Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS RELATING TO HYDROGEN
PEROXIDE AND SUPEROXIDE PRODUCTION BY ANTIBODIES
Field of the Invention
The invention relates to methods for the antibody-mediated generation of
superoxide free radical from ringlet oxygen. The invention also relates to the
generation of hydrogen peroxide from ringlet oxygen. Therapeutic methods are
based upon both enhancing and inhibiting these processes. Screening methods
relate to identifying modulators of antibody-mediated generation of hydrogen
peroxide and superoxide free radical through the respective increase or
decrease
in detectable hydrogen peroxide or superoxide. The invention further relates
to a
simplified immunoassay based on detecting hydrogen peroxide. The invention
also relates to therapeutic compositions that are engineered to increase the
production of hydrogen peroxide and superoxide free radical as well as
compositions that are engineered to prevent this production.
Back round
A relevant biological basis relating to varying disease mechanisms and
conditions is that of oxidative stress and the consequent production of free
radicals that paradoxically are both beneficial and detrimental to cellular
metabolism. Human metabolism is oxygen based. As such, the chemical
reactions relating to oxidative processes play a central role in cellular
homeostasis.
In the oxygen cascade, reactive oxygen species resulting from incomplete
reduction of oxygen include among others the free radicals, superoxide radical
(02 ) and hydroxyl radical (OH'), that have one or more unpaired electrons.
Superoxide spontaneously reacts with itself in a dismutation reaction to form
hydrogen peroxide (HZOz). The formed hydrogen peroxide, while not being a
free radical, under certain situations, e.g., in the absence of catalase,
becomes a
cytotoxic oxidant through the formation of hydroxyl radical and hypochlorous
acid (HOCI) (McCord, Amer. J. Med., 108:652-659 (2000)). Intracellularly,
most of the superoxide is generated as a result of mitochondrial respiration
(McCord, Amer. J. Med., 108:652-659 (2000)). At low superoxide
concentration, the conversion to hydrogen peroxide is catalyzed by superoxide
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dismutase, a process that helps maintain a lower steady-state concentration of
superoxide (Babior et al., Amer. J. Med., 109:33-34 (2000)).
Another highly reactive molecule involved in the oxygen cascade is
singlet oxygen (' OZ). Singlet oxygen results from irradiation by light of
metal-
s free porphyrin precursors that are present in the skin of porphyria
sufferers.
Singlet oxygen is also generated by neutrophils and is thought to be
responsible
for damage created by phagocytes on their targets (Babior et al., Amer. J.
Med.,
109:33-34 (2000)). Based on its high reactivity with biomolecules, singlet
oxygen has generally been considered to be an endpoint in the cascade of
oxygen-scavenging agents.
The reactive nature of free radicals causes them to have both positive and
negative effects on cells and on whole organisms. Methods to inhibit the
negative effects of these reactive species would be tremendously beneficial in
the treatment of many conditions. Also, methods to utilize the positive
effects of
these reactive species would be beneficial to control such things as cell
proliferation and infection. Accordingly, methods and agents are needed to
modulate the generation of free radicals and other reactive species.
Summary of the Invention
The invention provides methods for utilizing the newly discovered
abilities of an antibody to reduce singlet oxygen to superoxide. This
catalytic
reaction ultimately results in the formation of hydrogen peroxide. The
invention
also provides methods to utilize antibodies to produce hydrogen peroxide from
singlet oxygen by the oxidation of water. Hydrogen peroxide, under certain
biological conditions, itself generates reactive molecules. Thus, the
invention
generally provides methods to inhibit and facilitate these processes depending
on
the desired outcome. The invention further relates to screening methods to
identify agents that modulate the newly discovered antibody-mediated
processes.
The invention further contemplates ari improved immunoassay format based on
the direct detection of hydrogen peroxide that is produced by antibody
catalyzed
oxidation of water. The invention also provides an improved immunoassay
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based on hydrogen peroxide produced from antibody-generated superoxide in
the presence of singlet oxygen. The invention also contemplates therapeutic
compositions, preferably antibody compositions, that are engineered to exhibit
increased or decreased oxidative function.
Brief Description of the Drawings
Figure 1 illustrates the oxygen-dependent microbicidal action of phagocytes.
The interconversion of'Oz and OZ'-is indicated and is an intrinsic ability to
antibodies.
Figure 2 illustrates the amplex red assay.
Figure 3 shows the initial time course of H202 production in PBS (pH 7.4) in
the
presence (0) or absence (o) of murine monoclonal IgG EP2-1962 (20 ~.M).
Error bars show the range of the data from the mean.
Figure 4 shows the fluorescent micrograph of a single crystal of murine
antibody
1D4 Fab fragment after UV irradiation and HZOZ detection with the amplex red
reagent.
Figure 5 illustrates the (A) HP sensitization assay. Time course of H202
formation in PBS (pH 7.4) with HP (40 ~,M) and visible light, in the presence
(O) or absence (~) of 31127 (horse IgG, 20 ~,M). (B) Initial time course of
H20z
production with HP (40 ~.M) and visible light, in the presence of 31127 (horse
IgG, 6.7 ~.M) with no additive in PBS (pH 7.4) (~) or NaN3 in PBS (pH 7.4) (O,
100 ~,M) or in a D20 solution of PBS (pH 7.4) (0). (C) Protein concentration
(31127, horse IgG) versus rate of HZO2 formation. (D) Oxygen concentration on
the rate of H20z generation with 31127 (horse IgG, 6.7 ~M). All points are
mean values of at least duplicate experimental determinations. Error bars are
the
range of experimentally measured values from the mean.
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Figure 6 is a bar graph showing the measured initial rate of HZOZ formation
for a
panel of proteins and comparison with antibodies (data from Table I). All
points
are mean values of at least duplicate experimental determinations. Error bars
are
the range of experimentally measured values form the mean. OVA, chick-egg
ovalbumin; SOD, superoxide dismutase.
Figure 7 shows (A) the rate of Hz02 formation by UV irradiation of horse IgG
(6.7 wM) in PBS (pH 7.4). (B) simultaneous fluorescence emission of the horse
IgG, measured at 326 nm (excitation = 280 nm).
Figure 8 shows H202 production. (A) Production of HZOZ by immunoglobulins
and non-immunoglobulin proteins. Assays were performed by near-UV
irradiation (312 nm, 800 ~,W cm z) of individual protein samples (100 ~,L, 6.7
~,M) in phosphate-buffered saline (PBS) [10 mM sodium phosphate, 150 mM
NaCl (pH 7.4)] in a sealed glass vial on a transilluminator (Fischer Biotech)
under ambient aerobic conditions at 20°C. Aliquots (10 ~.L) were
removed
throughout the assay. HZO~ concentration was determined by the amplex red
method. Each data point is reported as the mean ~ SEM of at least duplicate
measurements: [~ polyclonal (poly) immunoglobulin (Ig) G, human; O
polyIgG, horse; ~ polyIgG, sheep; O monoclonal (m) IgG (WDl-6G6), murine;
o polyIgM, human; o mIgG (92H2), murine; ~ (3-galactosidase ((3-gal); ~ chick
ovalbumin (OVA); ~ a-lactalbumin (a-lact); ~ bovine serum albumin (BSA)].
(B) Long-term production of HZOz by sheep polyIgG (6.7 wM, 200 ~,L).
Near-UV irradiation for 8 hours in PBS in a sealed well of a 96-well quartz
plate. HZOZ concentration was measured as described in (A). (C) A solution of
PCP-21H3, mIgG (murine) (6.7 ~M, 200 ~,L), was irradiated in PBS in a sealed
well of a 96 well quartz plate for 510 min. The HZOZ was assayed by the amplex
red assay and then destroyed by addition of catalase (10 mg, 288 mU)
immobilized on Eupergit C. The catalase was removed by filtration and the
antibody solution re-irradiated for 420 min. Rate (0-510 min) = 0.368 , ~,M
min'
(r2 = 0.998); rate (511-930 min) = 0.398 ~,M miri' (r2 = 0.987). (D)
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Determination of ICSO of H202 on the photo-production of H202 by horse
polyIgG. A solution of horse IgG (6.7 ~,M) was incubated with varying
concentrations of H202 (0-450 ~M) and the initial rate of HZOZ formation
measured as described in (A). The graph is a plot of rate of H202 formation
versus H202 concentration and reveals an ICSO of 225 ~.M. (E) Long-term
inhibition of antibody photo-production of H202 by H202 and complete
re-establishment of activity. The assay involved an initial U.V. irradiation
of
horse polyIgG (6.7 mM in PBS pH 7.4) in the presence of HzOz (450 wM) for
360 min.
The H202 was then removed by catalase (immobilized on Eupergit C)
and the polyIgG sample was re-irradiated with UV light for a further 480
minutes. H202 formation throughout the assay was measured by the amplex red
assay. (F) A solution of a(3-TCR (6.7~M, 200 ~L) was irradiated as described
in
(C) for periods of 360, 367 and 389 min. The HZOZ generated during each
irradiation was assayed and destroyed as described in (C). Rate (0-360 min) _
0.693 ~.M miri' (r2 = 0.962). The curvature in the progress curve above 200 ~M
conforms to the expected inhibition by HzOZ (viele infra); rate (361-727 min)
_
0.427 ~.M miri' (r2 = 0.987); rate (728-1117 min) = 0.386 ~.M miri' (rz =
0.991).
Figure 9 illustrates the superposition of native 4C6 Fab (light blue and pink
in a
color photograph) and 4C6 Fab in the presence of H202 (dark blue and red in a
color photograph) (A). The native 4C6 crystals were soaked for 3 minutes in 4
mM H202, and immediately flash frozen for data collection at SSRL BL 9-1.
The overall structural integrity of the secondary and tertiary structure is
clearly
preserved in the presence of HZOZ (RMSD Ca = 0.33 ~, side chain = 0.49 ~).
The RMSD was calculated in CNS. (B) High resolution x-ray structures show
that Fab 4C6 is cross-reactive with benzoic acid. Superposition of the 4C6
combining site with and without H202 demonstrates that even the side chain
conformations within the binding site are preserved (light and dark colored
side
chains in a color photograph correspond to + and - H202 respectively).
Moreover, clear electron density for the benzoic acid underscores that the
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binding properties of Fab 4C6 remain unaltered in 4mM Hz02. The electron
density map is a 2f~ f° sigma weighted map contoured at l .5a, and the
figures
were generated in Bobscript.
Figure 10 shows the absorbance spectra of horse polyclonal IgG measured on a
diode array HP8452A spectrophotometer, AbsmaX 280 nm (A). (B) Action
spectra of horse polyclonal IgG, between 260 and 320 nm showing maximum
activity of Hz02 formation at 280 nm. The assay was performed in duplicate and
involved addition of an antibody solution [6.7 ~M in PBS (pH 7.4)] to a quartz
tube that was then placed in a light beam produced by a xenon arc lamp and
monochromator of an SLM spectrofluorimeter for 1 hour. HZOZ concentration
was measured by the amplex red assay.
Figure 11 shows the production of H202. (A) Production of HZOZ by tryptophan
(20 ~,M). The conditions and assay procedures were as described in Figure 8A.
(B) Effect of chloride ion on antibody-mediated photo-production of Hz02. A
solution of sheep polyIgG ~ (6.7 ~,M, 200 ~L) or horse polyIgG ~ (6.7 ~M, 200
~,L) was lyophilized to dryness and then dissolved in either deionized water
or
NaCI (aq.) such that the final concentration of chloride ion were (0-160 mM).
The samples were then irradiated, in duplicate, in sealed glass vials on a
transilluminator (800 ~,W cm Z) under ambient aerobic conditions at 20
°C.
Aliquots (10 ~L) were removed throughout the assay and the H202 concentration
determined by the amplex red assay. The rate of Hz02 formation is plotted as
the
mean ~ S.E.M. versus [NaCI] for each antibody sample. (C) Effect of dialysis
into EDTA-containing buffers on antibody-mediated photo-production of H20z.
The photo-production of HZOZ by two antibody preparations, mouse monoclonat
antibody PCP21 H3 and horse polyclonal IgG, were compared before and after
dialysis into PBS containing EDTA (20 mM). The conditions and assay
procedures were as described in Figure 8A. Each data point is reported as the
mean ~ SEM of at least duplicate measurements: [~ marine mIgG PCP21H3
before dialysis; ~ marine mIgG PCP21H3 after dialysis; ~ polyIgG, horse before
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dialysis; ~ polyIgG, horse after dialysis.
Figure 12 shows ESI (negative polarity) mass spectra of TCEP [(M-H)- 249] and
its oxide [(M-H)- 265 ('60) and (M-H)- 267 ('80)] produced by oxidation with
H202. (A) MS of TCEP and its oxides after irradiation of sheep polyIgG
(6.7/~.M) under '602 aerobic conditions in HZ'80 (98 %'80) PB. (B) MS of
TCEP and its oxides after irradiation of sheep polyIgG (6.7 ~.M) under
enriched
'80z (90 %'80) aerobic conditions in HZ'60 PB. (C) MS of TCEP and its oxides
after irradiation of the polyIgG performed under '602 aerobic concentration in
HZ'GO PB. The assay conditions and procedures were as described in the
methods and materials (Example II) with the exception that HZ'6 O replaced
Hz'$O. (D) MS of TCEP and its oxides after irradiation of sheep polyIgG (6.7
wM) and Hz'6O2 (200 ~M) under anaerobic (degassed and under argon)
conditions in HZ'$O PB for 8 hours at 20°C. Addition of TCEP was as
described
in the methods and materials (Example II). (E) MS of TCEP and its oxides after
irradiation of 3-methylindole (500 ~,M) under '602 aerobic conditions in Hz'g0
PB. The assay conditions and procedures were as described in the methods and
materials (Example II) with the exception that size-exclusion filtration was
not
performed because 3-methyl indole is of too low molecular weight. Therefore,
TCEP was added to the 3-methyl indole-containing PB solution. (F) MS of
TCEP and its oxides after irradiation of (3-gal (50 ~,M) under'GOz aerobic
conditions in HZ'$O PB. Assay conditions and procedures are as described in
the
methods and materials (Example II).
Figure 13 shows the Xe binding sites in antibody 4C6 as described in materials
and methods (Example II). (A) Standard side view of the Ca trace of Fab 4C6
with the light chain in pink and the heavy chain in blue in a color
photograph.
Three bound xenon atoms (green in a color photograph) are shown with the
initial Fo F~ electron density map contoured at 5 a. (B) Overlay of Fab 4C6
and
the 2C aJ3 TCR (PDB/TCR) around the conserved xenon site 1. The backbone
Ca trace of VL (pink in a color photograph) and side chains (yellow in a color
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photograph) and the corresponding Va of the 2C a(3 TCR (red and gold in a
color
photograph) are superimposed (figure generated using Insight2000).
Detailed Description of the Invention
The present invention concerns the discovery that antibodies, as a class
of molecules, have an inherent capability to intercept ringlet oxygen and
convert
it to either superoxide or hydrogen peroxide. This process acts to rescue and
recycle oxygen, particularly during phagocyte-mediated processes, thereby
contributing to microbicidal action of the immune system. These properties are
common to all antibodies and were not known prior to the present invention.
The common ability to convert ringlet oxygen to superoxide or hydrogen
peroxide,. regardless of source or antigenic specificity, is thought to link
the
previously appreciated recognition properties of antibodies with killing
events.
