green, EYFP, Topaz, Venus, yPet,
PhiYFP, mBanana, Kusabira Orange, mOrange, dTomato, DsRed-Monomer,
mTangerine, mStrawberry, Jred, mCherry, HcRed1, mRasberry, or
mPlum. Thus, the preceding description of the construction of the
REDantibody is merely exemplary; analogous constructs may be
designed using any suitable fluorescent protein domain.
[0117] Moreover, this platform may be applied to other existing
mAbs to create the next generation of diagnostic and therapeutic
molecules. The platform may also be used to create libraries of
V.sub.H and V.sub.L domains that may be readily accessed using high
throughput non-isotopic screening and not rely on phage or other
types of display selection technologies. In general, the use of
other pre-folded protein domains with N- and C-termini in the same
spatial plane with spacing close to 20-30 .ANG. may be used in this
modular approach to antibody engineering. Furthermore, the
monomeric fluorophore also may be engineered to alter the
excitation/emission spectra, thus enabling multicolored
antibody-based reagents for multi-analyte detection or
co-localization studies.
[0118] We engineered a scFv 4D5-8 fusion with mRFP1 based on the
X-ray crystal structure of 4D5-8 Fab (PDB 1N8Z; Cho et al., 2003
Nature 421(6924):756-760) and the modeled structure of mRFP1 based
on the DsRed dimer (PDB 1G7K; Yarbrough et al., 2001 Proc Natl Acad
Sci USA 98(2):462-467). We fused the C-terminus of 4D5-8 V.sub.H
chain to the N-terminus of mRFP1 and fused the 4D5-8 N-terminus of
V.sub.L to the C-terminus of mRFP1, with the addition of optimal
linkers separating both protein functions yet preserving the
correct spatial dimensions for V.sub.H/V.sub.L interaction and
fluorophore activity (FIG. 8A). With the addition of five amino
acid (Gly4Ser) linkers to either end of the mRFP1, the N-terminus,
C-terminus, or both can readily be extended to create a distance
of, for example, approximately 35{acute over (.ANG.)} between the
mRFP1 domain of the fusion and the V.sub.H/V.sub.L chains. The
predicted structure shows that the mRFP1 does not interfere with
the scFv binding site, which is fully available to contact the
target ligand. This spatial geometry permits the docking of mRFP1,
or any of its derivatives, between the antibody V.sub.H/V.sub.L or
V.sub.L/V.sub.H chains resulting in functional scFv-fluorophore
fusions.
Construction of the Expression p4D5-8mRFP1 Vector.
[0119] Construction of an exemplary vector expressing an exemplary
scFv-fluorophore fusion is shown in FIG. 8B. A 714 bp NcoI/NotI
fragment encoding the 4D5-8 scFv V.sub.H-V.sub.L orientation with a
five amino acid (Gly4Ser) linker incorporating an in-frame BamHI
(GGA TCC) restriction site (encoding amino acids Gly Ser) was
inserted into a modified pET-26b vector. Modified mRFP1 with
similar (Gly4Ser) linkers on both ends was cloned into the scFv
BamHI site.
Protein Expression and Purification.
[0120] The 52 kDa protein (4D5-8 V.sub.H-RFP-V.sub.L) was expressed
in E. coli BL21 (DE3) pRARE or Rosetta gami B(DE3). During the
growth and expression phase the bacteria had a distinct red
pigmentation. The protein was initially enriched by immobilized
metal ion affinity chromatography (IMAC) and fractions analyzed by
SDS-PAGE (FIG. 9a) and western blot (FIG. 9b), and then analyzed by
gel filtration chromatography compared with mRFP1 and standards
confirming the basically monomeric state (FIG. 9c). The fractions
with the red pigment corresponded to fractions 32-37. The purified
monomeric protein was further analyzed by SDS-PAGE (FIG. 9d).
Western blot analysis of the eluted fractions (FIG. 9b), revealed a
band above the expected size, and smaller products. The higher band
probably corresponds to the protein with the PelB leader intact,
recovered from the cell lysis and the lower bands are the breakdown
products due heat induced proteolysis of mRFP1 at the chromophore
site which has also been reported by others (Serebrovskaya et al.,
2009 Proc Natl Acad Sci USA 106(23):9221-9225). The proteolysis
occurs upon sample preparation for SDS-PAGE. A final yield of the
4D5-8 REDantibody was 5 mg/L of bacterial culture.
Flow Cytometry and Fluorescence Microscopy Analysis.
[0121] To further characterize the binding activity of 4D5-8
REDantibody we used flow cytometry and fluorescence microscopy. As
shown in FIG. 10, purified red antibody effectively recognized
SKBr-3 breast carcinoma cells that are characterized by high
expression levels of p185HER-2-ECD (FIG. 10a) and is used as +++
positive control in the FDA approved HercepTest (Dako North
America, Inc., Carpinteria, Calif.) and did not recognize the
MDA-MD-231 cancer cells (FIG. 10b) which do not express
p185HER-2-ECD and are used as a zero negative control in the same
test (Prichard et al., 2008 Clin Lab Med 28(2):207-222, vi).
Immunofluorescent staining of SKBr-3 breast cancer cells revealed
that purified 4D5-8 REDantibody effectively accumulates on the
surface of these cells after a 30-minute incubation at ambient
temperature as shown in FIG. 10c and FIG. 10d.
[0122] Few genetically encoded fluorescent antibodies have been
reported to date (Griep et al., 1999 J Immunol Methods
230(1-2):121-130; Lu et al., 2005 J Zhejiang Univ Sci B
6(8):832-837; Serebrovskaya et al., 2009 Proc Natl Acad Sci USA
106(23):9221-9225), in part because it has been proven difficult to
maintain native affinity activity of antibody domains and maintain
native fluorophore activity of the fluorophore domain. Efforts have
included omitting the antibody component and creating a GFP-based
biosensor possessing antibody-like properties using the GFP as a
scaffold and introducing two proximal binding loops (Pavoor et al.,
2009 Proc Natl Acad Sci USA 106(29):11895-11900). Conventional
antibodies, however, have six proximal binding loops that, combined
with subtle variations in the framework regions, can provide a much
greater range of possible affinities and specificities. On the
other hand, the rigid .beta.-barrel structure of fluorescent
proteins impose limits on the number of changes that can be
accommodated without altering the spectral properties (Abedi et
al., 1998 Nucleic Acids Res 26(2):623-630).
[0123] Ideally, a fluorescent antibody can possess as much native
ligand-binding activity as possible from its antibody domains and
as much of the native fluorophore spectral properties as possible
from its fluorophore domain.
[0124] One way to retain as much ligand-binding activity and as
much fluorophore activity as possible can involve a modular
approach where the two functionalities reside in distinct,
non-overlapping regions of a single molecule. However, recombinant
scFv with linkers can be prone to disassociation and aggregation
(Worn and Pluckthun, 2001 J Mol Biol 305(5):989-1010). Yet, in the
context of a Fab molecule, the V.sub.H/V.sub.L remain associated.
Nature provides two solutions for stabilizing V.sub.H/V.sub.L
pairs. In conventional antibodies, the CH1 and the constant light
chain orient and hold the V.sub.H and the V.sub.L in place for
optimal interface pairing. In the other case, the V.sub.L is not
required and only the V.sub.H is required for ligand binding, as is
the case in camel antibodies (Hamers-Casterman et al., 1993 Nature
363(6428):446-448). Although making direct fluorophore fusions with
engineered V.sub.H domains is possible, the vast majority of
characterized monoclonal antibodies consist of both V.sub.H and
V.sub.L domains. Thus, an approach that could conserve the Fab-like
V.sub.H/V.sub.L pairing and permit fusion with a fluorescent
protein can produce a fluorescent antibody that possesses reliable
spectral properties and high affinity and specificity
characteristics.
[0125] We have devised a novel modular approach that produces
efficient generation of a stable genetically-encoded intrinsically
labeled fluorescent antibody. We have inserted the .beta.-barrel
structure of the fluorophore between the variable domains. These
modular constructs not only introduce the fluorophore into the
fusion molecule, but also stabilizes the positional orientation of
the variable domain interfaces, resulting in Fab-like
V.sub.H/V.sub.L pairing and, therefore, Fab-like ligand
binding.
[0126] In the embodiment described in Example 1, the V.sub.H
C-terminus is fused to the N-terminus of mRFP1, replacing the CH1
domain. The C-terminus of the mRFP1 is fused to the N-terminus of
the V.sub.L, providing an alternative anchor for the V.sub.L and
thus eliminating a need for the CL. The exemplary 4D5-8 REDantibody
described in Example 1 has the following characteristics: it is
readily expressed and isolated from E. coli, with a molecular
weight similar to the Fab fragment; the fluorophore activity
remains unaltered; it is monomeric; it is stable; it retains the
specificity of the parental antibody; and it is simple to use as a
single reagent in immunohistochemistry, flow cytometry, and/or
molecular imaging.