The present invention provides methods that relate to the ability of an
antibody to reduce ringlet oxygen ('OZ) to superoxide radical (OZ ) and
hydrogen peroxide. In view of the critical nature and role of oxygen
metabolism in an aerobic organism, the identification of this biological
process
provides multiple and varied methods as described herein. The detailed
determination and characterization of the antibody-mediated reduction of
ringlet
oxygen is described in examples I and II.
As demonstrated in the examples, these properties are universal abilities
of all antibodies.
Superoxide Production by Antibodies
The ability to produce superoxide from singlet oxygen is present in both
intact immunoglobulins and well as Fab and F(ab')2 fragments (see examples).
The activity does not reside in molecules, including RNaseA, superoxide
dismutase, and Bowman-Birk inhibitor protein, that can be oxidized (example I
and Table 1). Also, the activity is not associated with the presence of
disulfides
in a molecule, even though they are sufficiently electron rich that they can
be
oxidized (Bent et al., J. Am. Chem. Soc., 87:2612-2619 (1975)). Rather, the
activity resides in an aromatic amino acid such as tryptophan that can be
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oxidized by singlet molecular oxygen via electron transfer (Grossweiner, Curr.
Top. Radiat. Res. Q., 11:141-199 (1976)). The activity is further attributed
to
the indole component of the tryptophan residue. Thus, the indole acts as a
reductive center in connection with the redox reaction. The indole portion
becomes oxidized to form a radical cation in the course of reducing singlet
molecule oxygen to superoxide free radical. In the same context, the antibody
is called a reluctant because it is oxidized in providing an electron to
singlet
molecular oxygen. It is believed that oxidized antibody interaction with an in
vivo antioxidant completes the catalytic cycle and returns the antibody to
neutrality. The ability of an antibody to generate superoxide from singlet
oxygen is abolished if the antibody is denatured. This indicates that the
location
of the oxidized molecules in the reactive center of the antibody is relevant
to the
reduction process used to generate superoxide. In particular, the reduction of
singlet molecular oxygen is primarily due to the two tryptophan residues that
are buried in the molecule rather than the solvent-exposed ones (example I).
Such buried aromatic residues in proteins, including antibodies, are generally
considered to contribute to structure stability (Burley, et al., Science,
229:23-28
(I985)). Furthermore, two aromatic tryptophan resides are conserved, referred
to as TRP-36 and TRP-47, and are both deeply buried (Rabat, et al., Sequences
of Proteins of Immunological Interest, U.S. Department of Health and Human
Services, Public Health Service, National Institutes of Health, Bethesda, MD
( 1991 )). The ability of antibodies as a class of proteins to reduce singlet
molecular oxygen to superoxide anion is thus based on the presence of the
conserved buried aromatic tryptophan residues.
Hydro~en Peroxide Production by Antibodies
The ability to produce hydrogen peroxide in an efficient and long term
manner from singlet oxygen is present in immunoglobulins and in the T-cell
receptor (example II, Figure 1F). The T-cell receptor shares a similar
arrangement of its immunoglobulin fold domains with antibodies (Garcia et al.,
Science, 274:209 (1996)). However, possession of this structural motif does
not
appear necessary to confer a hydrogen peroxide-generating ability on proteins.
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(32-macroglobulin, a member of the immunoglobulin superfamily having this
structural motif, does not generate hydrogen peroxide (Welinder et al., Mol.
Immunol., 28:177 (1991)). Structural studies suggest that a conserved
tryptophan residue found in both the T-cell receptor and in antibodies may
play
a role in the oxidation of water. The catalytic role of the tryptophan
conserved
in antibodies and in the T-cell receptor is further supported by the
observation
that the (3z-macroglobulin lacks the conserved residue as well as the
catalytic
activity. Furthermore, the sequence and structure surrounding the conserved
tryptophan residue is highly conserved between antibodies and the T-cell
receptor indicating that it may also play a role in allowing catalysis of
singlet
oxygen to hydrogen peroxide.
Information relating the structure to the function of immunoglobulins
and the T-cell receptor allows molecules to be designed that will catalyze the
oxidation of water. This information also provides many new methods and
treatment schemes that may be utilized based on existing molecules.
Definitions
Abbreviations: (HP) hematoporphyrin; (PBS) phosphate buffered saline;
(OVA) chick-egg ovalbumin; (SOD) superoxide dismutase; (PO) peroxidase
enzymes; (phox) phagocyte oxidase; (HRP) horseradish peroxidase; (MS) mass
spectroscopy; (AES) ICP-atomic emission spectroscopy; (MS) mass-spectral,
(QC) quantum chemical.
The term "agent" herein is used to denote a chemical compound, a
mixture of chemical compounds, a biological macromolecule, or an extract
made from biological materials such as bacteria, plants, fungi, or animal
(particularly mammalian) cells or tissues. Agents are evaluated for potential
activity as antibody modulatory agents by inclusion in screening assays as
described herein.
The term "antibody" as used in this invention includes intact molecules
as well as fragments thereof, such as Fab, F(ab')Z, and Fv which are capable
of
binding an epitope. These antibody fragments retain some ability to
selectively
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bind with its antigen or receptor and are defined as follows:
(1) Fab, the fragment which contains a monovalent antigen-binding
fragment of an antibody molecule can be produced by digestion of whole
antibody with the enzyme papain to yield an intact light chain and a portion
of
F
one heavy chain;
(2) Fab', the fragment of an antibody molecule can be obtained by
treating whole antibody with pepsin, followed by reduction, to yield an intact
light chain and a portion of the heavy chain; two Fab' fragments are obtained
per antibody molecule;
(3) (Fab')2, the fragment of the antibody that can be obtained by
treating whole antibody with the enzyme pepsin without subsequent reduction;
F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, defined as a genetically engineered fragment containing the
variable region of the light chain and the variable region of the heavy chain
expressed as two chains; and
(5) Single chain antibody ("sFv"), defined as a genetically
engineered molecule containing the variable region of the light chain, the
variable region of the heavy chain, linked by a suitable polypeptide linker as
a
genetically fused single chain molecule.
The preparation of polyclonal antibodies is well-known to those skilled
in the art. See, for example, Green, et al., Production of Polyclonal
Antisera, in:
Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press); Coligan,
et al., Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters,
in: Current Protocols in Immunolo~y, section 2.4.1 (1992), which are hereby
incorporated by reference.
The preparation of monoclonal antibodies is also conventional. See, for
example, I~ohler & Milstein, Nature, 256:495 (1975); Coligan, et al., sections
2.5.1-2.6.7; and Harlow, et al., in: Antibodies: A LaboratorX Manual, page 726
(Cold Spring Harbor Pub. (1988)), which are hereby incorporated by reference.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques include
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affinity chromatography with Protein-A Sepharose, size-exclusion
chromatography, and ion-exchange chromatography. See, e.g., Coligan, et al.,
sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al., Purification
of
Immunoglobulin G (IgG), in: Methods in Molecular Biolo~y, Vol. 10, pages
79-104 (Humana Press (1992).
Methods of in vitro and in vivo manipulation of monoclonal antibodies
are well known to those skilled in the art. One particular manipulation
involves
the process of humanizing a monoclonal antibody by recombinant means to
generate antibodies containing human specific and recognizable sequences.
See, for review, Holmes, et al., J. Immunol., 158:2192-2201 (1997) and
Vaswani, et al., Annals Aller;w, Asthma & Immunol., 81:105-115 (1998).
Methods of making antibody fragments are known in the art (see for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York, (1988), incorporated herein by reference).
Antibody fragments of the present invention can be prepared by proteolytic
hydrolysis of the antibody or by expression in E. coli of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies conventional methods. For example, antibody fragments can
be produced by enzymatic cleavage of antibodies with pepsin to provide a SS
fragment denoted F(ab')z. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfliydryl groups
resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent
fragments. Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab ' fragments and an Fc fragment directly. These methods are
described, for example, in US Patents No. 4,036,945 and No. 4,331,647, and
references contained therein. These patents are hereby incorporated in their
entireties by reference.
Other methods of cleaving antibodies, such as separation of heavy
chains to form monovalent light-heavy chain fragments, further cleavage of
fragments, or other enzymatic, chemical, or genetic techniques may also be
used, so long as the fragments bind to the antigen that is recognized by the
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intact antibody. For example, Fv fragments comprise an association of VH and
VL chains. This association may be noncovalent or the variable chains can be
linked by an intermolecular disulfide bond or cross-linked by chemicals such
as
glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains
connected by a peptide linker. These single-chain antigen binding proteins
(sFv) are prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is subsequently
introduced into a host cell such as E. coli. The recombinant host cells
synthesize a single polypeptide chain with a linker peptide bridging the two V
domains. Methods for producing sFvs are described, for example, by Whitlow,
et al., Methods: a Companion to Methods in Enzymolo~y, Vol. 2, page 97
(1991); Bird, et al., Science, 242:423-426 (1988); Ladner, et al, US Patent
No.
4,946,778; and Pack, et al., Bio/Technolo~y, 11:1271-77 (1993).
Another form of an antibody fragment is a peptide coding for a single
complementarity-determining region (CDR). CDR peptides ("minimal
recognition units") can be obtained by constructing genes encoding the CDR of
an antibody of interest. Such genes are prepared, for example, by using the
polymerase chain reaction to synthesize the variable region from RNA of
antibody-producing cells. See, for example, Larrick, et al., Methods: a
Companion to Methods in Enzymolo~y, Vol. 2, page 106 (1991).
The terms "effective amount", "effective reducing amount", "effective
ameliorating amount", "effective tissue injury inhibiting amount",
"therapeutically effective amount" and the like terms as used herein are terms
to
identify an amount sufficient to obtain the desired physiological effect,
e.g.,
treatment of a condition, disorder, disease and the like or reduction in
symptoms
of the condition, disorder, disease and the like. Such an effective amount of
an
antioxidant in the context of therapeutic methods is an amount that results in
reducing, reversing, ameliorating, inhibiting, and the like improving
directions,
the effects of an oxidant generated by an antibody.
An "engineered molecule" is a polypeptide that has been produced
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through recombinant techniques. Such molecules can include a reactive center
that can catalyze the production of superoxide or hydrogen peroxide from
singlet oxygen. Such engineered molecules may have a reactive indole
contained within a polypeptide structure. The indole of such a molecule may be
present as a tryptophan residue. Engineered molecules may also contain non-
natural amino acids and linkages as well as peptidomimetics. Engineered
molecules also include antibodies that are modified to eliminate the reaction
center such that they are no longer able to generate superoxide or hydrogen
peroxide.
As used herein, the term "epitope" means any antigenic determinant on
an antigen to which the paratope of an antibody binds. Epitopic determinants
usually consist of chemically active surface groupings of molecules such as
amino acids or sugar side chains and usually have specific three dimensional
structural characteristics, as well as specific charge characteristics.
Antigens
can include polypeptides, fatty acids, lipoproteins, lipids, chemicals,
hormones
and the like. In some embodiments, antigens include, but are not limited to,
proteins from viruses such as human immunodeficiency virus, influenza virus,
herpesvirus, papillomavirus, human T-cell leukemia virus and the like. In
other
embodiments, antigens include, but are not limited to, proteins expressed on
cancer cells such as lung cancer, prostate cancer, colon cancer, cervical
cancer,
endometrial cancer, bladder cancer, bone cancer, leukemia, lymphoma, brain
cancer and the like. Antigens of the invention also include chemicals such as
ethanol, tetrahydrocanabinol, LSD, heroin, cocaine and the like.
The term "modulate" refers to the capacity to either enhance or inhibit a
functional property of an antibody or engineered molecule of the invention,
such as production of superoxide or hydrogen peroxide.
A "non-natural" amino acid includes D-amino acids as well as amino
acids that do not occur in nature, as exemplified by 4-hydroxyproline, ~y-
carboxyglutarnate, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-
methylhistidine, 5-hydroxylysine and other such amino acids and imino acids.
The term "peptidomimetic" or "peptide mimetic" describes a peptide
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analog, such as those commonly used in the pharmaceutical industry as non-
peptide drugs, with properties analogous to those of the template peptide.
(Fauchere, J., Adv. Dru.~ Res., 15: 29 (1986) and Evans et al., J. Med. Chem.,
30:1229 (1987)). Generally, peptidomimetics are structurally similar to a
paradigm polypeptide (i.e., a polypeptide that has a biochemical property or
pharmacological activity), but have one or more peptide linkages optionally
replaced by a linkage such as, --CH2NH--, --CHZS--, --CHz -CHZ--, --CH=CH--
(cis and trans), --COCHZ -, --CH(OH)CHZ -, and --CHzSO--, by methods known
in the art. Advantages of peptide mimetics over natural polypeptide
embodiments may include more economical production, greater chemical
stability, altered specificity, reduced antigenicity, and enhanced
pharmacological properties such as half life, absorption, potency and
efficacy.
As used herein, the terms "pharmaceutically acceptable",
"physiologically tolerable" and grammatical variations thereof, as they refer
to
compositions, carriers, diluents and reagents, are used interchangeably and
represent that the materials are capable of administration to or upon a mammal
without the production of undesirable physiological effects such as nausea,
dizziness, gastric upset and the like.
The terms "protein" and "polypeptide" are used to describe a native
protein, fragments, or analogs of a polypeptide sequence. These terms may be
used interchangeably.
Antibodies
The invention provides therapeutic antibodies. All antibody molecules
belong to a family of plasma proteins called immunoglobulins. Their basic
building block, the immunoglobulin fold or domain, is used in various forms in
many molecules of the immune system and other biological recognition
systems. A typical immunoglobulin has four polypeptide chains, contains an
antigen binding region known as a variable region, and contains a non-varying
region known as the constant region. An antibody contemplated for use in the
present invention can be in any of a variety of forms, including a whole
immunoglobulin, Fv, Fab, other fragments, and a single chain antibody that
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includes the variable domain complementarity'determining regions (CDR), or
other forms. All of these terms fall under the broad term "antibody" as used
herein. The present invention contemplates the use of any specificity of an
antibody, polyclonal or monoclonal, and is not limited to antibodies that
recognize and immunoreact with a specific antigen. In preferred embodiments,
in the context of both the therapeutic and screening methods described herein,
an antibody or fragment thereof is used that is immunospecific for an antigen.
The preparation of a therapeutic antibody of this invention can be
accomplished by recombinant expression techniques as well as protein
synthesis, methods of which are well known to one of ordinary skill in the
art.
For recombinant approaches, mutation of a nucleic acid that encodes an
antibody or fragment thereof can be conducted by a variety of means, but is
most conveniently conducted using mutagenized oligonucleotides that are
designed to introduce mutations at predetermined sites that then encode an
altered amino acid sequence in the expressed molecule. Such alterations
include substitutions, additions, and/or deletions of particular nucleotide
sequences that similarly encode substitutions, additions, and/or deletions of
the
encoded amino acid residue sequence. Site-directed mutagenesis, also referred
to as oligonucleotide-directed mutagenesis and variations thereof, and the
subsequent cloning of the altered genes are well known techniques (Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Chapter 15, Cold
Spring Harbor Laboratory Press, (1989)). Another recombinant approach
includes synthesizing the gene encoding a therapeutic molecule of this
invention
by combining long oligonucleotide strands that are subsequently annealed and
converted to double-stranded DNA suitable for cloning and expression (Ausebel
et al., Current Protocols in Molecular Biolo~y, Units 10 and 15, Wiley and
Sons, Inc. (2000)). Such techniques can be used to create engineered molecules
that contain a reduction center and are able to generate hydrogen peroxide or
superoxide from singlet oxygen. It is contemplated that such engineered
molecules can be designed based on antibody structure and on the T-cell
receptor, in the case of hydrogen peroxide.