[0127] The 4D5-8 REDantibody molecule may be used, for example, for
the development of a diagnostic test for Her2-positive breast
cancer in biopsy samples by immunohistochemistry or possibly for
circulating cancer cells by FACs. Using this platform it should be
possible to create panels of REDantibodies against a range of
cancer and other biomarkers. Moreover, since the palette of
monomeric fluorescent proteins (mHoneydew to mPlum) are based on
the basic architecture of mRFP1 used in this study, it should be
possible to exchange the red fluorophore for these other colored
proteins that have similar 11 .beta.-sheet barrel-like structure
(Zimmer, 2002 Chem Rev 102(3):759-781). The combination of
different antibody specificities each with a distinct color
spectrum opens up the possibility of antibody mediated multiplexed
analysis by fluorescence microscopy and FACs analysis. Exemplary
modular fluorescent antibodies that produce colors other than the
red color produces by the 4D5-8 REDantibody are described in
Example 3.
[0128] Recently, p185HER-2-ECD expressing cancer cells have been
targeted using 4D5 scFv fused to the N-termini of KillerRed
(dimeric) reporter (Serebrovskaya et al., 2009 Proc Natl Acad Sci
USA 106(23):9221-9225). Upon light irradiation in oxygen rich
environment the KillerRed produces highly reactive oxygen species
(ROS) which oxidise molecules in close proximity resulting in
p185HER-2-ECD expressing cancer cell damage and death
(Serebrovskaya et al., 2009 Proc Natl Acad Sci USA
106(23):9221-9225). These properties have also been assigned to
other fluorescent proteins and it has been experimentally confirmed
that during formation of the chromophore one molecule of H2O2 is
generated for each molecule of fluorescent protein (Zhang et al.,
2006 J Am Chem Soc 128(14):4766-4772). Therefore the 4D5-8mRFP1
described could also generates ROS upon exposure to ambient light,
thus it may also have therapeutic potential in targeted cell
ablation and photodynamic therapy. Another advantage of mRFP1 is
its far-red spectrum which has a greater tissue penetration
permitting use for in vivo imaging (Yurchenko et al., 2007
Transgenic Res 16(1):29-40). Antibody targeted mRFP1 may also be
evaluated for use in vivo in diagnostic and/or therapeutic
applications. This dual modular assembly permits improvements to be
incorporated on the scFv and the fluorophore, resulting in
opportunities for further combinatorial optimised binding and more
desirable spectral properties. Similar REDantibody constructs based
on the anti-sialyl-Tn and anti-sialylated Lewis (Le)a antibody
B72.3 (Brady et al., 1991 J Mol Biol 219(4):603-604) and CA19.9
(Koprowski et al., 1979 Somatic Cell Genet 5(6):957-971)
respectively have also been assembled.
[0129] Assembling the REDantibody using the genetically encoded
fluorophore as a bridge results in a stabilized, stoichiometric
molecule with each binding site having a single reporter molecule,
allowing quantitative analysis. This platform may also be applied
to other existing mAbs to create the next generation of diagnostic
imaging and sorting molecules. The modular platform may also be
used to combine natural and synthetic immunoglobulin V.sub.H and
V.sub.L domains with libraries of fluorophores based on mRFP1 and
GFP to create a palette of highly specific colored binding
molecules that retain optimal binding and spectral properties.
[0130] For any method disclosed herein that includes discrete
steps, the steps may be conducted in any feasible order. And, as
appropriate, any combination of two or more steps may be conducted
simultaneously.
[0131] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
EXAMPLES
Example 1
Materials and Methods
Molecular Design and Visualization
[0132] Structure of B72.3 and 4D5 antibodies were downloaded from
PDB database (PBD: 1BBJ and 1FVC respectively). RFP structure was
predicted using Swiss-Model Workspace server. Further modeling was
performed using MIFit+ software version 2009.09-1 (Rigaku Americas
Corp., The Woodlands, Tex.) and protein models were viewed using
PyMOL software version 1.1 (DeLano Scientific LLC, Palo Alto,
Calif.).
Plasmids, Primers and Synthetic DNA
[0133] Plasmid pBAK1 and pA were previously constructed in our
laboratory is based on pET-26b and pET-32a vectors, respectively
(EMD Chemicals Inc., Gibbstown, N.J.). All primers were purchased
from Invitrogen Corp. (Carlsbad, Calif.). Synthetic DNA sequences
of B72.3 and CA19.9 antibody variable domains in V.sub.H-V.sub.L
orientation were codon optimized for E. coli expression and
purchased from Epoch Biolabs, Inc. (Missouri City, Tex.) as
plasmids pBSK-B72.3 and pBSK-CA19.9 respectively, with NcoI, BamHI
and NotI restriction sites to facilitate construction of the
expression vectors. Plasmid pAK19 encoding the 4D5 V.sub.H and
V.sub.L was provided by Dan Yansura (Genentech, Inc., South San
Francisco, Calif.). Plasmids pMTRFP and pMTBFP, with mRFP1 and BFP
genes, respectively, were a gift from Professor Ray St. Ledger
(University of Maryland, College Park, Md.). Synthetic DNA
sequences of mCitrine and Cerulean were codon optimized for E. coli
expression and purchased from Epoch Biolabs, Inc. (Missouri City,
Tex.) as plasmids pBSK-mCit and pBSK-Cer respectively, with
flanking BamHI restriction sites to facilitate insertion into the
expression vectors.
Bacterial Strains, Growth Media and Recombinant DNA Technique
[0134] XL1-Blue Escherichia coli strain (Stratagene, Agilent
Technologies, Inc., Santa Clara, Calif.) was used for plasmid
construction steps. To express recombinant antibodies BL21 (DE3) or
Rosetta gami B(DE3) strain of E. coli (EMD Chemicals Inc.,
Gibbstown, N.J.) were used. E. coli cells were grown in Lysogeny
Broth (LB) (Bertani, 2004) or LB agar plates. Kanamycin sulfate,
carbenicillin, and chloramphenicol were used at 30 .mu.g/mL, 100
.mu.g/mL, and 34 .mu.g/mL final concentration, respectively.
Plasmid DNA was isolated using QIAprep Spin Miniprep Kit (Qiagen
Inc.--USA, Valencia, Calif.) and DNA from gel was purified using
QIAquick Gel Extraction Kit (Qiagen Inc.--USA, Valencia, Calif.).
Escherichia coli cells were transformed using standard heat shock
methods. Restriction and modification enzymes were purchased from
New England Biolabs, Inc. (Ipswich, Mass.). Final plasmid
constructs were confirmed by DNA sequence analysis.
Construction of the Expression Plasmids
[0135] Antibody scFv encoding fragments were either digested
directly from pBSK-B72.3, pBSK-CA19.9 or assembled from V.sub.H and
V.sub.L domains encoded by pASK19 plasmids respectively and
inserted into NcoI, and NotI restriction sites of previously
digested plasmid pBAK1 to make pBAK1B72.3, pBAK1CA19.9 and pBAK14D5
respectively. The competent E. coli XL1 Blue cells were transformed
using ligation mixtures and the clones were selected on the LB
plates containing kanamycin. Positive clones were confirmed by DNA
sequencing. To make RFP chimeras in V.sub.H-RFP-V.sub.L
orientation, plasmids pBAK1B72.3, pBAK1CA19.9 and pBAK14D5 were
digested with BamHI restriction enzyme and PCR product of mRFP1
gene obtained using pMT-RFP plasmid template and oligonucleotide
primers RFPBamF and RFPBamR (Table 1), inserted to produce
pBAK1B72.3RFP, pBAK1CA19.9RFP and pBAK14D5RFP respectively.
Colonies were initially screened by colony PCR using primers T7F
and RFPBamR (Table 1) and selected clones confirmed by plasmid DNA
sequencing.
[0136] Antibody scFv encoding fragments of 41)5-8 were digested
from plasmid pBAK14D5 and inserted into pA plasmid to make pA4D5.
The competent E. coli XL1 Blue cells were transformed using
ligation mixtures and the clones were selected on the LB plates
containing carbenicillin. Positive clones were confirmed by DNA
sequencing. To make RFP, Citrine, and Cerulean chimeras in
V.sub.H-FP-V.sub.L orientation, this plasmid was digested with
BamHI and the mRFP1 gene obtained using pMT-RFP plasmid template
and oligonucleotide primers RFPBamF and RFPBamR (Table 1), was
inserted to produce pA4D5RFP. The BamHI fragment encoding the mRFP
was replaced by mCit and mCer to generate pA4D5mCit and pA4D5mCer
respectively. Colonies were initially screened by colony PCR using
primers T7F and RFPBamR (Table 1) or CITBamR selected clones
confirmed by plasmid DNA sequencing.
Antibody Expression in E. coli Periplasm.
[0137] To express scFv and REDantibody chimeras, E. coli BL21 (DE3)
pRARE (Phage-resistant derivative of BL21(DE3) (EMD Chemicals Inc.,
Gibbstown, N.J.) cells were transformed with the appropriate
plasmid and plated onto LB agar supplemented with kanamycin sulfate
(30 .mu.g/mL final concentration) and chloramphenicol (34 .mu.g/mL
final concentration). The cells were allowed to grow at 37.degree.
C. for 18 hours and the following day, five fresh colonies were
inoculated into 10 mL of LB media (with antibiotics) and grown at
37.degree. C. (with shaking at 250 rpm) for 16 hours. Next day, 200
mL of pre-warmed LB media, prepared in 1 L conical flasks (with
antibiotics) were inoculated with 10 mL of the overnight culture
and grown at 37.degree. C. (with shaking at 250 rpm) until the
optical density at 600 nm had reached 0.5, then the cells were
placed on ice for 30 minutes and Isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) added (final concentration
0.3 mM) to the cultures and the cells were grown at 20.degree. C.
for an additional 20 hours with shaking at 250 rpm. Bacterial cells
were pelleted by centrifugation for 20 minutes, 5,000 rpm at
4.degree. C. (using Sorvall SuperT 21 bench top centrifuge, with
SL-250T rotor) the supernatant discarded and the pellets retained
for periplasmic protein extraction.
Antibody Expression in E. coli Cytoplasm.
[0138] To express scFv and REDantibody chimeras, in the cytoplasm
of E. coli Rosetta gami (DE3) (EMD Chemicals Inc., Gibbstown, N.J.)
cells were transformed with the appropriate plasmid and plated onto
LB agar supplemented with carbenicillin and chloramphenicol (100
.mu.g/mL and 34 .mu.g/mL final concentration respectively). The
cells were allowed to grow at 37.degree. C. for 18 hours and the
following day, five fresh colonies were inoculated into 10 mL of LB
media (with antibiotics) and grown at 37.degree. C. (with shaking
at 250 rpm) for 16 hours. Next day, 200 mL of pre-warmed LB media,
prepared in 1 L conical flasks (with antibiotics) were inoculated
with 10 mL of the overnight culture and grown at 37.degree. C.
(with shaking at 250 rpm) until the optical density at 600 nm had
reached 0.5, then the cells were placed on ice for 30 minutes and
Isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) added (final
concentration 1 mM) to the cultures and the cells were grown at
20.degree. C. for an additional 20 hours with shaking at 250 rpm.
Fifty mL aliquots of bacterial cultures were pelleted by
centrifugation for 20 minutes, 5,000 rpm at 4.degree. C. (using
Sorvall SuperT 21 bench top centrifuge, with SL-250T rotor) the
supernatant discarded and the pellets retained for cytoplasmic
protein extraction.
Antibody Purification from E. coli Periplasm.
[0139] Bacterial cell pellets from 200 mL culture were resuspended
in 10 mL of periplasmic buffer (30 mM Tris-base, pH 8.0, 20%
sucrose and 1 mM EDTA) supplemented to a final concentration of 0.1
mM phenylmethylsulfonyl fluoride (PMSF). The cells were incubated
on ice for 10 minutes and centrifuged at 9000 rpm for 10 minutes at
4.degree. C. (using Sorvall SuperT 21 bench top centrifuge, with
SL-50T rotor). The supernatants were collected and stored on ice,
whilst cell pellets were resuspended in 7 mL of 5 mM MgCl2
(4.degree. C.). After incubation for five minutes on ice, bacterial
cells were pelleted by centrifugation as described before, and the
supernatants combined to give the periplasmic fraction.
Antibody Purification from E. coli Cytoplasm.
[0140] Bacterial cell pellets from 50 mL culture were resuspended
in 3 mL of periplasmic buffer (20 mM Tris-base, pH 8.0, 0.5 M NaCl,
10 mM imidazole 0.1% Triton-X-100) supplemented to a final
concentration of 0.1 mM phenylmethylsulfonyl fluoride (PMSF),
lysozyme 0.2 mg/mL and sonicated 6.times.30 second bursts. The
cells were incubated on ice for 10 minutes and centrifuged at 13000
rpm for 10 minutes at 4.degree. C. (using Biofuge). The
supernatants were collected to give the cytoplasmic fraction.
Ni-NTA Purification of Recombinant Proteins.
[0141] Recombinant proteins were purified using a 1 mL HisTrap HP
column (GE Healthcare) fitted to an AKTAprime.TM. plus (GE
Healthcare, Piscataway, N.J.) liquid chromatography system. First,
the 1 mL column was equilibrated with five column volumes of
equilibration buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM
imidazole). Following this, 17 mL E. coli periplasmic fraction
(diluted two times with 20 mM Tris-HCl, pH 8.0, 500 mM NaCl buffer)
or 3 mL of the cytoplasmic extract was loaded onto the column and
the column was washed with five column volumes of wash buffer (20
mM Tris-HCl, pH 8.0, 500 Mm NaCl, 20 mM imidazole). The protein was
eluted with five column volumes of elution buffer (20 mM Tris-HCl,
pH 8.0, 500 mM NaCl, 500 mM imidazole) and collected in 1 mL
aliquots. Protein elution was monitored at 280 nm. Eluted fractions
1-5 were analysed by SDS-PAGE.
Desalting and Anion Exchange Chromatography
[0142] Recombinant antibody obtained by Ni-NTA purification were
desalted using 40 mL Sephadex G25 column (GE Healthcare,
Piscataway, N.J.) fitted to an AKTAprime.TM. plus (GE Healthcare,
Piscataway, N.J.) liquid chromatography system in 20 mM Tris-HCl,
pH 8.0 buffer and further purified using 1 mL HiTrap Q Sepharose FF
column (GE Healthcare, Piscataway, N.J.) and eluted by increasing
buffer salt concentration. First, a 1 mL column was equilibrated
with five column volumes of equilibration buffer (20 mM Tris-HCl,
pH 8.0). Following this, 5 mL of desalted antibody fraction was
loaded onto the column and protein was eluted by increasing
concentration of NaCl in 20 mM Tris-HCl, pH 8.0 buffer and
collected into 1 mL fractions. Eluted antibodies were concentrated
and the buffer exchanged to 20 mM Tris-HCl, pH 8.0 and 150 mM NaCl
using Ultracel YM-10 Amicon centrifugal devices from Millipore.
Protein concentration was determined using Bradford assay kit.
(Bio-Rad Laboratories, Hercules, Calif.) (Bradford, 1976 Anal
Biochem 72:248-54).
Size Exclusion Chromatography SEC was performed on an AKTA Prime
Plus using a HiLoad 16/60 Superdex 200 size-exclusion column (GE
Healthcare, Amersham, UK) equilibrated with degassed phosphate
buffered saline (PBS). The purified proteins were separated by
loading 1 mL of the sample at a concentration of 100-200 .mu.g/mL.
The flow rate was 1 mL/min, and the absorbance of the eluted
protein was monitored at 280 nm. The column was calibrated with the
following protein standards: .beta.-amylase (200 kDa), bovine serum
albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c
(12.4 kDa).
SDS-PAGE
[0143] The fractions eluted from the HisTrap column (1-5) were
analysed by SDS-PAGE using 12% Tris-Glycine gels. Proteins were
stained with Coomassie Blue R250 as follows: after electrophoresis
gel was submerged in plastic container into 50 mL of 0.025%
Coomassie Blue in 10% acetic acid solution and heated in the
microwave until boiling (approximately 1 minute), cooled down for
two minutes on bench and destained in 50 mL of 10% acetic acid by
repeating previous procedure of heating in microwave and cooling
down. Finally, the gel was kept in 10% acetic acid before
scanning.
Optical Properties Determination
[0144] The fluorescent measurements were carried out using
SpectraMAX Gemini EM fluorescence microplate reader with Gemini EM
software (Molecular Devices, Inc., Sunnyvale, Calif.) using
Optiplex 96F microtitre plates (PerkinElmer, Inc., Waltham Mass.)
with mRFP, mCIT, CER, and BFP 4D5-8 antibodies, 50 .mu.g in 0.1
mL/well in phosphate buffered saline (137 mM NaCl, 2.5 mM KCl, 10
mM Na.sub.2HPO.sub.4 and 1.8 mM KH.sub.2PO.sub.4) pH7.4. Excitation
and emission spectra were determined at 1 nm intervals and the
arbitrary fluorescence units (AFU) recorded. The concentration
dependant fluorescence with excitation 584 nm and emission 607 nm
was determined for ten-fold serial dilutions of REDantibody 4D5-8
from a stock solution of 95 nmoles.
Surface Plasmon Resonance (SPR) Measurements
[0145] The SPR measurements were carried out on the BIAcore 3000
following the procedure previously described to determine the
binding of 4D5-8 scFv with p185HER2-ECD (Worn and Pluckthun, 1998
FEBS Lett 427:357-61). The p185HER2-ECD antigen (Sino Biological
Inc; Beijing, China) 100 .mu.g/mL in 20 mM citrate buffer pH 4.0
was coupled to the CM-5 research grade sensor chip using an amine
coupling kit (GE Healthcare, Piscataway, N.J.). The 4D5-8
REDantibody was applied at 50 .mu.g/mL, 100 .mu.g/mL, 175 .mu.g/mL,
250 .mu.g/mL, and 500 .mu.g/mL to the chip at a flow rate of 20
.mu.L/min at 20.degree. C. The surface was regenerated by injection
of 45 .mu.L of 0.1 M glycine-HCL, pH 2.2, 0.5 M NaCl. Data were
analyzed using the global fit in the BIAevaluation program version
4.1
Immunofluorescent Confocal Microscopy
[0146] Wild-type T. cruzi epimastigote four-days-old cultures were
fixed with 2% paraformaldehyde in phosphate buffer and allowed to
adhere onto glass slides. Adhered cells were washed with PBS and
non-specific antigen-binding sites were blocked with 2% BSA in PBS,
pH 8.0, for 1 hour at room temperature. Samples were incubated for
60 minutes at room temperature with recombinant REDantibodies.