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Thus, the present invention contemplates an antibody that has been
engineered to generate more superoxide free radical or hydrogen peroxide in a
desired location. The antibody is engineered to contain additional reductive
centers, as described in examples I and II herein, that increase the reduction
of
singlet molecular oxygen to superoxide free radical or hydrogen peroxide. The
invention also contemplates an antibody that has been engineered to have at
least a diminished capacity to generate superoxide free radical or hydrogen
peroxide from singlet oxygen. In that context, the antibody lacks at least one
of
its reductive centers and preferably is substantially free of a reductive
center.
Such antibody compositions are readily prepared with methods well known to
one of ordinary skill in the art.
If desired, polyclonal or monoclonal antibodies prepared for use as
therapeutic compositions or in the methods of invention can be further
purified,
for example, by binding to and elution from a matrix to which the polypeptide
or a peptide to which the antibodies were raised is bound. Those of skill in
the
art will know of various techniques common in the immunology arts for
purification and/or concentration of polyclonal antibodies, as well as
monoclonal antibodies (Coligan, et al., Unit 9, Current Protocols in
Immunolo~y, Wiley Interscience, (1991)).
1. Therapeutic Methods
Because aerobic organisms rely on oxygen metabolism in a
chemical environment where the toxicity of oxygen and metabolites thereof are
paramount consequence, these organisms have evolved a multitude of
mechanisms to maintain homeostasis and the overall health of the organism.
The toxic potential of oxygen is attributed to the formation, in vivo, of
reactive
free radicals. To become toxic, oxygen must be activated, a process that
occurs
either by photoactivation resulting in singlet oxygen production or by
reduction
followed by the formation of hydrogen peroxide and the hydroxyl radical. The
latter process is accelerated by the presence of transition metals, such as
iron
and copper, and/or specific enzymes such as monooxygenase. These processes
occur in cellular compartments including mitochondria, microsomes,
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peroxysomes and the cytoplasmic membrane. See, Sahnoun et al., Theranie,
52:251-270 (1997).
The free radicals that result from oxygen activation are by definition
chemical species that possess one or several mismatched electrons. Free
radicals are generated when a single electron is removed from the molecule.
This results in a molecule that has at least one of its electrons unpaired to
another electron. The resultant free radical is reactive since it seeks out
available electrons from other molecules, the process of which can create a
second reactive molecule thereby setting off a chain reaction.
Free radicals, also referred to as oxidants herein include superoxide,
hydroxyl radical, halogenated oxygens and nitrogen containing molecules.
Superoxide radical generated from the antibody-mediated reduction of singlet
oxygen is itself an oxidant and also provides for the production of hydrogen
peroxide. The latter, which while not itself an oxidant or reactive molecule,
can
generate reactive oxygen species that include hydroxyl radical, its secondary
products such as carbon, oxygen, nitrogen or sulfur, which can react with
other
compounds to produce yet other free radicals creating a free radical chain
reaction. (Babior et al., Am. J. Med., 109:33-44 (2000)). Other reactive
species
that are a consequence of the oxygen cascade include oxidized halogens, such
as
hypochlorous acid (HOCI), the HOCl-generated reactive species chloramine
(NHZCl) and aldehydes, and reactive nitrogen species.
A potential consequence of uncontrolled reactivity of free radicals is
damage to DNA, RNA, membrane lipids, lipoproteins or enzymes, ultimately
affecting the body. An end result is poor cell function leading to disease and
even tissue death. Paradoxically, free radicals aid the process of riding the
body
of unwanted bacteria or viruses. However, when the production of radicals is
excessive or in the wrong location, acute and chronic cellular and tissue
injury
can occur.
To counteract these reactions, aerobic organisms have evolved certain
built-in mechanisms to keep the equilibrium of oxygen metabolism in check.
These mechanisms broadly include inhibition of oxygen activation processes as
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well as neutralization of free radicals already formed. Neutralizing processes
include 1) enyzmes such as superoxide dismutase and catalase that together
produce peroxidases and 2) molecules such as tocopherols, carotenoids,
ubiquinones, flavonoids, ascorbic acid, uric acid and similar molecules that
serve as a source of electrons that are provided to free radicals without
damaging cellular components. Such processes are considered beneficial to the
well being of an organism. For example, the authors of a recent paper have
correlated an increase in life span in an animal with exposure to superoxide
dismutase/catalase mimetics (Melov et al., Science, 289:1567-1569 (2000)). In
the context of the present invention, any molecule that inhibits the antibody
mediated generation of hydrogen peroxide or superoxide that ultimately leads
to
hydrogen peroxide formation is referred to as an antioxidant: Such preferred
antioxidants of this invention are described below.
When the balance of oxidants to antioxidants tips in favor of the former,
the oxidative state is generally referred to as "oxidative stress". This
situation
occurs in the presence of an excess production of oxidants or free radicals
and a
diminishing of the control antioxidant mechanisms. Advantages of the present
invention are that the discovery of the role an antibody plays in the
generation
of oxidants in the oxygen cascade provides the basis for therapeutic methods
that are useful in maintaining oxygen balance and control of oxygen
metabolism, depending on the desired outcome. In other words, the methods of
this invention provide 1 ) for the production of oxidants when their
production is
warranted, such as in promoting wound healing, lysing bacteria, eliminating
viruses, targeting cancer cells for oxidant-induced lysis and the like
processes,
and 2) for the inhibition of antibody generated oxidants by exposure of
antioxidants when the inhibition of antibody generated oxidants is warranted,
such as in inflammation, heart conditions, diabetes and unwanted cellular
proliferation. For example, one may want to use antibody mediated generation
of superoxide or hydrogen peroxide to supplement the local concentration of
superoxide concentration generated by phagocytic neutrophils to combat a
bacterial infection in a wound. Here the neutrophil that contains NADPH
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oxidase produces superoxide radical in the presence of molecular oxygen. The
superoxide in effect acts as bactericidal agent destroying the bacteria and
ultimately the neutrophil in the process. Thus, to enhance this process, one
would use the method of this invention to provide an antibody composition to
the area to cause an increase in the local concentration of superoxide. On the
other hand, neutrophil-generated superoxide is deleterious in inflamed joints
such as in patients with rheumatoid arthritis who are concomitantly undergoing
intensive humoral antibody-mediated immune responses. In such conditions,
one would want to employ the opposing therapeutic method of this invention in
providing an antioxidant to control the production of damaging oxidants
produced by both neutrophils and antibodies in the local environment. The
decision to use the methods of this invention to inhibit or promote the
antibody-
mediated generation of superoxide and hydrogen peroxide and their derivatives
(i.e., molecules derived therefrom) products and/or their effects is thus
dependent on the desired outcome.
A. Inhibiting Antibody Activity
According to the invention, certain therapeutic methods for
affecting "antibody mediated production of hydrogen peroxide" have been
developed. Thus, the term "antibody mediated production of hydrogen
peroxide" encompasses the reactive species that are both precursor and
derivative to the generation of hydrogen peroxide.
The use of molecules that effect the antibody mediated production of
hydrogen peroxide is applicable to any situation in which unwanted,
deleterious,
damaging production of reactive oxidant species that are generated by
antibodies. The molecules that are useful in these situations are referred to
generally as "antioxidants", defined as any molecule that has an antagonist
effect to an oxidant. An antioxidant so defined includes 1) inhibitors of an
antibody thereby inhibiting superoxide generation, 2) inhibitors of hydrogen
peroxide generation, 3) inhibitors of the reactions converting hydrogen
peroxide
into derivative reactive oxidants; and 4) inhibitors of the reactive oxidants
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themselves. Preferred antioxidants include those that inhibit the activation
of
oxygen producing reactive oxidants as well as those neutralizing those already
formed. The antioxidant effect can occur by any mechanism, including
catalysis. Antioxidants as a category include oxygen scavengers or free
radical
scavengers. Antioxidants may be of different types so they are available if
and
when they are needed. In view of the presence of oxygen throughout an aerobic
organism, antioxidants may be available in different cellular, tissue, organ
and
extracellular compartments. The latter include extracellular fluid spaces,
intraocular fluids, synovial fluid, cerebrospinal fluid, gastrointestinal
secretions,
interstitial fluid, blood and lymphatic fluid. Antioxidants are present within
an
organism but are also provided by supplementing the diet and in the methods of
this invention. Particularly preferred antioxidants include but are not
limited to
ascorbic acid, a-tocopherol, y-glutamylcysteinylglycine, 'y-glutamyl
transpeptidase, a-lipoic acid, dihydrolipoate, -acetyl-5-methoxytryptamine,
flavones, flavonenes, flavanols, catalase, peroxidase, superoxide dismutase,
metallothionein, and butylated hydroxytoluene. A further preferred molecule
that has the capacity to function as an antioxidant in the context of the
methods
of this invention is an engineered antibody in which the ability to generate
superoxide free radical from reducing ringlet oxygen is diminished or
preferably absent altogether. Such antibody molecules are described herein.
The use of antioxidants is directed to situations in which an antioxidant
is required to prevent, control, minimize, reduce, or inhibit the damage of an
oxidant. Thus, the invention contemplates the use of an antioxidant for
reducing the antibody mediated production of hydrogen peroxide in a cell. In
such situations, without intervention, the cellular damage may be so extensive
that tissue injury results, for example, in inflammatory conditions, in trauma
conditions, in organ transplantation and the like. In the context of using an
engineered antibody as an antioxidant, the antibody, having diminished or
substantially no ability to generate superoxide or hydrogen peroxide since it
lacks the reductive centers that reduce ringlet oxygen, provides a therapeutic
benefit in promoting a desired immune response without inducing additional
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tissue damage resulting from excess superoxide production. Preferred
engineered therapeutic antibody compositions retain their antigen binding site
so that targeting to a particular antigen is achieved in concert with the
desired
therapeutic benefits.
The present invention further contemplates a method of ameliorating
oxidative stress in a subject as well as alleviating a symptom in a subject
where
the symptom is associated with production of oxidant. Exemplary of conditions
in which the therapeutic methods of inhibiting the antibody mediated
production of hydrogen peroxide with an antioxidant of the present invention
include but are not limited to inhibiting aberrant smooth muscle disorder,
inhibiting liver disease, proliferation of cancer cells, inhibiting
inflammation in
cancer patients receiving radiotherapy, inflammatory diseases (arthritis,
vasculitis, glomerulonephritis, systemic lupus erythematosus, and adult
respiratory distress syndrome), ischemic diseases (heart disease, stroke,
intestinal ischemia, and reperfusion injury), hemochromatosis, acquired
immunodeficiency syndrome, emphysema, organ transplantation, gastric ulcers,
hypertension, preeclampsia, neurological diseases (multiple sclerosis,
Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and
muscular dystrophy) alcoholism and smoking-related diseases.
Cells in which oxidative stress is deleterious include but are not limited
to endothelial, interstitial, epithelial, muscle (smooth, skeletal or
cardiac),
phagocytic (including neutrophils and macrophages), white blood cells,
dendritic, connective tissue and nervous system cells. Effected tissues
include
but are not limited to muscle, nervous, skin, glandular, mesenchymal, splenic,
sclerous, epithelial and endothelial tissues.
The literature as well as patented inventions describe the use of
antioxidants and oxygen scavengers to treat various conditions induced by
oxidative stress, other than that relating to the generation of oxidants by an
antibody as described in the present invention. Thus, the disclosures of US
Patents 5,362,492; 5,599,712; 5,637,315; 5,647,315; 5,747,026; 5,848,290;
5,994,339; 6,030,61 l and 6,040,611 support the therapeutic uses of
antioxidants
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in the present invention, and as such, the disclosures of which patents are
hereby incorporated by reference.
The oxidants and oxygen scavengers of the invention may be formulated
into a variety of acceptable compositions. In cases where compounds are
sufficiently basic or acidic to form stable nontoxic acid or base salts,
administration of the compounds as salts may be appropriate. Examples of
pharmaceutically acceptable salts are organic acid addition salts formed with
acids that form a physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, -
ascorbate, a-ketoglutarate, and a-glycerophosphate. Suitable inorganic salts
may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate,
and
carbonate salts.
Pharmaceutically acceptable salts are obtained using standard
procedures well known in the art, for example by reacting a sufficiently basic
compound such as an amine with a suitable acid affording a physiologically
acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or
alkaline earth metal (for example calcium) salts of carboxylic acids also are
made.
The oxidants and oxygen scavengers may be formulated as
pharmaceutical compositions and administered to a mammalian host, such as a
human patient in a variety of forms adapted to the chosen route of
administration, i.e., orally or parenterally, by intravenous, intramuscular,
topical
or subcutaneous routes.
Thus, the present compounds may be systemically administered, e.g., .
orally, in combination with a pharmaceutically acceptable vehicle such as an
inert diluent or an assimilable edible carrier. They may be enclosed in hard
or
soft shell gelatin capsules, may be compressed into tablets, or may be
incorporated directly with the food of the patient's diet. For oral
therapeutic
administration, the oxidants and oxygen scavengers may be combined with one
or more excipients and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such
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compositions and preparations should contain at least 0.1 % of active
compound.
The percentage of the compositions and preparations may, of course, be varied
and may conveniently be between about 2 to about 60% of the weight of a given
unit dosage form. The amount of oxidants and oxygen scavengers in such
therapeutically useful compositions is such that an effective dosage level
will be
obtained.
The tablets, troches, pills, capsules, and the like may also contain the
following: binders such as gum tragacanth, acacia, corn starch or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such as corn
starch, potato starch, alginic acid and the like; a lubricant such as
magnesium
stearate; and a sweetening agent such as sucrose, fructose, lactose or
aspartame
or a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring
may be added. When the unit dosage form is a capsule, it may contain, in
addition to materials of the above type, a liquid earner, such as a vegetable
oil
or a polyethylene glycol. Various other materials may be present as coatings
or
to otherwise modify the physical form of the solid unit dosage form. For
instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac
or
sugar and the like. A syrup or elixir may contain the active compound, sucrose
or fructose as a sweetening agent, methyl and propylparabens as preservatives,
a
dye and flavoring such as cherry or orange flavor. Of course, any material
used
in preparing any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the active
compound may be incorporated into sustained-release preparations and devices.
The active compound may also be administered intravenously or
intraperitoneally by infusion or injection. Solutions of the active compound
or
its salts may be prepared in water, optionally mixed with a nontoxic
surfactant.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols,
triacetin, and mixtures thereof and in oils. Under ordinary conditions of
storage
and use, these preparations contain a preservative to prevent the growth of
microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can
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include sterile aqueous solutions or dispersions or sterile powders comprising
the active ingredient that are adapted for the extemporaneous preparation of
sterile injectable or infusible solutions or dispersions, optionally
encapsulated in
liposomes. In all cases, the ultimate dosage form should be sterile, fluid and
stable under the conditions of manufacture and storage. The liquid carrier or
vehicle can be a solvent or liquid dispersion medium comprising, for example,
water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid
polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and
suitable mixtures thereof. The proper fluidity can be maintained, for example,
by the formation of liposomes, by the maintenance of the required particle
size
in the case of dispersions or by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. In many cases, it will be preferable to include
isotonic
agents, for example, sugars, buffers or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the oxidants
and oxygen scavengers in the required amount in the appropriate solvent with
various of the other ingredients enumerated above, as required, followed by
filter sterilization. In the case of sterile powders for the preparation of
sterile
injectable solutions, the preferred methods of preparation are vacuum drying
and the freeze drying techniques, which yield a powder of the oxidants and
oxygen scavengers plus any additional desired ingredient present in the
previously sterile-filtered solutions.
For topical administration, the oxidants and oxygen scavengers may be
applied in pure form, i.e., when they are liquids. However, it will generally
be
desirable to administer them to the skin as compositions or formulations, in
combination with a dermatologically acceptable carrier, which may be a solid
or
a liquid.
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Useful solid carriers include finely divided solids such as talc, clay,
microcrystalline cellulose, silica, alumina and the like. Useful liquid
carriers
include water, alcohols or glycols or water-alcohol/glycol blends, in which
the
present compounds can be dissolved or dispersed at effective levels,
optionally
with the aid of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the properties for a
given use. The resultant liquid compositions can be applied from absorbent
pads, used to impregnate bandages and other dressings, or sprayed onto the
affected area using pump-type or aerosol sprayers.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and
esters, fatty alcohols, modified celluloses or modified mineral materials can
also
be employed with liquid carriers to form spreadable pastes, gels, ointments,
soaps, and the like, for application directly to the skin of the user.
Examples of useful dermatological compositions that can be used to
deliver the oxidants and oxygen scavengers of the present invention to the
skin
are known to the art; for example, see Jacquet et al. (U.S. Pat. No.
4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No.
4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
Useful dosages of the oxidants and oxygen scavengers of the present
invention can be determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of effective dosages
in
mice, and other animals, to humans are known to the art; for example, see U.S.
Pat. No. 4,938,949.
Generally, the concentration of the oxidants and oxygen scavengers of
the present invention in a liquid composition, such as a lotion, will be from
about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a
semi-solid or solid composition such as a gel or a powder will be about 0.1-5
wt-%, preferably about 0.5-2.5 wt-%.
The amount of the oxidants and oxygen scavengers, or an active salt or
derivative thereof, required for use in treatment will vary not only with the
particular salt selected but also with the route of administration, the nature
of
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the condition being treated and the age and condition of the patient and will
be
ultimately at the discretion of the attendant physician or clinician.
In general, however, a suitable dose will be in the range of from about
0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mglkg of body weight
per day, such as 3 to about 50 mg per kilogram body weight of the recipient
per
day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the
range
of 15 to 60 mglkg/day.
The oxidants and oxygen scavengers are conveniently administered in
unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750
mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
Ideally, the oxidants and oxygen scavengers should be administered to
achieve peak plasma concentrations of the active compound of from about 0.5
to about 75 wM, preferably, about 1 to 50 ~,M, most preferably, about 2 to
about
30 wM. This may be achieved, for example, by the intravenous injection of a
0.05 to 5% solution of the oxidants and oxygen scavengers, optionally in
saline,
or orally administered as a bolus containing about 1-100 mg of the oxidants
and
oxygen scavengers. Desirable blood levels may be maintained by continuous
infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions
containing about 0.4-15 mg/kg of the oxidants and oxygen scavengers.
The desired dose may conveniently be presented in a single dose or as
divided doses administered at appropriate intervals, for example, as two,
three,
four or more sub-doses per day. The sub-dose itself may be further divided,
e.g., into a number of discrete loosely spaced administrations; such as
multiple
inhalations from an insufflator or by application of a plurality of drops into
the
eye.
In a preferred embodiment, an antioxidant enters the cell and reacts with
the hydrogen peroxide or its precursor oxygen molecules thereby reducing the
hydrogen peroxide concentration in the cell. In an alternative embodiment, an
antioxidant enters the cell or is present in the surrounding extracellular
milieu
and reacts with the oxidants generated from hydrogen peroxide.
The therapeutic compositions of this invention, the antioxidants
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described herein, antibodies that include both engineered antibodies and other
molecules containing additional reductive centers as described herein for
promoting antibody activity, are administered in a manner compatible with the
dosage formulation, and in a therapeutically effective amount. The quantity to
be administered and timing depends on the subject to be treated, capacity of
the
subject's system to utilize the active ingredient, and degree of therapeutic
effect
desired. Precise amounts of active ingredient required to be administered
depend on the judgement of the practitioner and are peculiar to each
individual.
However, suitable dosage ranges for various types of applications depend on
the
route of administration. Suitable regimes for administration are also
variable,
but are typified by an initial administration followed by repeated doses at
intervals to result in the desired outcome of the therapeutic treatment.
Antioxidants contemplated for use in the present invention are delivered
to the site of interest to mediate the desired outcome in a composition such
as a
liposome, the preparation of which is well known to one of ordinary skill in
the
art of liposome-mediated delivery. Alternative delivery means include but are
not limited to administration intravenously, topically, orally, by inhalation,
by
cannulation, intracavitally, intramuscularly, transdermally, and
subcutaneously.
Therapeutic compositions of the present invention contain a
physiologically tolerable carrier together with an antioxidant as described
herein
or an antibody as described herein for providing antibody activity, dissolved
or
dispersed therein as an active ingredient. In a preferred embodiment, the
therapeutic composition is not immunogenic when administered to a mammal
or human patient for therapeutic purposes.
The preparation of a pharmacological composition that contains active
ingredients dissolved or dispersed therein is well understood in the art and
need
not be limited based on formulation. Typically such compositions are prepared
as injectables either as liquid solutions or suspensions, however, solid forms
suitable for solution, or suspensions, in liquid prior to use can also be
prepared.
The preparation can also be emulsified.
The active ingredient can be mixed with excipients which are
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pharmaceutically acceptable and compatible with the active ingredient and in
amounts suitable for use in the therapeutic methods described herein. Suitable
excipients are, for example, water, saline, dextrose, glycerol, ethanol or the
like
and combinations thereof. In addition, if desired, the composition can contain
minor amounts of auxiliary substances such as wetting or emulsifying agents,
pH buffering agents and the like which enhance the effectiveness of the active
ingredient.
The therapeutic compositions of the present invention can include
pharmaceutically acceptable salts of the components therein. Pharmaceutically
acceptable salts include the acid addition salts (formed with the free amino
groups of the polypeptide) that are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as acetic,
tartaric, mandelic and the like. Salts formed with the free carboxyl groups
can
also be derived from inorganic bases such as, for example, sodium, potassium,
ammonium, calcium or fernc hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and
the like.
Physiologically tolerable carriers are well known in the art. Exemplary
of liquid carriers are sterile aqueous solutions that contain no materials in
addition to the active ingredients and water, or contain a buffer such as
sodium
phosphate at physiological pH value, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous Garners can contain more
than
one buffer salt, as well as salts such as sodium and potassium chlorides,
dextrose, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in addition to and to
the exclusion of water. Exemplary of such additional liquid phases are
glycerin,
vegetable oils such as cottonseed oil, and water-oil emulsions.
Other therapeutic conditions that would benefit from the antioxidant
inhibition of antibody mediated oxidant production in a cell, tissue, or
organs as
well as extracellular compartments are well known to those of ordinary skill
in
the art and have been reviewed by McCord, Am. J. Med., 108:652-659 (2000)
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and Babior et al., Am. J. Med., 109:33-44 (2000), the disclosures of which are
hereby incorporated by reference.
B. Providing Antibody Activity
The present invention also generally contemplates the use of any
antibody to generate superoxide radical or hydrogen peroxide in a situation
where the production of superoxide or hydrogen peroxide is warranted. The
present invention also contemplates the use of engineered molecules including
engineered antibodies that have been altered to contain a reductive center,
the
presence of which provides for the capability to generate superoxide or
hydrogen peroxide from singlet oxygen when such production is desired. In the
case of superoxide, the use of engineered molecules having more than two
reductive centers compared to a non-engineered antibody having the two
conserved tryptophan residues is warranted when enhanced production of
superoxide is needed. Thus, for the therapeutic methods that benefit from a
production of superoxide free radical, also called superoxide, the present
invention contemplates the use of antibodies as defined above that contain the
naturally occurring buried tryptophan residues as well as the engineered
antibodies and other molecules described herein. In the case of hydrogen
peroxide, the use of engineered molecules having additional reductive centers
is
warranted when enhanced production of hydrogen peroxide is needed. Thus,
for the therapeutic methods that benefit from a production of hydrogen, the
present invention contemplates the use of antibodies as defined above that
contain naturally occurring tryptophan residue as well as the engineered
antibodies and other molecules described herein.
The conditions under which hydrogen peroxide or superoxide radical
and its consequent production of hydrogen peroxide is generated by an antibody
is more completely described in examples I and II. The minimum requirement
for generating hydrogen peroxide or superoxide is the presence of oxygen,
i.e.,
aerobic conditions. The biological reduction of singlet oxygen to hydrogen
peroxide or superoxide radical that results in hydrogen peroxide occurs both
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visible light and ultraviolet irradiation conditions. In the former, the
production
of hydrogen peroxide is enhanced in the presence of photosensitizer molecules
such as hematoporphyrin. Moreover, ultraviolet light irradiation is not
essential
for the antibody mediated reduction events. In the absence of light, antibody
mediated production of superoxide or hydrogen peroxide occurs when aerobic
conditions are present along with a superoxide or hydrogen peroxide generating
amount of photosensitizer.
In view of the minimal requirements for the antibody mediated
generation of hydrogen peroxide or superoxide that results in hydrogen
peroxide
production, the present invention contemplates the therapeutic use of an
antibody to create an superoxide or hydrogen peroxide environment where one
does not exist or enhance an already existing one. Such conditions are well
known to practitioners in the art of oxygen cascade chemistry and the
generation of oxidants to provide a desired beneficial outcome such as those
described herein.
In one embodiment, the invention contemplates a method for exposing
an antigen to superoxide and hydrogen peroxide where the antigen is contacted
with a composition including an antibody able to generate hydrogen peroxide or
superoxide from singlet oxygen. As previously discussed, the method is
successful with either nonspecific or immunospecific (antigen directed )
intact
antibody, fragments derived therefrom and further including single chain
antibodies as well as the engineered molecules and antibodies described
herein.
Exemplary concentrations of antibody at the cell surface range from 1 to 5
micromolar. However, the concentration may vary depending on the desired
outcome where the amount of antibody provided is that amount of antibody that
is sufficient to obtain the desired physiological effect, i.e, the generation
of
hydrogen peroxide or superoxide radical and its derivative oxidants to
generate
oxidative stress. Dosing and timing of the therapeutic treatments with
antibody
compositions are compatible with those described for antioxidants above. The
antigen is preferably presented on a cell but need not be so limited. The
antigen
can be any antigen that is present in a cell, tissue or organ including
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extracellular fluids where the presence of superoxide and the antibody
mediated
process of producing it is warranted. In a preferred embodiment, the antigen
is
a fatty acid, a low density lipoprotein, an antigen associated with
inflammation,
a cancer cell antigen, a bacterial antigen or a similar molecule.
Cells on which antigens are associated include but are not limited to
endothelial, interstitial, epithelial, muscle, phagocytic, blood, dendritic,
connective tissue and nervous system cells. Particularly preferred target
cells
for the present therapeutic approach are neutrophils or macrophages.
The invention further contemplates exposing a target cell to irradiation
with either ultraviolet, infrared or visible light in the method of generating
antibody superoxide or hydrogen peroxide.
To enhance the production of superoxide or hydrogen peroxide, a
superoxide or hydrogen peroxide generating amount of a photosensitizer, also
referred to as a sensitizer, is utilized in the therapeutic methods described
herein. As defined herein, a sensitizer is any molecule that induces or
increases
the concentration of singlet oxygen. Sensitizers are generally used in the
presence of irradiation, the process of which includes exposure to
ultraviolet,
infrared or visible light for a period sufficient to activate the sensitizer.
Exemplary exposures are described in examples I and II. A superoxide or
hydrogen peroxide generating amount of sensitizer is the amount of sensitizer
that is sufficient to obtain the desired physiological effect, e.g.,
generation of
superoxide or hydrogen peroxide from singlet oxygen mediated by an antibody
in any situation where superoxide or hydrogen peroxide presence and the
derivatives thereof is warranted. In a preferred embodiment, a sensitizer is
conjugated to the antibody. In a particularly preferred embodiment, a
sensitizer
conjugated antibody is capable of binding to a antigen, i.e., retains an
active
antigen binding site, allowing for antigen recognition and complexing to
occur.
Exemplary sensitizers include but are not limited to pterins, flavins,
hematoporphyrin, tetrakis(4-sulfonatophenyl)porphyrin, bipyridyl
ruthemium(II) complexes, rose Bengal dye, quinones, rhodamine dyes,
phtalocyanine, and hypocrellins.
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In a further embodiment, the generation of superoxide or hydrogen
peroxide is enhanced by administering a means to enhance the production of
ringlet oxygen. Reduced ringlet oxygen is the source of superoxide or
hydrogen peroxide as previously discussed. Such reduction can occur through
the action of an antibody or molecule containing greater than two reductive
centers. One preferred means is referred to as a prodrug that is any molecule,
compound, reagent and the like that is useful in generating ringlet oxygen. A
preferred prodrug is endoperoxide, that is administered at a time subsequent
to
the administering or contacting of an antibody with a desired target cell,
tissue
or organ as described below. In this context, endoperoxide is preferably
delivered after a superoxide or hydrogen peroxide producing antibody or
molecule has immunoreacted with its target antigen forming an antibody-
antigen complex. A preferred concentration of endoperoxide to achieve at the
antibody-antigen complex site is about 10 micromolar. This embodiment has
particular advantages. For example, the ability to create an increased local
accumulation of ringlet oxygen provides the necessary reactant to be reduced
to
the therapeutically desirable superoxide or hydrogen peroxide at a desired
site
or location.
Preferred therapeutic methods based on the use of an antibody including
an engineered antibody or molecule having reductive centers to generate
superoxide or hydrogen peroxide from ringlet oxygen includes a method for
killing a cancer cell where the cancer cell is contacted with a composition
including an antibody capable of generating superoxide or hydrogen peroxide
from ringlet oxygen. In a preferred embodiment, the antibody recognizes and
immunoreacts with an antigen expressed on the cancer cell. Such methods are
therapeutically useful for a subject with lung cancer, prostate cancer, colon
cancer, cervical cancer, endometrial cancer, bladder cancer, bone cancer,
leukemia, lymphoma, or brain cancer. In one aspect, the cancer cell is removed
from a subject with cancer and cultured ex vivo for exposing to an antibody,
and
can further be exposed to ultraviolet light, infrared light or visible light
for the
cell to then be returned to the subject.
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In other aspects, the antibody composition is delivered in vivo to a
subject with cancer. Preferred ifa vivo delivery methods include
administration
intravenously, topically, by inhalation, by cannulation, intracavitally,
intramuscularly, transdermally, subcutaneously or by liposome containing the
antibody.
In still further aspects, the antibody is a recombinant antibody, that is
provided as above or alternatively is expressed from an expression vector
delivered to the cell. The expression vector in this context can also express
a
sensitizer molecule.
Therapeutic compositions in pharmaceutically acceptable excipients and
pharmaceutically effective amounts as described for antioxidant containing
compositions are applicable to the use of antibody containing compositions.