REDantibody 4D5 was used as a negative control. Slides were mounted
in Npropylgalate (Sigma P130, Sigma-Aldrich, St. Louis, Mo.) to
reduce photobleaching and observed on a Leica SP2 Confocal Laser
Scanning Microscope, using 543-nm He--Ne laser. All images were
analysed using IMARIS software version 6.3 (Bitplane). Formaldehyde
fixed Trypanosoma cruzi epimastigotes TC1 strain were a gift from
Professor Michael Miles and Michael Lewis.
Example 2
Monomeric Recombinant Fluorescent Antibody Platform for Cell
Sorting and Molecular Imaging
Methods
Molecular Modelling.
[0147] X-ray structures of human Her2 with HERCEPTIN (Genentech,
Inc., South San Francisco, Calif.) antibody (PDB 1N8Z) and DsRed
(PDB 1GyK) were downloaded from RCSB Protein data bank. A mRFP1
model was generated by homology-modeling using server Swiss-Model.
MIFit program (Rigaku Americas Corp., The Woodlands, Tex.) was used
to obtain a molecular 3D model of human Her2 with HERCEPTIN
(Genentech, Inc., South San Francisco, Calif.) Fv domains and
mRFP1. The 3D model of the proposed refined structure 4D5-8mRFP1
was generated using PyMOL software.
Construction of the Expression p4D5-8mRFP1 Vector.
[0148] Genetic engineering manipulations, plasmid preparation, cell
culture, protein expression and cell lysis followed the standard
protocols.
[0149] Primers are shown in Table 3.
TABLE-US-00002 TABLE 3 SEQ ID Primer Sequence NO: 4D5BAMF
5'-GTCCCTTCTCGCTTCTCTGGGTCCAGA SEQ ID TCTGGGACGGATTTCAC-3' NO: 9
4D5BAMR 5'-GTGAAATCCGTCCCAGATCTGGACCCA SEQ ID GAGAAGCGAGAAGGGAC-3'
NO: 10 PAKVLBAM 5'-CAGCGGCGGAGGCGGATCCGATATCCA SEQ ID
GATGACCCAGTC-3' NO: 11 PAKVLNOT 5'-TCGAGTGCGGCCGCATCCGCGCGTTTG SEQ
ID ATCTCCACCTTGGTAC-3' NO: 12 PAKVHBAM
5'-TCGGATCCGCCTCCGCCCGAGGAGACG SEQ ID GTGACCAGGGTTC-3' NO: 13
PAKVHNCO 5'-TAGGCCATGGCCGAGGTTCAGCTGGTG SEQ ID GAG-3' NO: 14
RFPBamF 5'-CAGTGGATCCGAGGACGTCATCAAGGA SEQ ID GTTC-3' NO: 15
RFPBamR 5'-CAGTGGATCCGCCTCCGCCTGTGCGGC SEQ ID CCTCGGCGCGCTCGTAC-3'
NO: 16 CITBamR CAGTGGATCCGCCGCCGCCGGTAATGCCCG SEQ ID CCGCGGTCAC NO:
17
All primers were purchased from Invitrogen Corp. (Carlsbad,
Calif.). Plasmids pAK19 was provided by Dan Yansura (Genentech,
Inc., South San Francisco, Calif.), pMT-RFP and pMT-BFP were
provided by Ray St. Ledger (University of Maryland, College Park,
Md.). Restriction nuclease NcoI, NotI and BamHI, T4 DNA ligase, CIP
were purchased from New England Biolabs, Inc. (Ipswich, Mass.). The
mRFP1 was amplified by PCR from pMT-RFP plasmid using primers
RFPBamF and RFPBamR. The V.sub.H and V.sub.L domains of the
humanized humAb 4D5-8 antibody were amplified by PCR from the
plasmid pAK19 using PAKVHNCO/PAKVHBAM and PAKVLBAM/PAKVLNOT
respectively. To obtain the scFv of the humanized 4D5-8 antibody
and simultaneously introduce BamHI restriction site between V.sub.H
and V.sub.L chains, PCR amplified V.sub.H and V.sub.L chains were
joined by splice-overlapping PCR using PAKVHNCO and PAKVLNOT.
Internal BamHI site present in the original V.sub.L chain of
humAb4D5-8 was eliminated by two-step mutagenesis PCR using primers
4D5BAMF and 4D5BAMR. Final product of scFv 4D5-8 was digested with
NcoI and NotI restriction enzymes and inserted into the same sites
of pBAK1 plasmid in frame with pelB leader sequence and an octa
His-tag to make p4D5-8Bam plasmid. This plasmid was subsequently
digested with BamHI and mRFP1 DNA inserted to obtain p4D5-8mRFP1
plasmid. All constructs were verified by DNA sequencing.
Protein Expression.
[0150] Transformants from strain BL21 (DE3) pRARE (Phage-resistant
derivative of BL21(DE3), with pRARE plasmid encoding rare codon
tRNAs, chloramphenicol-resistant) were inoculated into 200 mL
Luria-Bertani (LB) medium plus kanamycin (30 .mu.g/mL) and
chloramphenicol (34 .mu.g/mL), and grown overnight at 37.degree. C.
This culture was used to inoculate a 12 L fermentor (1:60 dilution)
containing LB medium plus kanamycin (30 .mu.g/mL) and
chloramphenicol (34 .mu.g/mL), and grown for approximately 5 hours
at 37.degree. C. until optical density (OD.sub.600nm) of the
culture reached 0.6. Then Isopropyl-.beta.-D-thio-galactoside
(IPTG) at the final concentration of 0.3 mM was added to the
culture to induce protein expression. Fermentations were performed
at 20.degree. C. for 20 hours at 350 rpm, four gas volume flow per
unit of liquid volume per minute (vvm) aeration at pH 7.2.
Protein Purification and Analysis.
[0151] Protein was prepared from the whole cell lysates, purified
on 1 mL HisTrap HP Ni Sepharose.TM. column using AKTAprime.TM. plus
purification system (GE Healthcare, Piscataway, N.J.) and analyzed
by two 12% SDS-PAGE Tris-Glycine gels. One gel was stained with
Coomassie and the other was used for Western immunoblots. The cells
were harvested, washed with PBS, centrifuged, and the pellet was
resuspended in lysis buffer (0.01 M Tris HCl pH 8.0, containing 0.5
M NaCl, 10 mM imidazole and 0.1% (vol/vol) Triton X100) and
sonicated on ice. The lysate was then centrifuged at 22,000.times.g
for 30 minutes at 4.degree. C. The supernatant was used for the
purification of His-tagged proteins using immobilized metal ion
affinity chromatography (IMAC) and proteins were eluted with 500 mM
imidazole. The metal affinity-enriched proteins were loaded onto
Sephadex G200 gel filtration column (GE Healthcare, Piscataway,
N.J.) run using 0.02 M Tris HCl pH 7.5, containing 0.15 M NaCl,
with a flow rate of 1 mL/min, collecting 1 mL fractions. Fractions
32-37 were pooled and concentrated using Microcon concentrators
(Millipore, Billerica, Mass.) with a molecular mass cutoff of 10
kDa, and verified by SDS-PAGE and Western blot analysis. SDS/PAGE
analyses were performed according to the standard protocols using
12% polyacrylamide gels. Immunoblots on nitrocellulose membrane
(Millipore, Billerica, Mass.) were carried out according to the
manufacturer's instructions using monoclonal anti-poly
histidine-alkaline phosphatase antibody clone His-1 (Sigma A5588,
Sigma-Aldrich, St. Louis, Mo.) were visualized using an
5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium
(NBT) (Sigma B1911, Sigma-Aldrich, St. Louis, Mo.).
Flow Cytometry and Fluorescence Microscopy Analysis.