Additional therapeutic methods based on using an antibody that is able
to generate superoxide or hydrogen peroxide from singlet oxygen are 1) for
inhibiting proliferation of a cancer cell, 2) for targeting and killing a
cancer cell
in a patient where the antibody recognizes and immunoreacts with an antigen
expressed on the cancer cell, 3) for inhibiting tissue injury associated with
neutrophil mediated inflammation in a subject, for example where the
inflammation results from a bacterial infection or when the subject has an
autoimmune disease, 4) for enhancing the bactericidal effectiveness of a
phagocyte in a subject, 5) for promoting wound healing in a subject having a
open wound where the superoxide or hydrogen peroxide stimulates fibroblast
proliferation and/or the immune response that further includes lymphocyte
proliferation, 6) for stimulating cell proliferation, such as stimulating
fibroblast
proliferation in a wound in a subject, and the like situations. For wound
healing, topical application to a wound on a subject is a preferable delivery
approach such as with a bandage containing an antibody. Other therapeutic
conditions that would benefit from the creation or enhancement of superoxide
or hydrogen peroxide iri a cell, tissue, organ or extracellular compartment
are
well known to those of ordinary skill in the art and have been reviewed by
McCord, Am. J. Med., 108:652-659 (2000), the disclosure of which are hereby
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incorporated by reference.
2. Screening Methods
The invention further contemplates screening methods that are
based on the newly discovered antibody reduction of singlet oxygen to
hydrogen peroxide or superoxide radical.
Thus, in one embodiment, the invention contemplates a method for
identifying an agent that modulates antibody mediated production of hydrogen
peroxide or superoxide. A modulator is a molecule that either inhibits or
promotes the production of superoxide or hydrogen peroxide. Either type of
modulator is identifiable with the same method. In a preferred embodiment, the
method includes the steps of:
a) contacting a composition comprising an antibody capable of
generating superoxide or hydrogen peroxide with an agent to form an admixture
in an assay solution in the presence of molecular oxygen;
b) irradiating the admixture to generate singlet oxygen from
molecular oxygen, wherein the singlet oxygen is reduced to hydrogen peroxide
or superoxide by the antibody, wherein the superoxide dismutates to form
hydrogen peroxide;
c) detecting the formed hydrogen peroxide; and
d) comparing the detected hydrogen peroxide with a suitable
control, thereby determining how the agent modulates the production of
hydrogen peroxide or superoxide.
The irradiating step is performed with either ultraviolet light or visible
light. With the latter form, a sensitizer as previously described can be added
with the antibody composition.
The formed hydrogen peroxide is detected through reaction directly with
a hydrogen peroxide where the reacted substrate is detected with a fluorescent
means, such as with fluorescent microscopy or fluorescent spectrometry. In
fluorescent spectrometry, detection is ELISA based or with done with a
standard cuvette. Exemplary assay methods are performed as described in
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examples I and II.
In a separate screening method of the present invention, a method for
performing an immunoassay to detect antibody immunoreactivity with an antigen
is
also contemplated based on the discovery of antibody generated superoxide or
hydrogen peroxide. The method comprises the steps of:
a) contacting in a singlet oxygen-generating medium a substrate having
immobilized thereon a composition comprising a first reagent comprising an
antigen
or an antibody, with a second composition comprising an antigen or an antibody
that
is reactive with the first reagent to form an immobilized antigen-antibody
complex,
wherein the antibody generates superoxide or hydrogen peroxide from singlet
oxygen in the presence of oxygen; and
b) detecting the antibody-generated superoxide or hydrogen peroxide,
thereby detecting the antibody immunoreactivity with the antigen.
The reaction and detection means are those as described herein. In one
aspect, the first composition is an antigen and the second composition is an
antibody.
In the opposite aspect, the first composition is an antibody and the second
composition is an antigen.
The invention further contemplates a similar method for performing an
immunoassay to detect antibody immunoreactivity with an antigen where an
antigen
is immobilized and contacted with an antibody composition.
Such immunoassay methods are an improvement over those that are well
known as methods to assess antigen-antibody immunoreactivity and to identify
antigens and/or antibodies. The advantage of the present method over previous
other
immunoassay methods lies in the present elimination of at least one method
step
and/or the incorporation of a secondary labeled immunoreactive molecule, the
labeling either being a radioactive or enzymatic compound.
In the present invention, the minimum requirements are oxygen, an antibody
reagent, an antigen reagent, and a detectable reactant that reacts with
hydrogen
peroxide generated from the antibody. A preferred reactant is a fluorogenic
substrate. One such reactant used as described in examples I and II is called
AMPLEXTM Red. It is a commercially available reagent sold by Molecular Probes
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(Eugene, Oregon) for reacting antibody generated hydrogen peroxide in the
immunoassay. It is sold in a kit that provides a one-step fluorometric method
for
measuring hydrogen peroxide using a fluorescent microplate or fluorimeter for
detection. The assay is based on the detection of hydrogen peroxide using 10-
acetyl-
3,7-dihyroxyphenoxazine, a highly sensitive and stable probe for hydrogen
peroxide.
In the presence of horseradish peroxidase, the AMPLEXTM Red reagent reacts
with
hydrogen peroxide in a 1:1 stoichiometry to produce highly fluorescent
resorufin,
that provides a detection mechanism to detect as little as 10 picomoles of
hydrogen
peroxide in a 200 microliter volume.
In contrast, prior immunoassay techniques, including radioimmunoassays
(RIA), enzyme-immunoassays (EIA), and the classic enzyme-linked immunosorbent
assay (ELISA), all require either the use of a radioactively labeled
immunoreactive
molecule as in RIA or an additional labeled immunoreactive molecule. The
present
invention neither requires potentially harmful radioactive isotopes to label a
molecule or requires an additional immunoreactive reagent that generally is
referred
to as a secondary antibody that is usually conjugated with an enzyme to allow
for the
detection of the complex formed with the first antibody with the antigen. In
the
latter assays, the reaction of the secondary antibody with the formed antigen-
antibody complex (generally through an anti-first antibody specificity
immunoreactivity) is detected through a color-producing substrate solution
specific
for the conjugated enzyme. In summary, in the present invention, the antibody
mediated generation of hydrogen peroxide is detected with high detection
capacity
without radioactive agents, without requiring an additional reagent and/or
admixing
step such as those practiced in US Patents 3,905,767; 4,016,043; USRE032696;
and
4,376,110, the disclosures of which are hereby incorporated by reference.
3. Therapeutic Compositions
The present invention contemplates therapeutic compositions useful
in practicing the therapeutic methods as described above. Antibodies, as a
class of
proteins, are now known to act as reductants in reducing singlet molecular
oxygen
(also referred to herein as singlet oxygen) to generate superoxide free
radical (also
referred to herein as superoxide). As a result of the redox reaction, the
antibody
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becomes oxidized. The oxidation of the antibody is now known to occur at the
two
buried tryptophan residues as further discussed in example I. The activity is
further
ascribed to the indole component of the tryptophan residue. Thus, in view of
the
redox reaction where the indole portion becomes oxidized forming a radical
cation in
the reaction of reducing singlet molecule oxygen to superoxide free radical,
the
indole is referred to as a reductive center. A reductive center as defined in
the
present invention as having the ability to reduce singlet oxygen to superoxide
and
becoming oxidized in the process. Preferably, a reductive center is more
efficient if
it is not solvent-exposed, i.e., is buried within the therapeutic composition
defined
herein.
Therapeutic compositions may also be produced and used according to the
therapeutic methods described above with antibodies and engineered molecules
that
produce hydrogen peroxide through oxidation of water. Antibodies, as a class
of
proteins, are now known to catalyze the oxidation of water to produce hydrogen
peroxide. The activity is ascribed to a conserved tryptophan residue.
Thus, the present invention contemplates therapeutic compositions that are
useful in either acting to reduce the local concentration of hydrogen peroxide
or
superoxide production or in the alternative useful in acting to enhance it.
Such
compositions contain reagents referred to generally as being "engineered",
defined
herein to connote a reagent, such as an antibody or fragment thereof as
defined
herein, or other molecule, that has been altered in some form to either
increase or
decrease the number of reductive centers as defined herein.
The invention thus contemplates an antibody that has been engineered to
have at least a diminished capacity to generate hydrogen peroxide or
superoxide free
radical from singlet oxygen. In that context, the antibody lacks at least one
of its
reductive centers and preferably is substantially free of a reductive center.
Such
antibody compositions are readily prepared with recombinant expression methods
well known to one of ordinary skill in the art. In preferred embodiments, the
antibody retains the same amino acid residue number but the reductive center
has
been replaced or substituted with a component that lacks the ability to reduce
singlet
oxygen. In such aspects, the reductive center comprises a buried indole and
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preferably, in the case of superoxide, two buried indoles. In particularly
preferred
embodiments, the reductive center comprises an indole on a tryptophan residue
that
is substituted by another amino acid that does not have reductive capacity.
Such
preferred substitutions includes the amino acids phenylalanine and alanine. In
other
aspects, the present invention also contemplates deletion of the tryptophan
without
replacement or substitution thereof as long as the desired antibody activity,
particularly antigen binding activity, is not adversely affected. As
previously
discussed, an engineered antibody having reduced or absent reductive centers
while
retaining antigen targeting ability provides the therapeutic advantage of
providing an
antibody to stimulate a desired immune response in particular situations while
reducing or eliminating altogether the undesirable production of hydrogen
peroxide
or superoxide and its byproducts that can further damage cells and tissues.
Methods
for making an engineered antibody that functions as an antioxidant in the
context of
the therapeutic methods described herein are well known in the art, such as
site-
directed mutagenesis of a nucleotide sequence encoding the antibody of
interest as
previously discussed.
Engineered antibodies that function as an antioxidant according to the
methods of the invention are contemplated for any of the methods as described
herein.
The present invention also contemplates engineered therapeutic molecules
including engineered antibodies that have been altered to contain a reductive
center
where they were in an insufficient amount to effect adequate production of
superoxide or hydrogen peroxide, or where they are needed to increase the
number
of reductive centers to a number in excess of those that were naturally
occurring in
the molecule or antibody. Introduction of a reductive center in a engineered
molecule or antibody is accomplished by methods well known to one of ordinary
skill in the art. Preferred means including recombinant expression methods and
well
as direct protein synthesis methods have been previously described. The choice
of
method is necessarily dependent on the length of the molecule being
engineered.
Regardless of the methods employed, the positioning, i.e., the location, of
the
engineered reductive center is based upon the ability of the engineered
molecule to
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exhibit reducing activity on singlet oxygen. Preferably, the incorporation of
reductive centers are positioned such that they are deeply buried in the
folded
molecule allowing a retention of structural ability without comprising
superoxide or
hydrogen peroxide production. In one embodiment, in an antibody where it is
desired to retain antigen binding function, the location of an engineered
reductive
center is adjacent to a variable binding domain. In certain aspects, one
reductive
center is contemplated. In other aspects, two reductive centers are
contemplated.
Still, in other aspects, more than three reductive centers are contemplated.
Preferably, the reductive centers comprise indole. Also contemplated are
reductive
centers comprising indole present in tryptophan residue. Any technique to
engineer
such reductive centers in a molecule or antibody is contemplated for use in
the
present invention. In a preferred embodiment, the reductive centers are
introduced
by site-directed mutagenesis of nucleotide sequences encoding the engineered
antibody such that the substituted nucleotides encode tryptophan residues at
predetermined locations in the encoded molecule.
In the embodiment of preparing an engineered molecule such as an antibody
to include desired reductive centers, such molecule that is produced by
recombinant
technology is also contemplated to be in the form of a fusion conjugate, where
the
conjugate provides a sensitizer molecule as previously described for use in
therapeutic methods as described herein.
Engineered antibodies or other molecules, which can be any protein or
polypeptide such that they contain reductive centers that function according
to the
methods of the invention, are contemplated for any of the methods as described
herein.
The invention is further described in detail by reference to the non-limiting
examples that follow. While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be understood that
modifications and variations are within the spirit and scope of that which is
described and claimed.
Examble I
Antibodies have the intrinsic cayacity to destroy anti~Lens
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Materials and Methods
Antibodies: The following whole antibodies were obtained from
PharMingen: 49.2 (mouse IgGzb x), 6155-178 (mouse IgGza x), 107.3 (mouse IgG,
x), A95-1 (rat IgGZb), 6235-2356 (hamster IgG), R3-34 (rat IgG x), R35-95 (rat
IgG2a x), 27-74 (mouse IgE), A110-1 (rat IgG~ ~,), 145-2C11 (hamster IgG
groupl
x), M18-254 (mouse IgA x), and MOPC-315 (mouse IgA ~.). The following were
obtained from Pierce: 31243 (sheep IgG), 31154 (human IgG), 31127 (horse IgG),
and 31146 (human IgM).
The following F(ab')2 fragments were obtained from Pierce: 31129 (rabbit
IgG), 31189 (rabbit IgG), 31214 (goat IgG), 31165 (goat IgG), and 31203 (mouse
IgG). Protein A, protein G, trypsin-chymotrypsin inhibitor (Bowman-Birk
inhibitor), (3-lactoglobulin A, a-lactalbumin, myoglobin, (3-galactosidase,
chicken
egg albumin, aprotinin, trypsinogen, lectin (peanut), lectin (Jacalin), BSA,'
superoxide dismutase, and catalase were obtained from Sigma. Ribonuclease I A
was obtained from Amersham Pharmacia. The following immunoglobulins were
obtained in-house using hybridoma technology: OB2-34C12 (mouse IgG~ x),
SHO1-4169 (mouse IgG, x), OB3-14F1 (mouse IgG2a x), DMP-15612 (mouse
IgG2a x), AD1-1961 (mouse IgG2b x), NTJ-92C12 (mouse IgG~ x), NBA-SG9
(mouse IgGI x), SPF-12H8 (mouse IgG2a x), TIN-6C11 (mouse IgG2a x), PRX-1B7
(mouse IgGZa x), HAS-19A11 (mouse IgG2a x), EP2-1962 (mouse IgG~ x), GNC-
92H2 (mouse IgG~ x), WD1-666 (mouse IgG~ x), CH2-SH7 (mouse IgGZb x), PCP-
21H3 (mouse IgG~ x), and TM1-87D7 (mouse IgG, x). DRB polyclonal (human
IgG) and DRB-bl2 (human IgG) were supplied by Dennis R. Burton (The Scripps
Research Institute). 1 D4 Fab (crystallized) was supplied by Ian A. Wilson
(The
Scripps Research Institute).
All assays were carried out in PBS (10 mM phosphateJ160 mM sodium
chloride, pH 7.4). Commercial protein solution samples were dialyzed into PBS
as
necessary. Amplex Red hydrogen peroxide assay kits (A-12212) were obtained
from Molecular Probes.
Antibody/Protein Irradiation. Unless otherwise stated, the assay solution
(100 ~.1, 6.7 ~,M protein in PBS, pH 7.4) was added to a glass vial, sealed
with a
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screw-cap, and irradiated with either UV (312 nm, 8000 ~Wcm Z Fischer-Biotech
transilluminator) or visible light.
Quantitative Assay for Hydro~en Peroxide. An aliquot (20 ~,l) from the
protein solution was removed and added into a well of a 96-well microtiter
plate
(Costar) containing reaction buffer (80 ~,l). Working solution (100 x.1/400
~.M
Amplex Red reagent 1 /2 units/ml horseradish peroxidase) was then added, and
the
plate was incubated in the dark for 30 min. The fluorescence of the well
components
was then measured using a CytoFluor Multiwell Plate Reader (Series 4000,
PerSeptive Biosystems, Framingham, MA; Ex/Em: 530/580 nm). The hydrogen
peroxide concentration was determined using a standard curve. All experiments
were run in duplicate, and the rate is quoted as the mean of at least two
measurements.
Sensitization and Ouenchin~ Assay. A solution of 31127 (100 ~1 of horse
IgG, 6.7 ~.M) in PBS (pH 7.4, 4% dimethylformamide) and hematoporphyrin IX (40
~M) was placed in proximity to a strip light. Hydrogen peroxide concentration
was
determined as described herein. The assay was also performed in the presence
of
NaN3 (100 mM) or PBS in D20.
Oxygen Dependence. A solution of 31127 ( 1.6 ml, horse IgG, 6.7 ~,M) in
PBS (pH 7.4) was rigorously degassed using the freeze/thaw method under argon.
Aliquots (100 ~,l) were introduced into septum-sealed glass vials that had
been
purged with the appropriate OZ/Ar mixtures (0-100%) via syringe. Dissolved
oxygen concentrations were measured with an Orion 862A dissolved oxygen meter.
These solutions were then vortexed vigorously, allowed to stand for 20 min,
and
then vortexed again. A syringe containing the requisite OZ/Ar mixture was used
to
maintain atmospheric pressure during the course of the experiment. Aliquots
(20 ~,l)
were removed using a gas-tight syringe and hydrogen peroxide concentration
measured as described herein. The data from three separate experiments were
collated and analyzed using the Enzyme Kinetics v1 .l computer program (for
determination of VmaX and Km parameters).
Antibody Production of Hydro~en Peroxide in the Dark Using a Chemical
'-OZ Source. A solution of sheep IgG 31243 (100 ~l, 20 wM) in PBS- (pH 7.4)
and the
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endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate (25 mM in DZO)
was placed in a warm room (37°C) for 30 min in the dark. Hydrogen
peroxide
concentration was determined as described herein.
Hydrogen Peroxide Formation by the FablD4 C ,rte. A suspension of
crystals of the Fab fragment of 1D4 (2 ~l) was diluted with PBS (198 ~.1, pH
7.4) and
vortexed gently. Following centrifugation, the supernatant was removed, and
the
washing procedure was repeated twice further. The residual crystal suspension
was
then diluted into PBS, pH 7.4 (100 ~.1), and added into a well of a quartz
ELISA
plate. Following UV irradiation for 30 min, Amplex Red working solution (100
~.l)
was added, and the mixture was viewed on a fluorescence microscope.
Antibody Fluorescence Versus H~~en Peroxide Formation. A solution of
31127 (1.0 ml of horse IgG, 6.7 ~,M) in PBS (pH 7.4) was placed in a quartz
cuvette
and irradiated with LTV light for 40 min. At 10-min intervals, the
fluorescence of the
solution was measured using an SPF-SOOC spectrofluorimeter (SLM-Aminco,
Urbana, IL; Ex/Em, 280/320). At the same time point, an aliquot (20 ~l) of the
solution was removed, and the hydrogen peroxide concentration was determined
as
described herein.
Consumption of Hydrogen Peroxide by Catalase. A solution of EP2-19612
(100 ~,1 ofmouse IgG, 20 wM in PBS, pH 7.4) was irradiated with UV light for
30
min, after which time the concentration of hydrogen peroxide was determined by
stick test (EM Quant Peroxide Test Sticks) to be 2 mg/liter. Catalase [1 ~,1,
Sigma,
3. 2 M (NH4)ZS04, pH 6.0] was added, and after 1 min, the concentration of
H202
was found to be 0 mg/liter.
Denaturation. IgG 19612 (100 ~,1, 6.7 ~,M) was heated to 100°C in
an
Eppendorf tube for 2 min. The resultant solution was transferred to a glass,
screw-
cap vial and irradiated with UV light for 30 min. The concentration of HZOZ
was
determined after 30 min.
Results and Discussion
Research throughout the last century has led to a consensus as to the strategy
of the humoral component of the immune system. The essence is that, for
killing,
the antibody molecule activates additional systems that respond to antibody-
antigen
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union. It is now reported that the immune system has a previously unrecognized
chemical potential intrinsic to the antibody molecule itself. All antibodies
studied,
regardless of source or antigenic specificity, can convert molecular oxygen
into
hydrogen peroxide, thereby potentially aligning recognition and killing within
the
same molecule. Aside from pointing to a new chemical arm for the immune
system,
these results are thought to be important to the understanding of how
antibodies
evolved and what role they may play in human diseases.
The antibody is a remarkable adaptor molecule, having evolved both
targeting and effector functions that place it at the frontline of vertebrate
defense
against foreign invaders (Burton, D. R., Trends Biochem. Sci., 15, 64-69
(1990)). In
terms of the effector mechanism, the central idea is that antibodies
themselves do not
possess destructive ability but mark foreign substances for removal by the
complement cascade and/or phagocytosis (Arlaud et al., Immunol. Today, 8, 106-
111 (1987); Sim & Reid, Immunol. Today, 12, 307-311 (1991)).
The advent of antibody catalysis has demonstrated that antibodies are capable
of much more complex chemistry than simple binding (Wentworth & Janda, Curr.
Opin. Chem. Biol., 2, 138-144 (1998)). This has inevitably led to the question
as to
whether more sophisticated chemical mechanisms are part of the strategy of the
antibody molecule itself. Thus far, there has been no evidence to support this
idea,
and we are left with the notion that just because antibodies are capable of
complex
chemistry, it does not mean that they use it in host defense. However, it is
now
reported that a hitherto unremarked capacity of antibodies to convert
molecular
oxygen into hydrogen peroxide, thereby effectively linking recognition and
killing
events.
The preliminary step in the phagocytic oxidative burst is the single electron
reduction of ground-state molecular oxygen (302) by the NADPH-dependent
transmembrane phagocyte oxidase enzyme system that generates superoxide anion
(OZ'-) (Figure 1) (Klebanoff, S. J. in Enc~pedia of Immunolo~y, eds. Delves,
P. J.
& Roitt, I. M. (Academic, San Diego), pp. 1713-1718 (1998); Rosen, H. &
Klebanoff, S. J., J. Biol. Chem., 252, 4803-4810 (1997)).
Superoxide anion occupies a critical position in the cycling of oxygen-
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dependent micxobicidal agents in vivo because although it is not itself
considered to
be cytotoxic (Fee, J. A. in International Conference on Oxy~en and Oxygen-
Radicals, eds. Rodgers, M. A. J. & Powers, E. L. (Academic, San Diego, and
University of Texas at Austin), pp. 205-239 (1981)), it is a direct precursor
of
hydrogen peroxide and the toxic derivatives it spawns, such as hydroxyl
radical
(HO') and hypohalous acid (HOCl). In addition, when iron concentrations are
limiting, OZ'- is a vital reducing agent that regenerates Fe2+, thus
facilitating the
iron-catalyzed Haber-Weiss reaction, or the so-called superoxide-driven Fenton
reaction that produces HO' (Esq. 1 and 2). Therefore, processes that
facilitate the
generation of OZ'- will ultimately perpetuate and potentiate oxygen-dependent
microbicidal action.
Fe3+ + Oz'- --~ Fez+ + OZ [ 1 ]
Fez+ + HzOz --~ Fe3+ + -OH + HO' [2]
Another key component of the oxygen-scavenging cascade is singlet
molecular oxygen (' OZ). This particularly reactive species is an excited
state of
molecular oxygen in which both outer shell electrons are spin-paired (Kearns,
D. R.,
Chem. Rev., 71, 395-427 (1971)). It is~important in pathological biological
systems
and has a very short life-time (ca. 4 ~,s) isz vivo (Foote, C. S. in Free
Radicals in
Biolo~y, ed. Pryor, W. A. (Academic, New York), pp. 85-133 (1976)). Generation
of'OZ during microbicidal processes is either direct, via the action of
flavoprotein
oxidases (Allen, R. C., Stjernholm, R. L., Benerito, R. R. & Steele, R. H.,
eds.
Cormier, M. J., Hercules, D. M. & Lee, J. (Plenum, New York), pp. 498-499
(1973);
I~lebanoff, S. J. in The Pha _~ocytic Cell in Host Resistance (National
Institute of
Child Health and Human Development, Orlando, FL) (1974)), or indirect, via the
nonenzymatic disproportionation of OZ'- in solutions at low pH, as found in
the
phagosome (Eq. 3) (Stauff, J., Sander, U. & Jaeschke, W., Chemiluminescence
and
Bioluminescence, eds., Williams, R. C. & Fudenberg, H. H. (Intercontinental
Medical Book Corp., New York), pp. 131-141 (1973); Allen, R. C., Yevich, S.
J.,
Orth, R. W. & Steele, R. H., Biochem. Biophys. Res. Commun., 60, 909-917
(1974)).
Oz'- + 2H0'2 -~ 'OZ + HZOZ [3]
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The high reactivity of'Oz with biomolecules has meant that it is generally
considered to be an endpoint in the cascade of oxygen-scavenging agents.
However,
it has been found that antibodies, as a class of proteins, have the intrinsic
ability to
intercept'02 and efficiently reduce it to Oz'-, thus offering a mechanism by
which
oxygen can be rescued and recycled during phagocyte action, thereby
potentiating
the microbial action of the immune system.
The measured values for the initial rate of formation of hydrogen peroxide by
a panel of intact immunoglobulins and antibody fragments are collected in
Table 1.
It is believed that Ig-generated OZ'- dismutates spontaneously into HzOz,
which is
then utilized as a cosubstrate with N acetyl-3,7-dihydroxyphenazine 1 (Amplex
Red)
for horseradish peroxidase, which produces the highly fluorescent resorufin 2
(excitation maxima 563 nm, emission maxima 587 nm) (Figure 2) (Zhou, M., Diwu,
Z., Panchuk-Voloshina, N. & Haugland, R. P., Anal. Biochem., 253, 162-168 '
(1997)). To confirm that irradiation of the buffer does not generate OZ'' and
that the
antibodies are not simply acting as protein dismutases (Petyaev, I. M. & Hunt,
J. V.,
Redox Report, 2, 365-372 (1996)), the enzyme superoxide dismutase was
irradiated
in PBS. Under these conditions, the rate of hydrogen peroxide generation is
the
same as irradiation of PBS alone.
Table 1. Production of hydrogen peroxide* by immunoglobulins
Entry Clone Source Isotype Rate,t
nmol/min/mg
1 CH25H7 Mouse IgG2b, x 0.25
2 WD16G6 Mouse IgGl, x 0.24
3 SHO-14169 Mouse IgGl, x 0.26
4 OB234C12 Mouse IgGl, x 0.22
5 OB314F1 Mouse IgG2a, K 0.23
6 DMP15G12 Mouse IgG2a, x 0.18
7 AD19G1 Mouse IgG2b, x 0.22
8 NTJ92C12 Mouse IgGl, x 0.17
9 NBA5G9 Mouse IgGl, x 0.17
10 SPF12H8 Mouse IgG2a, x 0.29
11 TIN6C11 Mouse IgG2a, x 0.24
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Entry Clone Source Isotype Rate,
nmol/min/mg
12 PRX1B7 Mouse IgG2a, x 0.22
13 HA519A4 Mouse IgGl, x 0.20
14 92H2 Mouse IgGl, x 0.41
15 1962 Mouse IgGl, x 0.20
16 PCP-21H3 Mouse IgGl, x 0.97
17 TMl-87D7 Mouse IgGl, x 0.28
18 49.2 Mouse IgG2b, x 0.24
19 27-74 Mouse IgE, std. isotype0.36
20 M18-254 Mouse IgA, x 0.39
1021 MOPC-315 Mouse IgA, ~, 0.39
22 31203 Mouse F(ab')Z 0.21
23 b 12 Human IgG 0.45
24 polyclonal Human IgG 0.34
25 . 31154 Human IgG 0.18
1526 31146 Human IgM 0.22
27 R3-34 Rat IgGl, x 0.27
28 R35-95 Rat IgG2a, x 0.17
29 A95-1 Rat IgG2b 0.15
30 A110-1 Rat IgGl, ~, 0.34
2031 6235-2356 Hamster IgG 0.24
32 145-2C11 Hamster IgG, gp 1, x 0.27
33 31243 Sheep IgG 0.20
34 31127 Horse IgG 0.18
35 polyclonal Horse IgG 0.34
2536 31229 Rabbit F(ab')Z 0.19
37 31189 Rabbit F(ab')2 0.14
38 31214 Goat F(ab')Z 0.24
39 31165 Goat F(ab')Z 0.25
30 ~ Assay conditions are described in Materials a~cd Methods.
fi Mean values of at least two determinations. The background rate of HZOZ
formation is 0.005 nmol/min in PBS and 0.003 nmlmin in PBS with SOD.
35 The rates of hydrogen peroxide formation were linear for more than 10% of
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_q.8_
the reaction, with respect to the oxygen concentration in PBS under ambient
conditions (275 ~,M). With sufficient oxygen availability, the antibodies can
generate at least 40 equivalents of H20z per protein molecule without either a
significant reduction in activity or structural fragmentation. An example of
the
initial time course of hydrogen peroxide formation in the presence or absence
of
antibody 1962 is shown in Figure 3A. This activity is lost following
denaturation of
the protein by heating.
The data in Table 1 reveal a universal ability of antibodies to generate HZOZ
from'O2. This function seems to be shared across a range of species and is
independent of the heavy and light chain compositions investigated or antigen
specificity. The initial rates of hydrogen peroxide formation for the intact
antibodies
is highly conserved, varying from 0.15 nmol/min/mg [clone A95-1 (rat IgG2b)]
to
0.97 nmol/min/mg (clone PCP-21 H3, a murine monoclonal IgG) across the whole
panel. Although the information available is more limited for the component
antibody fragments, the activity seems to reside in both the Fab and F(ab')z
fragments.
If this activity were due to a contaminant, it would have to be present in
every antibody and antibody fragment obtained from diverse sources. However,
to
further rule out contamination, crystals of the murine antibody 1 D4 Fab from
which
high-resolution x-ray structures have been obtained (at 1.7 A) were
investigated for
their ability to generate Hz02 (Figure 4). Reduction of'OZ is clearly observed
in
these crystals.
Investigations into this antibody transformation support ringlet oxygen as the
intermediate being reduced. No formation of hydrogen peroxide occurs with
antibodies under anaerobic conditions either in the presence or absence of W
irradiation. Furthermore, no generation of hydrogen peroxide occurs under
ambient
aerobic conditions without irradiation. Irradiation of antibodies with visible
light in
the presence of a known photosensitizer of 30z in aqueous solutions (Kreitner,
M.,
Alth, G., Koren, H., Loew, S. & Ebermann, R., Anal. Biochem., 213, 63-67
(1993)),
hematoporphyrin (HP), leads to hydrogen peroxide formation (Figure SA). The
curving in the observed rates is due to consumption of oxygen from within the
assay
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mixture. Concerns that the interaction between photoexcited HP and oxygen may
be
resulting in OZ~- formation (Beauchamp, C. & Fridovich, L, Anal. Biochem., 44,
276-287 (1971); Srinivasan, V. S., Podolski, D., Westrick, N. J. & Neckers, D.
C., J.
Am. Chem. Soc., 100, 6513-6515 (1978)) were largely discounted by suitable
background experiments with the sensitizer alone (data shown in Figure SA).
The
efficient formation of H202 with HP and visible light both reaffirm the
intermediacy
of'OZ and show that IJV radiation is not necessary for the Ig to perform this
reduction.
Furthermore, incubation of sheep antibody 31243 in the dark at 37°C,
with a
chemical source of'Oz [the endoperoxide of 3',3'-(1,4-
naphthylidene)dipropionate]
leads to hydrogen peroxide formation.
The rate of formation of HZOz, by horse IgG with HP (40 ~,M) in visible
light, is increased in the presence of Dz0 and reduced with the'02 quencher
NaN3
(40 mM) (Figure SB) (Hasty, N., Merkel, P. B., Radlick, P. & Kearns, D. R.