[0152] The SKBr-3 and MDA-MB-231 cell lines were maintained in RPMI
medium 1640, supplemented with 10% FCS and 2 mM L-glutamine in
culture flasks. For flow cytometry analysis, the adherent SKBr-3
and MDA-MB-231 cells were carefully detached with trypsin in PBS
(pH 8.0) containing 5 mM EDTA. After a brief wash with cold PBS the
cells were incubated with 0.2% paraformaldehyde in PBS for five
minutes and again washed with cold PBS, counted, incubated with
4D5-8mRFP1 30 minutes on ice, washed twice with cold buffer
Dulbecco's PBS containing 1% FCS, 2 mM EDTA (pH 7.2) (MACS), and
analyzed by CyAn.TM. ADP flow cytometer (Dako North America, Inc.,
Carpinteria, Calif.). Results were analyzed using Summit software
from Beckman Coulter. For immunofluorescent analysis, cells were
plated on glass cover slips at density of 4.times.10.sup.3 cells
per well and cultured overnight at 37.degree. C. in a 5% CO2
atmosphere. Cells were fixed in 0.2% paraformaldehyde in PBS for
five minutes, washed with PBS 3.times., permeabilized with 0.1%
Triton-X 100 in PBS for five minutes, washed as before, incubated
with 4D5-8mRFP1 (10 .mu.g/mL) and GelGreen.TM. nuclear stain
(Biotium, Inc., Hayward, Calif.) for 30 minutes, washed as before
and analyzed using fluorescence microscope Axioscop 50 (Carl Zeiss
Microlmaging, LLC; Thornwood, N.Y.). Images were captured by using
a CCD camera (PowerShot digital camera, Canon U.S.A., Inc., Lake
Success, N.Y.) and AxioVision software (Carl Zeiss Microlmaging,
LLC; Thornwood, N.Y.).
Molecular Design and Visualisation
[0153] Structure of B72.3 and 4D5 antibodies were downloaded from
PDB database (PBD: 1BBJ and 1FVC respectively). RFP structure was
predicted using Swiss-Model Workspace server. Further modeling was
performed using MIFit+ software version 2009.09-1 (Rigaku Americas
Corp., The Woodlands, Tex.) and protein models were viewed using
PyMOL software version 1.1 (DeLano Scientific LLC, Palo Alto,
Calif.).
Plasmids, Primers and Synthetic DNA
[0154] Plasmid pBAK1, previously constructed in our laboratory is
based on pET-26b vector (EMD Chemicals Inc., Gibbstown, N.J.). All
primers were purchased from Invitrogen Corp. (Carlsbad, Calif.).
Synthetic DNA sequences of B72.3 and CA19.9 antibody variable
domains in V.sub.H-V.sub.L orientation were codon optimized for E.
coli expression and purchased from Epoch Biolabs, Inc. (Missouri
City, Tex.) as plasmids pBSK-B72.3 and pBSK-CA19.9, respectively,
with NcoI, BamHI and NotI restriction sites to facilitate
construction of the expression vectors. Plasmid pAK19 encoding the
4D5-8 V.sub.H and V.sub.L was provided by Dan Yansura (Genentech,
Inc., South San Francisco, Calif.). Plasmid pMT-RFP with mRFP1 gene
was a gift from Professor Ray St. Ledger.
Bacterial Strains, Growth Media and Recombinant DNA Technique
[0155] XL1-Blue Escherichia coli strain (Stratagene, Agilent
Technologies, Inc., Santa Clara, Calif.) was used for plasmid
construction steps. To express recombinant antibodies BL21 (DE3)
strain of E. coli (EMD Chemicals Inc., Gibbstown, N.J.) were used.
E. coli cells were grown in Lysogeny Broth (LB) (Bertani, 2004 J
Bacteriol 186:595-600) or LB agar plates. Kanamycin sulfate and
carbenicillin were used at 30 .mu.g/mL and 100 .mu.g/mL final
concentrations respectively. Plasmid DNA was isolated using QIAprep
Spin Miniprep Kit (Qiagen Inc.--USA, Valencia, Calif.) and DNA from
gel was purified using QIAquick Gel Extraction Kit (Qiagen
Inc.--USA, Valencia, Calif.). E. coli cells were transformed using
standard heat shock methods. Restriction and modification enzymes
were purchased from New England Biolabs, Inc. (Ipswich, Mass.).
Final plasmid constructs were confirmed by DNA sequence
analysis.
Construction of the Expression Plasmid
[0156] Antibody scFv encoding fragments were either digested
directly from pBSK-B72.3, pBSK-CA19.9 or assembled from V.sub.H and
V.sub.L domains encoded by pASK19 plasmids respectively and
inserted into NcoI, and NotI restriction sites of previously
digested plasmid pBAK1 to make pBAK1B72.3, pBAK1CA19.9 and pBAK14D5
respectively. The competent E. coli XL1 Blue cells were transformed
using ligation mixtures and the clones were selected on the LB
plates containing kanamycin. Positive clones were confirmed by DNA
sequencing. To make RFP chimeras in V.sub.H-RFP-V.sub.L
orientation, plasmids pBAK1B72.3, pBAK1CA19.9 and pBAK14D5 were
digested with BamHI restriction enzyme and PCR product of mRFP1
gene obtained using pMT-RFP plasmid template and oligonucleotide
primers RFPBamF and RFPBamR (Table 1), inserted to produce
pBAK1B72.3RFP, pBAK1CA19.9RFP and pBAK14D5RFP respectively.
Colonies were initially screened by colony PCR using primers T7F
and RFPBamR (Table 1) and selected clones confirmed by plasmid DNA
sequencing.
Antibody Expression in E. coli.
[0157] To express scFv and REDantibody chimeras, E. coli BL21 (DE3)
(EMD Chemicals Inc., Gibbstown, N.J.) cells were transformed with
the appropriate plasmid and plated onto LB agar supplemented with
kanamycin sulfate (30 .mu.g/mL final concentration). The cells were
allowed to grow at 37.degree. C. for 18 hours and the following
day, five fresh colonies were inoculated into 10 mL of LB media
(with antibiotics) and grown at 37.degree. C. (with shaking at 250
rpm) for 16 hours. Next day, 200 mL of pre-warmed LB media,
prepared in 1 L conical flasks (with antibiotics) were inoculated
with 10 mL of the overnight culture and grown at 37.degree. C.
(with shaking at 250 rpm) until the optical density at 600 nm
(OD.sub.600) had reached 0.5, then the cells were placed on ice for
30 minutes and Isopropyl .beta.-D-1-thiogalactopyranoside (IPTG)
added (final concentration 0.3 mM) to the cultures and the cells
were grown at 20.degree. C. for an additional 20 hours with shaking
at 250 rpm. Bacterial cells were pelleted by centrifugation for 20
minutes, 5,000 rpm at 4.degree. C. (using Sorvall SuperT 21 bench
top centrifuge, with SL-250T rotor), the supernatant discarded, and
the pellets retained for periplasmic protein extraction.
Antibody Purification from E. coli Periplasm.
[0158] Bacterial cell pellets from 200 mL culture were resuspended
in 10 mL of periplasmic buffer (30 mM Tris-base, pH 8.0, 20%
sucrose and 1 mM EDTA) supplemented to a final concentration of 0.1
mM phenylmethylsulfonyl fluoride (PMSF). The cells were incubated
on ice for 10 minutes and centrifuged at 9000 rpm for 10 minutes at
4.degree. C. (using Sorvall SuperT 21 bench top centrifuge, with
SL-50T rotor). The supernatants were collected and stored on ice;
cell pellets were resuspended in 7 mL of 5 mM MgCl.sub.2 (4.degree.
C.). After incubation for five minutes on ice, bacterial cells were
pelleted by centrifugation as described above, and the supernatants
combined to give the periplasmic fraction.
Ni-NTA Purification of Recombinant Proteins.
[0159] Recombinant proteins were purified using a 1 mL HisTrap HP
column (GE Healthcare, Piscataway, N.J.) fitted to an AKTAprime.TM.
plus (GE Healthcare, Piscataway, N.J.) liquid chromatography
system. First, the 1 mL column was equilibrated with five column
volumes of equilibration buffer (20 mM Tris-HCl, pH 8.0, 500 mM
NaCl, 10 mM imidazole). Following this, 17 mL E. coli periplasmic
fraction (diluted two times with 20 mM Tris-HCl, pH 8.0, 500 mM
NaCl buffer) was loaded onto the column and the column was washed
with five column volumes of wash buffer (20 mM Tris-HCl, pH 8.0,
500 mM NaCl, 20 mM imidazole). The protein was eluted with five
column volumes of elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM
NaCl, 500 mM imidazole) and collected in 1 mL aliquots. Protein
elution was monitored at 280 nm. Eluted fractions 1-5 were analyzed
by SDS-PAGE.
Desalting and Anion Exchange Chromatography
[0160] Recombinant antibody obtained by Ni-NTA purification were
desalted using 40 mL Sephadex G25 column (GE Healthcare,
Piscataway, N.J.) fitted to an AKTAprime.TM. plus (GE Healthcare,
Piscataway, N.J.) liquid chromatography system in 20 mM Tris-HCl,
pH 8.0 buffer and further purified using 1 mL HiTrap Q Sepharose FF
column (GE Healthcare, Piscataway, N.J.) and eluted by increasing
buffer salt concentration. First, a 1 mL column was equilibrated
with five column volumes of equilibration buffer (20 mM Tris-HCl,
pH 8.0). Following this, 5 mL of desalted antibody fraction was
loaded onto the column and protein was eluted by increasing
concentration of NaCl in 20 mM Tris-HCl, pH 8.0 buffer and
collected into 1 mL fractions. Eluted antibodies were concentrated
and the buffer exchanged to 20 mM Tris-HCl, pH 8.0 and 150 mM NaCl
using Ultracel YM-10 Amicon centrifugal devices (Millipore,
Billerica, Mass.). Protein concentration was determined using
Bradford assay kit (Bio-Rad Laboratories, Hercules, Calif.)
(Bradford, 1976 Anal Biochem 72:248-54).
Size Exclusion Chromatography
[0161] Size exclusion chromatography was performed on an AKTA Prime
Plus using a HiLoad 16/60 Superdex 200 size-exclusion column (GE
Healthcare, Piscataway, N.J.) equilibrated with degassed phosphate
buffered saline (PBS). The purified proteins were separated by
loading 1 mL of the sample at a concentration of 100-200 .mu.g/mL.
The flow rate was 1 mL/min, and the absorbance of the eluted
protein was monitored at 280 nm. The column was calibrated with the
following protein standards: .beta.-amylase (200 kDa), bovine serum
albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c
(12.4 kDa).
SDS-PAGE
[0162] The fractions eluted from the HisTrap column (1-5) were
analysed by SDS-PAGE using 12% Tris-Glycine gels. Proteins were
stained with Coomassie Blue R250 as follows: after electrophoresis
gel was submerged in plastic container into 50 mL of 0.025%
Coomassie Blue in 10% acetic acid solution and heated in the
microwave until boiling (approximately 1 min), cooled down for two
minutes on the bench and destained in 50 mL of 10% acetic acid by
repeating previous procedure of heating in microwave and cooling
down. Finally, the gel was kept in 10% acetic acid before
scanning.
Example 3
[0163] Delivery of Recombinant P. agglomerans to the Cibarium of H.
vitripennis
[0164] For this experiment, one can use adult H. vitripennis
collected from citrus orchards at the Agricultural Operations at UC
Riverside. Experimental group plants can be coated with recombinant
P. agglomerans containing a plasmid encoding a fluorescent antibody
such as, for example, the REDantibody described herein. Control
group plants can be coated with bacteria-free growth medium.
[0165] To verify that sharpshooters from the Agricultural
Operations are free of recombinant P. agglomerans in their natural
state, one can analyze P. agglomerans cibarial tissues using PCR
and fluorescence microscopy. For each trial, one can use 10 potted
Chardonnay grape vines of 3 years' age that exhibit new growth. The
shoots of each plant can be hand painted with liquid cultures of
recombinant P. agglomerans at a concentration of 10.sup.8 CFU/mL to
achieve coverage of 100 percent surface area. Application of
recombinant P. agglomerans cultures can occur with 1.0 mL/cm.sup.2
of plant surface area (shoots only). In the set-up of this trial,
one can trim the 10 plants to allow approximately 200 cm.sup.2 of
shoot surface area. Thus, one delivers approximately 200 mL or
close to 2.times.10.sup.10 CFU of recombinant P. agglomerans to
each plant.
[0166] Sharpshooters (n=100) can be introduced into the cage and
allowed to feed for 5 days. At this early stage, one observes
little mortality and one can remove all sharpshooters from the cage
and place them in a new cage containing five untreated plants. At
transfer, 20 sharpshooters can be sacrificed and cibarial tissues
can be dissected out. Analysis of colonization by recombinant P.
agglomerans can be conducted using three methods: (1) confocal
microscopy to detect Red Fluorescent Protein at 608 nm (2) qtPCR
with primers specific for P. agglomerans, and (3) standard light
microscopy for visualization of red bacteria. The remaining 80
insects can be serially analyzed over the next 20 days for
retention of recombinant P. agglomerans in the cibarium.
[0167] Recombinant P. agglomerans will colonize the cibarium of H.
vitripennis. In the initial 20 sharpshooters that are removed after
five days, uniform colonization of the anterior mouthparts of H.
vitripennis will be observed. The outcome of cibarial colonization
can be measured by fluorescence/confocal microscopy, qtPCR, and/or
light microscopy for visualization of red pigment. Two specific
outcomes can be measured: (1) percent of sharpshooters that carry
recombinant P. agglomerans in the cibarium, and (2) relative
microbial CFU in the sharpshooters that are positive for
recombinant P. agglomerans. Other environmental bacteria may
colonize the mouthparts of H. vitripennis and microbial competition
can occur. One can measure total bacterial load by performing qtPCR
with universal 16S RNA primers and measure the fraction of total
bacterial burden that is attributed to recombinant P. agglomerans.
The experimental parameters of this study--i.e., 100 percent
coverage of shoots with 10.sup.8 CFU of recombinant P. agglomerans
per mL of applied culture--are established to optimize conditions
for colonization. Statistical significance of recombinant P.
agglomerans colonization in experimental group sharpshooters can be
determined by comparing qtPCR values with control insects by chi
squared analysis.
[0168] The second part of this study, persistence of recombinant P.
agglomerans after initial exposure can reveal colonization of the
cibarium in experimental group sharpshooters. There may be some
decrease in CFU over the 20-day period, but one can calculate rates
of decay via qtPCR. The decay rate can adjust for plasmid loss of
the engineered recombinant P. agglomerans.
Transmission Blocking Effect of REDantibody in GWSS
[0169] One can assess the effect of REDantibody, expressed by
recombinant P. agglomerans in paratransgenic H. vitripennis, in:
(1) rendering the arthropod refractory to challenge infection by X.
fastidiosa, and (2) preventing transmission of the pathogen to
healthy grape vines. Three groups of fourth-instar GWSS can be
used: (1) an experimental group can be colonized with recombinant
P. agglomerans as above (n=100), (2) a control group of GWSS can be
exposed to P. agglomerans transformed to express a marker antibody
(rDB3) that has no activity against Xylella (N=100), (3) a second
control can consist of GWSS exposed to untransformed P.
agglomerans.
[0170] For these studies, we can use freshly molted adults to
increase likelihood of Xylella transmission. Sharpshooters can be
exposed to P. agglomerans-coated Chardonnay grape plants for five
days. Ten insects from each group can be sacrificed at day 5 to
confirm cibarial colonization by P. agglomerans and expression of
antibody (ELISA, Western blot). Using the protocol of Almeida and
Purcell (Almeida, R. P. and Purcell, A. H., Transmission of Xylella
fastidiosa to grapevines by Homalodisca coagulata (Hemiptera:
Cicadellidae). J Econ Entomol, 2003. 96:264-71), remaining insects
can be challenged with X. fastidiosa. Briefly, GWSS that have been
exposed to P. agglomerans can be transferred to closed containers
containing Xylella-infected Chardonnay grape (confirmed by culture
and PCR of plant issues). Seedlings with succulent growth can be
used throughout to maximize feeding and transmission. The
Acquisition Access Period (AAP) can be two days. This can permit
acquisition of Xylella by the nymphs and prevent excessive loss of
the recombinant P. agglomerans. Following the two-day AAP, 10
insects from each group can be sacrificed and cibarial tissues can
be examined by both culture and PCR for X. fastidiosa. Since
presence of Xylella alone in GWSS is not correlated to subsequent
transmission, we can assess infectivity of the three groups of
insects by transferring them to fresh, uninfected grape plants with
succulent growth for an Inoculation Access Period (IAP) of 7-21
days, until molting occurs. This can allow ample time for
transmission to occur and, possibly, cycles of transmission to
occur between plants. Plants from the previous step can be stored
separately and monitored for a minimum of eight weeks to assess for
symptoms of Pierce's Disease. All plants can be examined by culture
and PCR to verify X. fastidiosa infection.
[0171] Insects in the two control groups will be infected with
Xylella at rates of nearly 100 percent. Likewise, between 80-90%
transmission of the pathogen and disease in target grape vines are
seen in the control groups. Transmission of Xylella and infection
of grape plants after the IAP, however, will be reduced by
approximately 30%, preferably by approximately 50%, in the
experimental group of sharpshooters.
Example 4
[0172] Survival and Dispersal of pSCR-189b-Transformed
(Recombinant) P. agglomerans in the Rhizosphere of Chardonnay Grape
Vines
[0173] One can measure soil CFU of recombinant P. agglomerans
before and after each 25-day trial for each of the experimental
pots containing recombinant P. agglomerans-treated grape plants.
Five separate soil samples (1.0 cm.sup.3) can be taken from the
base of the grape vine at days 1, 12, and 25. Soil analysis for
recombinant P. agglomerans can be conducted using one or more of
three techniques: (1) culture on selection agar with PCR
identification of colonies (gold standard), (2) fluorescence
microscopy to detect RFP, and/or (3) direct visualization of
colonies on agar for red color. Analytic techniques can evaluate
not only the persistence and dispersal of recombinant P.
agglomerans into the rhizosphere but also the accuracy of direct
color visualization of bacterial colonies to identify recombinant
bacteria.
[0174] No recombinant P. agglomerans are present in the rhizosphere
on day 1. Probing of shoots by GWSS, physical detachment of
bacteria from shoots and movement of bacteria through watering
activities may to contribute to rhizosphere invasion by recombinant
P. agglomerans. Therefore, CFU counts of recombinant P. agglomerans
may increase by day 12. Values on day 25 can reflect a balance of
bacterial replication and gene decay. Comparison of values with
control pots using chi-squared analysis can yield measures of
statistical significance.