Tetrahedron Lett., 49-52 (1972)). The substitution of D20 for H20 is known to
promote'OZ mediated processes via an increase of approximately 10-fold in its
lifetime (Merkel, P. B., Nillson, R. ~z Kearns, D. R., J. Am. Chem. Soc., 94,
1030-
1031 (1972)).
The rate of hydrogen peroxide formation is proportional to IgG concentration
between 0.5 and 20 ~M but starts to curve at higher concentrations (Figure
SC). The
lifetime of'OZ in protein solution is expected to be lower than in pure water
due to
the opportunity for reaction. It is therefore thought that the observed
curvature may
be due to a reduction in the lifetime of'OZ due to reaction with the antibody.
Significantly, the effect of oxygen concentration on the observed rate of HZOZ
production shows a significant saturation about 200 ~,M of oxygen (Figure SD).
Therefore, the mechanism of reduction may involve either one or more oxygen
binding sites within the antibody molecule. By treating the raw rate data to
nonlinear regression analysis and by fitting to the Michaelis-Menten equation,
a
Kmapp(OZ) of 187 wM and a VmaXapp of 0.4 nmollmin/mg are obtained. This
antibody rate is equivalent to that observed for mitochondrial enzymes that
reduce
molecular oxygen in vivo.
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The mechanism by which antibodies reduce'OZ is still being determined.
However, the participation of a metal-mediated redox process has been largely
discounted because the activity of the antibodies remains unchanged after
exhaustive
dialysis in PBS containing EDTA (4 mM). This leaves the intrinsic ability of
the
amino acid composition of the antibodies themselves. Aromatic amino acids such
as
tryptophan (Trp) can be oxidized by'OZ via electron transfer (Grossweiner, L.
L,
Curr. Top. Radiat. Res. Q., 11, 141-199 (1976)). In addition, disulfides are
sufficiently electron rich that they can also be oxidized (Bent. D. V. &
Hayon, E., J.
Am. Chem. Soc., 87, 2612-2619 (1975)). ~ Therefore, there is the potential
that Trp
residues and/or the intrachain or interchain disulfide bonds homologous to all
antibodies are responsible for'OZ reduction. To both investigate to what
extent this
ability of antibodies is shared by other proteins and to probe the mechanism
of
reduction, a panel of other proteins was studied (Figure 6).
Whereas other proteins can convert'OZ into OZ'-, in contrast to antibodies it
is by no means a universal property. RNase A and superoxide dismutase, which
do
not possess Trp residues but have several disulfide bonds, do not reduce'O2.
Similarly, the Bowman-Birk inhibitor protein (Voss, R.-H., Ermler, U., Essen,
L.-O.,
Wenzl, G., Kim, Y.-M. & Flecker, P., Eur. J. Biochem., 242, 122-131 (1996);
Baek,
J. & Kim, S., Plant Ph, sue, 102, 687 (1993)) that has seven disulfide bonds
and
zero Trp residues does not reduce'OZ. In contrast, chick ovalbumin, which has
only
2 Trp residues (Feldhoff, R. & Peters, T. J., Biochem. J., 159, 529-533
(1976)), is
one of the most efficient proteins at reducing'O2.
Given the loss of antibody activity upon denaturation, the location of key
residues in the protein is likely to be more critical than their absolute
number.
Because the majority of aromatic residues in proteins are generally buried to
facilitate structural stability (Burley, S. K. & Petsko, G. A., Science, 229,
23-28
(1985)), the nature of the reduction process was explored in terms of relative
contribution of surface and buried residues by fluorescence-quenching
experiments.
Aromatic amino acids in proteins are modified by the absorption of ultraviolet
light,
especially in the presence of sensitizing agents such as molecular oxygen or
ozone
(Foote, C. S., Science, 162, 963-970 (1968); Foote, C. S., Free Radicals
Biol., 2, 85-
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133 (1976); Gollnick, K., Adv. Photochem., 6, 1-122 (1968)). Trp reacts
with'OZ
via a [2 + 2] cycloaddition to generate N formylkynurenine or kynurenine,
which are
both known to significantly quench the emission of buried Trp residues (Mach,
H.,
Burke, C. J., Sanyal, G., Tsai, P.-K, Volkin, D. B. & Middaugh, C. R. in
S Formulation and Delivery of Proteins and Peptides, eds. Cleland, J. L. &
Langer, R.
(American Chemical Society, Denver, CO) (1994)). The intrinsic fluorescence of
horse IgG is rapidly quenched to 30% of its original value during a 40-min
irradiation, whereas hydrogen peroxide generation is linear throughout (~ =
0.998)
(Figure 7). If the reduction of singlet oxygen is due to antibody Trp
residues, then
the solvent-exposed Trp seem to contribute to a lesser degree than the buried
ones.
This factor may help to explain why this ability is so highly conserved among
antibodies. In greater than 99% of known antibodies there are two conserved
Trp
residues, and they are both deeply buried: Trp-36 and Trp-47 (Kabat, E. A.,
Wu, T.
T., Perry, H. M., Gottesman, K. S. & Foeller, C., Sequences of Proteins of
Immunological Interest (U.S. Department of Health and Human Services, Public
Health Service, National Institutes of Health, Bethesda, MD) (1991)).
Throughout nature, organisms have defended themselves by production of
relatively simple chemicals. At the level of single molecules, this mechanism
has
thought to be largely abandoned with the appearance in vertebrates of the
immune
system. It was considered that once a targeting device had evolved, the
killing
mechanism moved elsewhere. The present results realign recognition with
killing
within the same molecule. In a certain sense this chemical immune system
parallels
the purely chemical defense mechanism of lower organisms, with the exception
that
a more sophisticated and diverse targeting element is added.
Given the constraints that an ideal killing system must use host molecules in
a localized fashion while minimizing self damage, one can hardly imagine a
more
judicious choice than'O2. Because one already has such a reactive molecule, it
is
important to ask what might be the advantage of its further conversion by the
antibody. The key issue is that by conversion of the transient singlet oxygen
molecule (lifetime 4 ~,s) into the more stable OZ'-, one now has access to
hydrogen
peroxide and all of the toxic products it can generate. In addition,
superoxide is the
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only molecular oxygen equivalent remaining at the end of the oxygen-scavenging
cascade. Therefore, this "recycling" may serve as a crucial mechanism for
potentiation of the microbicidal process. Another benefit of ringlet molecular
oxygen is that it is only present when the host is under assault, thereby
making it an
"event-triggered" substrate. Also, because there are alternative ways to
defend that
use accessory systems, this chemical arm of the immune system might be silent
under many circumstances. This said, however, there may be many disease states
where antibody and ringlet oxygen find themselves juxtaposed, thereby leading
to
cellular and tissue damage. Given that diverse events in man lead to the
production
of ringlet oxygen, its activation by antibodies may lead to a variety of
diseases
ranging from autoimmunity to reperfusion injury and atherosclerosis (Skepper
et al.,
Microsc. Res. Tech., 42, 369-385 (1998)).
Example II
Antibodies Catalyze the Oxidation of Water
Methods and Materials
C s~-' talog,_raphy: IgG 4C6 was digested with papain and the Fab' fragment
purified using standard protocols (Harlow and Lane). The Fab' was crystallized
from 13-18% PEG 8 K, 0.2 M ZnAc, 0.1 M cacodylate, pH 6.5. Crystals were
pressurized under xenon gas at 200 psi for two minutes (Soltis et al., J.
Appl. Cry,
30, 190, (1997)) and then flash cooled in liquid nitrogen. Data were collected
to 2.0
A resolution at SSRL BL9-2. The structure was solved by molecular replacement
using coordinates from the native 4C6 structure, and xenon atom sites were
identified from strong peaks in the difference Fourier map. Refinement of the
structure was done in CNS (Brunger et al., Acta. C ,r s~o~r., D54, 905 (1998))
to
final R = 23.1 % and Rfree = 25.7%. The occupancies of the two xenon atoms
were
refined after fixing their B values fifty percent higher than the B factors of
the
immediately surrounding protein. The figures were generated in Bobscript (R.M.
Esnouf, Acta Crystallo;~,, D55, 938 (1999)).
Scanning of the Kabat database: The Kabat database of human and mouse
sequences was analyzed to determine the number of Trp, Tyr, Cys, Met in their
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structures. Sequences were rejected if there were too many residue deletions
or
missing fragments. This allowed a high certainty analysis for 2068 of the 3894
sequences available. The values are reported as the mean totals with the range
in
parentheses of the CH, VH, CL and VL (x and ~,.) regions: Trp 15.5 (14 to 31),
Tyr
30.4 (13 to 47), Cys 19.3 (15 to 29), Met 11.6 (7 to 32), His 13.3 (8 to 28).
Grand
total = 90.1 (49 to 167).
Inductivelv coupled plasma atomic emission spectroscopy: Inductively
coupled plasma atomic emission spectroscopy (ICP-AES) of mAb PCP21 H3 was
performed on a Varian, Axial Vista Simultaneous ICP-AES spectrometer. Mouse
monoclonal antibody (PCP21H3) was exhaustively dialyzed into sodium phosphate
buffered saline (PBS, 50 mM pH 7.4) with 20 mM EDTA. In a typical assay 300
~.L
of a 10.5 % HN03 solution was added to 100 ~L of a 10 mg/mL antibody solution
and was incubated at 70°C for 14 hours. This solution was then diluted
to 2 mL with
MQH20 and then analyzed by comparison to standards. ICP-AES analysis results
are reported in parts per million (~,g/mL): Ag 0.0026 (0.0072 atoms per
antibody
molecule); Al 0.0098 (0.113 atoms per antibody molecule); As 0.0062 (0.025
atoms
per antibody molecule); Ba below level of detection; Ca 0.0355 (0.266 atoms
per
antibody molecule). The high Ca concentration is a result of contamination of
the
phosphate buffer system utilized in our assay system. To investigate the
effect of
Ca(II) on the rate of antibody-mediated HZOZ, the irradiation of antibody
samples
was performed using the assay procedure outlined in the legend of Figure 8A
with
the addition of varying concentrations of CaC 1 Z (0 - 100 ~,M). The process
was
found to be independent of Ca(II) concentration; Cd 0.0007 (0.0187 atoms per
antibody molecule); Ce 0.0012 (0.003 atoms per antibody molecule); Co 0.0013
(0.007 atoms per antibody molecule); Cr 0.0010 (0.006 atoms per antibody
molecule); Cu 0.0014 (0.007 atoms per antibody molecule); Fe 0.0089 (0.048
atoms
per antibody molecule); Gd 0.0008 (0.001 atoms per antibody molecule); K
0.0394
(0.302 atoms per antibody molecule); La 0.0007 (0.002 atoms per antibody
molecule); Li 0.0013 (0.056 atoms per antibody molecule); Mg 0.0027 (0.033
atoms
per antibody molecule); Mn 0.0007 (0.004 atoms per antibody molecule); Mo
0.0023 (0.007 atoms per antibody molecule); Na 102.0428 (1332 atoms per
antibody
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molecule); Ni 0.0007 (0.004 atoms per antibody molecule); P 14.3521 (138.9
atoms
per antibody molecule); Pb below level of detection; Rb 0.0007 (0.002 atoms
per
antibody molecule); Se below level of detection; V 0.0109 (0.019 atoms per
antibody molecule); W 0.0119 (0.019 atoms per antibody molecule); Zn 0.0087
(0.040 atoms per antibody molecule).
Oxygen isotope experiments: In a typical experiment, a solution of antibody
(6.7 wM, 100 ~,L) or non-immunoglobulin protein (50 wM, 100 ~,L) in PB (160 mM
phosphate; pH 7.4) was lyophilized to dryness and then dissolved in Hz02 (100
wL,
98 %). Sodium chloride was excluded to minimize signal suppression in the MS.
The higher concentration of non-immunoglobulin protein was necessary to
generate
a detectable amount of HZOZ for the MS assay. This protein solution was
irradiated
on a UV-transilluminator under saturating'60z aerobic conditions in a sealed
quartz
cuvette for 8 hours at 20°C. The HzOz concentration was determined
after 8 hours
using the Amplex Red assay (Zhou et al., Anal. Biochem., 253, 162 (1997)). The
sample was then filtered by centrifugation through a microcon (size-exclusion
filter)
to remove the protein and the HZOZ concentration re-measured. TCEP (freshly
prepared 20 mM stock in HZ'80) was added (ca. 2 mol eq relative to HZOZ) and
the
solution was left to stand at 37°C for 15 minutes, after which time all
the HZOZ had
reacted. The TCEP solution in HZ'80 was prepared fresh prior to every assay
because'80 is slowly incorporated into the carboxylic acids of TCEP (over
days).
During the time course of the assay, no incorporation of'$O occurs due to this
pathway. Furthermore, there is no incorporation of'80 from Hz'$O into the'6O
phosphine oxide. The peak at 249 m/z is the (M-H)- of TCEP. The peak at 249 is
observed in all the MS because an excess of TCEP (twofold) relative to HZOZ is
used
in the assay.
The reproducibility of the'60/'80 ratio from protein samples lyophilized
together is reasonable (~10 %). However, problems with removing protein-bound
water molecules during the lyophilization process means that the observed
ratios can
vary between samples from different lyophilization batches by as much as 2:1
to 4:1
(when lyophilizing from HZ'6O). It is, therefore, important that rigorous
lyophilization and degassing procedures are followed. In this regard, the '
802 and
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HZ'60 experiments exhibit far less inter-assay variability due to the relative
ease of
removing protein-bound oxygen molecules.
Antibodies from different species give similar ratios within the experimental
constraints detailed below: '60:'80: WD1-6G6 mIgG (murine) 2.1:1; polyIgG
(horse) 2.2:1; polyIgG(sheep) 2.2:1; EP2-1962 mIgG (murine) 2.1: l; CH2-SH7
mIgG (murine) 2.0:1; polyIgG (human) 2.1:1. Ratios are based on the mean value
of
duplicate determinations except for polyIgG (horse) which is the mean value of
ten
measurements. All assays and conditions are as described above.
In a typical experiment, a solution of sheep or horse polyIgG (6.7 ~,M, 100
~L) in PB ( 160 mM phosphate; pH 7.4) was degassed under an argon atmosphere
for
30 min. This solution was then saturated with'80z (90 %) and irradiated as
described above. Assays and procedures are then as described herein.
Assa, for HzOz production as a fiznction of the efficienc,~2 formation
via 302 sensitization with hematoporph, '~: The assay is a modification of a
procedure developed by H. Sakai and co-workers, Proc. SPIE-Int. Soc. Opt.
En~.,
2371, 264 (1995). In brief, the horse polyIgG (1 mg/mL) in PBS (50 mM, pH 7.4)
and hematoporphyrin IX (40 ~.M) is irradiated with white light from a
transilluminator. Aliquots are removed (50 ~,L) and the concentration of HZOZ
and
3-aminophthalic acid measured simultaneously. H202 concentration was measured
by the amplex red assay (Zhou et al., Anal. Biochem., 253, 162 (1997)).
3-Aminophthalic acid concentration was measured by reversed-phase HPLC on a
Hitachi D4000 series machine with an Adsorbosphere-C 18 column, a LTV detector
at
254 nm, and a mobile phase of acetonitrile/water (0.1% TFA) of 18:82 at 1
mL/min
(retention time of luminol = 7.4 min and 3-aminophthalic acid 3.5 min). The
concentrations of luminol and 3-aminophthalic acid were determined by
comparison
of peak height and area to control samples. The experimental data yields the
amount
of'OZ formed by hematoporphyrin IX (being directly proportional to the amount
of
3-aminophthalic acid formed) and the amount of HZOZ formed by the antibody.