[0175] Definitive detection of recombinant P. agglomerans may be
performed using colony PCR derived from selection agar, but may be
a cumbersome and expensive method for field use to detect
paratransgenics. CFU counts of red bacteria can initially be
compared statistically to values obtained by colony PCR to
determine the accuracy of direct visualization of color. Similar
comparison can be made to fluorescent read-outs that detect mRFP.
Certain factors may decrease accuracy of visualization methods.
First, other soil bacteria may appear red on agar (e.g., Serratia
spp., Gordonia spp.). Second, subjective variability can confound
the readings. Third, intensity of red color can wane over time. All
readings that involve direct visualization of colonies on agar can
be repeated by a blinded observer to minimize bias. Concordance
between direct visual methods and PCR data can be used to determine
accuracy of this method.
Extent of Horizontal Gene Transfer Between Recombinant Pa and
Rhizosphere Bacteria
[0176] Populations of recombinant P. agglomerans can be exposed to
a variety of microbes commonly found in soil consortia to determine
the extent of horizontal gene transfer (HGT). HGT is a dynamic
process and accounts for significant portions of the genomes of
bacteria such as E. coli. Conjugative transfer of plasmids,
heterologous recombination of chromosomal material,
retrotransposons, and phage-mediated gene flow are some of the
mechanisms by which microbes exchange genetic material.
[0177] HGT may occur in this milieu and one can assess the ability
to monitor HGT events using the REDantibody platform. Though HGT
has contributed to plasticity of microbial genomes, individual
events are relatively infrequent and result from bacterial
encounters in the 10.sup.7-8 range. One can score events in these
experiments by counting CFUs of recipient bacteria that acquire the
transgene using one or more of: (1) selection and PCR-based assay
of REDantibody gene, and/or (2) direct visualization for red color.
Control exchanges can involve direct co-incubation of wild-type P.
agglomerans with consortium bacteria. Statistical significance of
the difference in events--e.g., HGT from control versus recombinant
P. agglomerans, and quantification of bacteria using PCR-based
analysis versus direct color visualization--can be calculated using
chi-squared analysis.
[0178] Recombinant P. agglomerans expressing bright red color can
be co-incubated with representative bacteria of the rhizosphere of
common agricultural plants. Though rhizosphere analysis reveals
highly varied structure and variability over time, one can choose
five representative organisms that are commonly found in soil
environments: Pseudomonas fluorescens, Arthrobacter globiformis,
Escherichia coli, Enterobacter agglomerans, and Acinetobacter sp.
For these studies, one may elect to include only Gram-negative
bacteria with similar lipid bilayer membranes as P. agglomerans.
Though Gram-positive bacteria with peptidoglycan membranes and
eukaryotic organisms such as yeast also may be present in the
rhizosphere, one can exclude them from this analysis since direct
visualization of red color is an endpoint.
[0179] One can first verify that the target organisms lack red
color. Recombinant P. agglomerans can serve as the donor organism
at a total CFU count of 10.sup.12 bacteria/reaction in liquid
culture. Recipient bacteria can be co-incubated at 22.degree. C. at
equal CFU count for time points of 24 hours, 48 hours, 96 hours, or
168 hours. Cultures for ampicillin-resistant recipient bacteria can
be done on selective agar and number of recombinant events can be
scored. Identity of recombinant bacteria can be determined as above
using 16S RNA-specific primers for PCR and compared to direct
visualization for color.
[0180] Both invasion of the rhizosphere by recombinant P.
agglomerans and HGT between recombinant P. agglomerans and
consortium members may be observed. Indeed, the large numbers of
recombinant P. agglomerans used in these trials, the protracted
nature of these experiments, and the favorable conditions for HGT
are designed to promote gene spread in the rhizosphere. One
endpoint is evaluation of accuracy of direct color visualization to
determine the extent of rhizosphere invasion and HGT in these
confined settings.
[0181] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the," include
plural referents unless expressly and unequivocally limited to one
referent. As used herein, the term "include" and its grammatical
variants are intended to be non-limiting, such that recitation of
items in a list is not to the exclusion of other like items that
can be substituted or other items that can be added to the listed
items.
[0182] Upon studying the disclosure, it will be apparent to those
skilled in the art that various modifications and variations can be
made in the devices and methods of various embodiments of the
invention. Other embodiments will be apparent to those skilled in
the art from consideration of the specification and practice of the
embodiments disclosed herein. It is intended that the specification
and examples be considered as examples only. The various
embodiments are not necessarily mutually exclusive, as some
embodiments can be combined with one or more other embodiments to
form new embodiments.
Sequence CWU 1
1
2011400DNAartificialplasmid sequence for VH, VL, and fluorophore of
B72.3RFP 1ccatggccca ggtgcagctg cagcagagcg atgcggaact ggtgaaaccg
ggcgcgagcg 60tgaaaattag ctgcaaagcg agcggctata cctttaccga tcatgcgatt
cattgggcga 120aacagaaacc ggaacagggc ctggaatgga ttggctatat
tagcccgggc aacgatgata 180ttaaatataa cgaaaaattt aaaggcaaag
cgaccctgac cgcggataaa agcagcagca 240ccgcgtatat gcagctgaac
agcctgacca gcgaagatag cgcggtgtat ttttgcaaac 300gcagctatta
tggccattgg ggccagggca ccaccctgac cgtgagcagc ggcggaggcg
360gatccgagga cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag
ggctccgtga 420acggccacga gttcgagatc gagggcgagg gcgagggccg
cccctacgag ggcacccaga 480ccgccaagct gaaggtgacc aagggcggcc
ccctgccctt cgcctgggac atcctgtccc 540ctcagttcca gtacggctcc
aaggcctacg tgaagcaccc cgccgacatc cccgactact 600tgaagctgtc
cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
660gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
tacaaggtga 720agctgcgcgg caccaacttc ccctccgacg gccccgtaat
gcagaagaag accatgggct 780gggaggcctc caccgagcgg atgtaccccg
aggacggcgc cctgaagggc gagatcaaga 840tgaggctgaa gctgaaggac
ggcggccact acgacgccga ggtcaagacc acctacatgg 900ccaagaagcc
cgtgcagctg cccggcgcct acaagaccga catcaagctg gacatcacct
960cccacaacga ggactacacc atcgtggaac agtacgagcg cgccgagggc
cgcacaggcg 1020gaggcggatc cgatattcag atgacccaga gcccggcgag
cctgagcgtg agcgtgggcg 1080aaaccgtgac cattacctgc cgcgcgagcg
aaaacattta tagcaacctg gcgtggtatc 1140agcagaaaca gggcaaaagc
ccgcagctgc tggtgtatgc ggcgaccaac ctggcggatg 1200gcgtgccgag
ccgctttagc ggcagcggca gcggcaccca gtatagcctg aaaattaaca
1260gcctgcagag cgaagatttt ggcagctatt attgccagca cttttggggc
accccgtata 1320cctttggcgg cggcacccgc ctggaaatta aacgcgcgga
tgcggccgca ctcgagcacc 1380accaccacca ccaccaccac
14002466PRTartificialamino acid sequence for VH, VL, and
fluorophore of B72.3RFP 2Met Ala Gln Val Gln Leu Gln Gln Ser Asp
Ala Glu Leu Val Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Asp His Ala Ile His Trp
Ala Lys Gln Lys Pro Glu Gln Gly Leu Glu 35 40 45 Trp Ile Gly Tyr
Ile Ser Pro Gly Asn Asp Asp Ile Lys Tyr Asn Glu 50 55 60 Lys Phe
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr 65 70 75 80
Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85
90 95 Phe Cys Lys Arg Ser Tyr Tyr Gly His Trp Gly Gln Gly Thr Thr
Leu 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Asp Val Ile
Lys Glu Phe 115 120 125 Met Arg Phe Lys Val Arg Met Glu Gly Ser Val
Asn Gly His Glu Phe 130 135 140 Glu Ile Glu Gly Glu Gly Glu Gly Arg
Pro Tyr Glu Gly Thr Gln Thr 145 150 155 160 Ala Lys Leu Lys Val Thr
Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp 165 170 175 Ile Leu Ser Pro
Gln Phe Gln Tyr Gly Ser Lys Ala Tyr Val Lys His 180 185 190 Pro Ala
Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe 195 200 205
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val 210
215 220 Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val
Lys 225 230 235 240 Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val
Met Gln Lys Lys 245 250 255 Thr Met Gly Trp Glu Ala Ser Thr Glu Arg
Met Tyr Pro Glu Asp Gly 260 265 270 Ala Leu Lys Gly Glu Ile Lys Met
Arg Leu Lys Leu Lys Asp Gly Gly 275 280 285 His Tyr Asp Ala Glu Val
Lys Thr Thr Tyr Met Ala Lys Lys Pro Val 290 295 300 Gln Leu Pro Gly
Ala Tyr Lys Thr Asp Ile Lys Leu Asp Ile Thr Ser 305 310 315 320 His
Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly 325 330
335 Arg Thr Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ala
340 345 350 Ser Leu Ser Val Ser Val Gly Glu Thr Val Thr Ile Thr Cys
Arg Ala 355 360 365 Ser Glu Asn Ile Tyr Ser Asn Leu Ala Trp Tyr Gln
Gln Lys Gln Gly 370 375 380 Lys Ser Pro Gln Leu Leu Val Tyr Ala Ala
Thr Asn Leu Ala Asp Gly 385 390 395 400 Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Gln Tyr Ser Leu 405 410 415 Lys Ile Asn Ser Leu
Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln 420 425 430 His Phe Trp
Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu 435 440 445 Ile
Lys Arg Ala Asp Ala Ala Ala Leu Glu His His His His His His 450 455
460 His His 465 3672DNAartificialnucleotide sequence for mRFP1
3ggatccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg
60aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
120accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga
catcctgtcc 180cctcagttcc agtacggctc caaggcctac gtgaagcacc
ccgccgacat ccccgactac 240ttgaagctgt ccttccccga gggcttcaag
tgggagcgcg tgatgaactt cgaggacggc 300ggcgtggtga ccgtgaccca
ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg 360aagctgcgcg
gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gactatgggc
420tgggaggcct ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg
cgagatcaag 480atgaggctga agctgaagga cggcggccac tacgacgccg
aggtcaagac cacctacatg 540gccaagaagc ccgtgcagct gcccggcgcc
tacaagaccg acatcaagct ggacatcacc 600tcccacaacg aggactacac
catcgtggaa cagtacgagc gcgccgaggg ccgcacaggc 660ggaggcggat cc
6724224PRTartificialamino acid sequence for mRFP1 4Gly Ser Glu Asp
Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg Met 1 5 10 15 Glu Gly
Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu 20 25 30
Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys 35
40 45 Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe
Gln 50 55 60 Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile
Pro Asp Tyr 65 70 75 80 Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp
Glu Arg Val Met Asn 85 90 95 Phe Glu Asp Gly Gly Val Val Thr Val
Thr Gln Asp Ser Ser Leu Gln 100 105 110 Asp Gly Glu Phe Ile Tyr Lys
Val Lys Leu Arg Gly Thr Asn Phe Pro 115 120 125 Ser Asp Gly Pro Val
Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser 130 135 140 Thr Glu Arg
Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys 145 150 155 160
Met Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys 165
170 175 Thr Thr Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr
Lys 180 185 190 Thr Asp Ile Lys Leu Asp Ile Thr Ser His Asn Glu Asp
Tyr Thr Ile 195 200 205 Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg Thr
Gly Gly Gly Gly Ser 210 215 220 5720DNAartificialnucleotide
sequence for mCitrine 5ggatccgatc cgatggtgag caaaggcgaa gaactgttta
ccggcgtggt gccgattctg 60gtggaactgg atggcgatgt gaacggccat aaatttagcg
tgagcggcga aggcgaaggc 120gatgcgacct atggcaaact gaccctgaaa
tttatttgca ccaccggcaa actgccggtg 180ccgtggccga ccctggtgac
cacctttggc tatggcctga tgtgctttgc gcgttatccg 240gatcacatga
aacagcatga tttttttaaa agcgcgatgc cggaaggcta tgtgcaggaa
300cgtaccattt tttttaaaga tgatggcaac tataaaaccc gtgcggaagt
gaaatttgaa 360ggcgataccc tggtgaaccg tattgaactg aaaggcattg
attttaaaga agatggcaac 420attctgggcc ataaactgga atataactat
aacagccata acgtgtatat tatggcggat 480aaacagaaaa acggcattaa
agtgaacttt aaaattcgtc ataacattga agatggcagc 540gtgcagctgg
cggatcatta tcagcagaac accccgattg gcgatggccc ggtgctgctg
600ccggataacc attatctgag ctatcagagc gcgctgagca aagatccgaa
cgaaaaacgt 660gatcacatgg tgctgctgga atttgtgacc gcggcgggca
ttaccggcgg cggcggatcc 7206240PRTartificialamino acid sequence for
mCitrine 6Gly Ser Asp Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr
Gly Val 1 5 10 15 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
Gly His Lys Phe 20 25 30 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
Thr Tyr Gly Lys Leu Thr 35 40 45 Leu Lys Phe Ile Cys Thr Thr Gly
Lys Leu Pro Val Pro Trp Pro Thr 50 55 60 Leu Val Thr Thr Phe Gly
Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro 65 70 75 80 Asp His Met Lys
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 85 90 95 Tyr Val
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 100 105 110
Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 115
120 125 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
His 130 135 140 Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile
Met Ala Asp 145 150 155 160 Lys Gln Lys Asn Gly Ile Lys Val Asn Phe
Lys Ile Arg His Asn Ile 165 170 175 Glu Asp Gly Ser Val Gln Leu Ala
Asp His Tyr Gln Gln Asn Thr Pro 180 185 190 Ile Gly Asp Gly Pro Val
Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr 195 200 205 Gln Ser Ala Leu
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 210 215 220 Leu Leu
Glu Phe Val Thr Ala Ala Gly Ile Thr Gly Gly Gly Gly Ser 225 230 235
240 7720DNAartificialnucleotide sequence for mCerulean 7ggatccgatc
cgatggtgag caaaggcgaa gaactgttta ccggcgtggt gccgattctg 60gtggaactgg
atggcgatgt gaacggccat aaatttagcg tgagcggcga aggcgaaggc
120gatgcgacct atggcaaact gaccctgaaa tttatttgca ccaccggcaa
actgccggtg 180ccgtggccga ccctggtgac caccctgacc tggggcgtgc
agtgctttgc gcgttatccg 240gatcacatga aacagcatga tttttttaaa
agcgcgatgc cggaaggcta tgtgcaggaa 300cgtaccattt tttttaaaga
tgatggcaac tataaaaccc gtgcggaagt gaaatttgaa 360ggcgataccc
tggtgaaccg tattgaactg aaaggcattg attttaaaga agatggcaac
420attctgggcc ataaactgga atataacgcg attagcgata acgtgtatat
taccgcggat 480aaacagaaaa acggcattaa agcgaacttt aaaattcgtc
ataacattga agatggcagc 540gtgcagctgg cggatcatta tcagcagaac
accccgattg gcgatggccc ggtgctgctg 600ccggataacc attatctgag
cacccagagc gcgctgagca aagatccgaa cgaaaaacgt 660gatcacatgg
tgctgctgga atttgtgacc gcggcgggca ttaccggcgg cggcggatcc
7208240PRTartificialamino acid sequence for mCerulean 8Gly Ser Asp
Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 1 5 10 15 Val
Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 20 25
30 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
35 40 45 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
Pro Thr 50 55 60 Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe
Ala Arg Tyr Pro 65 70 75 80 Asp His Met Lys Gln His Asp Phe Phe Lys
Ser Ala Met Pro Glu Gly 85 90 95 Tyr Val Gln Glu Arg Thr Ile Phe
Phe Lys Asp Asp Gly Asn Tyr Lys 100 105 110 Thr Arg Ala Glu Val Lys
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 115 120 125 Glu Leu Lys Gly
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 130 135 140 Lys Leu
Glu Tyr Asn Ala Ile Ser Asp Asn Val Tyr Ile Thr Ala Asp 145 150 155
160 Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile
165 170 175 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
Thr Pro 180 185 190 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
Tyr Leu Ser Thr 195 200 205 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
Lys Arg Asp His Met Val 210 215 220 Leu Leu Glu Phe Val Thr Ala Ala
Gly Ile Thr Gly Gly Gly Gly Ser 225 230 235 240
944DNAartificialsynthetic oligonucleotide primer 9gtcccttctc
gcttctctgg gtccagatct gggacggatt tcac 441044DNAartificialsynthetic
oligonucleotide primer 10gtgaaatccg tcccagatct ggacccagag
aagcgagaag ggac 441139DNAartificialsynthetic oligonucleotide primer
11cagcggcgga ggcggatccg atatccagat gacccagtc
391243DNAartificialsynthetic oligonucleotide primer 12tcgagtgcgg
ccgcatccgc gcgtttgatc tccaccttgg tac 431340DNAartificialsynthetic
oligonucleotide primer 13tcggatccgc ctccgcccga ggagacggtg
accagggttc 401430DNAartificialsynthetic oligonucleotide primer
14taggccatgg ccgaggttca gctggtggag 301531DNAartificialsynthetic
oligonucleotide primer 15cagtggatcc gaggacgtca tcaaggagtt c
311644DNAartificialsynthetic oligonucleotide primer 16cagtggatcc
gcctccgcct gtgcggccct cggcgcgctc gtac 441740DNAartificialsynthetic
oligonucleotide primer 17cagtggatcc gccgccgccg gtaatgcccg
ccgcggtcac 401831DNAartificialsynthetic oligonucleotide primer
18cagtggatcc gaggacgtca tcaaggagtt c 311944DNAartificialsynthetic
oligonucleotide primer 19cagtggatcc gcctccgcct gtgcggccct
cggcgcgctc gtac 442024DNAartificialsynthetic oligonucleotide primer
20gcagctaata cgactcacta tagg 24
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