N.B.
There is no significant amount of'02 formed by antibodies without
hematoporphyrin
IX in white light.
Any concerns that the amplex red assay may be detecting protein-
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hydroperoxide derivatives in addition to H20z have been discounted because the
apparent H202 concentration measured using this method is independent of
whether
irradiated protein is removed from the sample (by size-exclusion filtration).
Quantum Chemical Methods: All QC calculations were carried out with
Jaguar [Jaguar 4.0, Schrodinger, Inc. Portland, Oregon, 1998. See B. H.
Greeley, T.
V. Russo, D. T. Mainz, R. A. Friesner, J.-M. Langlois, W. A. Goddard III, R.
E.
Donnelly, J. Chem. Ph,~~s., 101, 4028 (1994)] using the B3LYP flavor of
density
functional theory (DFT) [J. C. Slater in Quantum Theory of Molecules and
Solids,
Vol. 4: The Self Consistent Field of Molecules and Solids, McGraw Hill, New
York,
(1974)], that includes the generalized gradient approximation and exact
exchange.
The 6-31G** basis set was used on all atoms. All geometries were fully
optimized.
Vibrational frequencies were calculated to ensure that each minimum is a true
local
minimum (only positive frequencies) and that each transition state (TS) has
only a
single imaginary frequency (negative eigenvalue of the Hessian). Such QC
calculations have been demonstrated to have an accuracy of ~3 kcal/mol for
simple
organic molecules. Non-closed shell molecules such as OZ and 30z are expected
to
have larger errors. However, such errors are expected to be systematic such
that the
mechanistic implications of the QC results should be correct. All energetics
are
reported in kcal/mol without correcting for zero point energy or temperature.
Results and Discussion
Antibodies are capable of generating hydrogen peroxide (H202) from singlet
molecular oxygen ('OZ). However, it was not known until now, as reported
herein,
that the process was catalytic and the source of electrons. It is now shown
that
antibodies are unique as a class of proteins in that they can produce up to
500 mole
equivalents of HZOZ from'O2, without a reduction in rate, in the absence of
any
discernible cofactor and electron donor. Based on isotope incorporation
experiments
and kinetic data, it is proposed that antibodies are capable of facilitating
an
unprecedented addition of HZO to'OZ to form H203 as the first intermediate in
a
reaction cascade that eventually leads to HZOz. X-ray crystallographic studies
with
xenon point to conserved oxygen binding sites within the antibody fold where
this
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chemistry could be initiated. This findings suggest a unique protective
function of
immunoglobulins against'OZ and raise the question of whether the need to
detoxify
'OZ has played a decisive role in the evolution of the immunoglobulin fold.
Antibodies, regardless of source or antigenic specificity, generate hydrogen
peroxide (H202) from singlet molecular oxygen ('Oz) thereby potentially
aligning
recognition and killing within the same molecule (Wentworth et al., Proc.
Natl.
Acad. Sci. U.S.A., 97, 10930 (2000)). Given the potential chemical and
biological
significance of this discovery, the mechanistic basis and structural location
within
the antibody of this process has been investigated. These combined studies
reveal
that, in contrast to other proteins, antibodies may catalyze an unprecedented
set of
chemical reactions between water and singlet oxygen.
Kinetic studies. Long term UV irradiation studies reveal that
antibody-mediated H202 production is a much more efficient process than is the
case
for the non-immunoglobulin proteins (Figure 8A). Typically antibodies exhibit
linearity in HzOz formation for up to 40 mole equivalents of H202 before the
rate
begins to decline asymptotically (Figure 8B). By contrast, non-immunoglobulin
proteins display a short 'burst' of H202 production followed by quenching as
photo-oxidation occurs (Figure 8A).
In contrast to other proteins, antibodies are able to resume photo-production
of HzOz at the same initial rate as at the start of the experiment if the H202
generated
during the assay is removed by catalase, as shown for murine monoclonal IgG
PCP21H3 (Figure 8C). This profile of continued linear production of HZOZ after
catalase-mediated destruction of Hz02 was conserved for all antibodies
assayed.
Thus, the HZOz that accumulates during the process is inhibiting (reversibly)
its own
formation. The apparent ICSO was estimated as 225 ~,M (Figure 8D). Inhibition
of
the catalytic function of an enzyme either by substrates, transition state
analogs or
reaction products is often taken as strong evidence for an active site
phenomenon. It
has already been noted that the antibody-mediated photo-production of H202 is
saturable with molecular oxygen (Kmapp(Oz 187 ~,M) (Wentworth et al., Proc.
Natl.
o Acad. Sci. U.S.A., 97, 10930 (2000)). This formal product inhibition of H202
provides further evidence for such a binding site phenomenon.
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An earlier report concerning the photo-production of HZOZ by antibodies did
not probe the maximum amount of HzOz that could be generated (Wentworth et
al.,
Proc. Natl. Acad. Sci. U.S.A., 97, 10930 (2000)). This issue has been examined
by
repetitive cycles of UV irradiation of antibody samples followed by removal of
the
S generated H202 by catalase (Figure 8C shows two such cycles). In one series
of
experiments, the cycle of LTV-irradiation and addition of catalase was carried
out for
up to 10 cycles (horse poly IgG in PBS, pH 7.4). During these experiments >
500
mole equivalents (equiv.) of H202 were generated, with only a slight reduction
in the
initial rate being observed. Beside antibodies, the only other protein that
was found
thus far to generate HZOZ in such an efficient and long-term maimer was the
a(3 T
cell receptor (a(3 TCR) (Figure 8F). Interestingly, the a[3 TCR shares a
similar
arrangement of its immunoglobulin fold domains with antibodies (Garcia et al.,
Science, 274, 209 (1996)). However, possession of this structural motif seems
not
necessarily to confer an H202-generating ability on proteins as demonstrated
by
j32-microglobulin which does not generate H20z even though it is a member of
the
immunoglobulin superfamily (Welinder et al., Mol. Immunol., 28, 177 (1991)).
The antibody structure is remarkably inert against the oxidizing effects of
H202. When exposed to standard UV irradiation conditions for 6 hours in the
presence of HZOz (at a concentration high enough to fully inhibit H202
production), a
polyclonal horse IgG antibody sample becomes fully active once the inhibitory
H202
has been destroyed by catalase (Figure 8E). The ability to continue H202
production
for long periods at a constant rate, even after exposure to HZOZ, reveals a
remarkable,
and hitherto unnoticed, resistance of the antibody structural fold to both
chemical
and photo-oxidative modifications suffered by other proteins. SDS-PAGE gel
2S analysis of antibody samples after UV irradiation under standard conditions
for 8
hours reveals neither significant fragmentation nor agglomeration of the
antibody
molecule. To ensure that there was no change in the protein structure in the
presence
of HzOZ (that may be contributing to the apparent inhibitory effect of H202)
even at
the level of side-chain position, x-ray crystal structures of Fab 4C6 were
determined
in the presence and absence of HZOz. Fab 4C6 was selected because its native
crystals diffract to a higher resolution than any other published antibody (~l
.3 A).
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The root mean square difference (RMSD) of key structural parameters were
compared for the 4C6 structure before and after a soak experiment with 3 mM
H20z_
RMSD of all atoms = 0.412 A, RMSD Ca atoms = 0.327 A, RMSD main chain
atoms = 0.328 A, RMSD side-chain atoms = 0.488 A. The overlayed.native and
H202-treated structures of murine Fab 4C6 (Li et al., J. Am. Chem. Soc., 117,
3308
(1995)) are superimposable, reinforcing the evidence of stability of the
antibody fold
to H202 (Figure 9).
An action spectrum of the antibody-mediated photo-production of HZOZ and
the corresponding absorbance spectrum of the antibody protein for the same
wavelength range (260 - 320 nm) are juxtaposed in figure 10. The two spectra
are
virtually superimposable with maximal efficiency of HZOZ production being
observed at an excitation wavelength that coincides with the UV absorbance
maxima
of tryptophan in proteins.
Probing the efficiency of H202 production by horse IgG as a function of the
efficiency of'OZ formation via 302 sensitization with hematoporphyrin IX (~A =
0.22
in phosphate buffer pH 7.0 and visible light reveals that for every 275 ~ 25
mole
equivalents of'OZ generated by sensitization, 1 mole equivalent of HzO~ is
generated
by the antibody molecule (Wilkinson et al., J. Phys. Chem. Ref. Data, 22, 113
(1993); Sakai et al., Proc. SPIE-Int. Soc. Opt. End., 2371, 264 (1995)).
The question of the electron source. The mechanism problem posed by the
antibody-mediated HZOZ production from singlet oxygen has to be sharply
divided
into two sub-problems: one referring to the electron source for the process
and the
other concerning the chemical mechanism of the process. Given that the
conversion
of'OZ to H202 requires two mole equivalents electrons, the fact that
antibodies can
generate > 500 equivalents of HZOZ per equivalent of antibody molecule raises
an
acute electron inventory problem. The search for this electron source began
with the
most distinct possibilities. Since electron transfer through proteins can
occur with
remarkable facility and over notably large distances (Winkler et al., Pure &
Ap~l.
Chem., 71, 1753 (1999); Winkler, Curr. Opin. Chem. Biol., 4, 192 (2000)), the
first
considered was that a collection of the residues implicated as electron donors
cited in
normal protein photo-oxidation processes might be involved. The nearly
constant
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rate of Ha02 production by antibodies and the a~3-TCR during the repetitive
cycles of
irradiation and catalase treatment (Figure 8C and 8E) argued against such a
mechanism because a marked reduction of rate would have to accompany HZOZ
production as the residues capable of being oxidized become exhausted. This
reduction of rate would be further exacerbated because the redox potentials of
the
remaining unoxidized residues would have to rise as the protein becomes more
positively charged.
Normal protein photo-oxidation is a complex cascade of processes that leads
to the generation of'Oz and other reactive oxygen species (ROS), such as
superoxide
anion (02 -), peroxyl radical (H02 ) and H202 (Foote, Science, 162, 963
(1968)).
Present mechanistic thinking links the sensitivity of proteins to photo-
oxidation with
up to five amino acids: tryptophan (Trp), tyrosine (Tyr), cysteine (and
cystine),
methionine (Met), and histidine (His) (Straight and Spikes, in Sin_lg et Oz,
A.A.
Frimer, Ed. (CRC Press, Inc., Boca Raton, Florida, 1985), vol IV9, pp. 91-143;
Michaeli and Feitelson, Photochem. Photobiol., 9 284 (1994)). The
photo-production of H202 by Trp and molecular oxygen is a well-characterized
process that involves, at least in part, the formation and reduction of'02 to
02 that
spontaneously dismutates into HZOZ and 30z (McMoi~rnick and Thompson, J. Am.
Chem. Soc., 100, 312 (1978)). Tryptophan, both as an individual amino-acid and
as
a constituent of proteins, is particularly sensitive to near-UV irradiation
(300-375
nm) under aerobic conditions, owing to its conversion to N'-formylkynurenine
(NFI~) that is a particularly effective near-UV (~.",ax 320 nm)
photosensitizer
(Walrant and Santus, Photochem. Photobiol., 19, 411 (1974)). However, Trp
photo-oxidation is accompanied by substoichiometric production of HzOz (ca.
0.5
mole equivalents) during near-UV irradiation (Figure 11A) (McMormick and
Thompson, J. Am. Chem. Soc., 100, 312 (1978)) and the most efficient
non-immunoglobulin protein at HZOZ photo-production, /3-galactosidase,
generates
only 5.9 mol eq. of HzOz from its 39 Trp residues (Figure 8A) (Fowler and
Zabin, J.
Biol. Chem., 253, 5521 (1978)).
Scanning of the Rabat database of human and mouse antibody heavy- and
light-chain sequences (2068 of 3894 sequences were analyzed) revealed that
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antibodies rarely have more than 15 Trp residues in their entire structure
(mean value
= 15.5 with a range of 14 to 31 Trp residues)(Kabat et al., Sequences of
Proteins of
Immunological Interest (US Department of Health and Human Services, Public
Health Service, NIH, ed. 5th, 1991); Martin, PROTEINS: Struct., Funct. and
Genet.,
25, 130 (1996)). In fact, even if all of the amino acids that are implicated
in protein
photo-oxidation processes viele supra are collectively involved in antibody-
mediated
H20z production, there is still an insufficient number of these residues (mean
value =
90.1 with a range of 49 to 167 reactive residues) to account for the 500 mole
equivalents of H202 generated.
The potential of chloride ion (present at 150 mM in PBS) as a reducing
equivalent was then investigated given that chloride ion is known to be a
suitable
electron source for photo-production of HZOZ via a triplet excited state of an
anthraquinone (Scharf and Weitz, Symp. Quantum Chem. Biochem., Jerusalem vol.
12 (fatal. Chem. Biochem.: Theory Exp.), pp. 355-365 (1979)). This possibility
was quickly discounted when the rate of HZOz production by immunoglobulins was
found to be independent of chloride ion concentration (Figure 11 B).
The possible role of metal ions was investigated. While such ions could
hardly be present in antibodies in such amounts that they could serve as an
electron
source, trace amounts of them might play a central role as catalytic redox
centers.
Experiments were performed that, for all practical purposes, allow the
implication of
trace metals in this process to be ruled out. The rate of antibody-mediated
photo-
production of HZOZ is unchanged before and after exhaustive dialysis of
antibody
samples with EDTA-containing buffer (Figure 11 C). After EDTA treatment of
antibody samples, ICP-atomic emission spectroscopy (AES) revealed the presence
of
trace metal ions remaining in amounts that are far below parts per million.
For a
trace metal to be implicated in this reaction it must be common to all
antibodies
because all antibodies assayed have this intrinsic ability. It is generally
accepted that
metal-binding is not an implicit feature of antibodies and is consistent with
our own
analysis of antibody crystals as well as the approximate 300 antibody
structures
available on the Brookhaven database.
All of the observations thus far forcibly pointed towards the need to identify
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an electron source that would not imply a deactivation of the protein catalyst
and that
could account for the high turnover numbers and hence, for a guasi unlimited
source
of electrons. A more broad consideration of the chemical potential of'OZ was
done.
The participation of this energized form of molecular oxygen in the antibody-
mediated mechanism was clearly inferred from a previous report (Wentworth et
al.,
Proc. Natl. Acad. Sci. U.S.A., 97, 10930 (2000)). In brief, the antibody-
mediated
rate of HZOZ photo-production is increased in D20 and reduced in the presence
of the
'OZ quencher, sodium azide. Furthermore, antibodies have been shown to
generate
H202 via sensitization of 302 with hematoporphyrin IX in visible light, and in
the
dark with the endoperoxide of disodium 3',3'-(1,4-naphthylidene) dipropionate
(a
chemical'OZ source). The involvement of'Oz is also in line with the close
similarity
of the action spectrum of antibody-mediated H202 production and the absorbance
spectrum of antibody constituent tryptophans (Figure 10).
Given that the known chemistry of'OZ can be conceptualized as the
chemistry of the super-electrophile "dioxa-ethene" (Foote, Acc. Chem. Res., 1,
104
(1968), the heretofore unknown possibility was considered that a molecule of
water
may, in the presence of an antibody, add as a nucleophile to ' OZ and form
H203 as an
intermediate. Thus, water becoming oxidized to HZOZ would fulfil the role of
the
electron source.
Oxygen isotope experiments were undertaken to test the hypothesis of an
antibody-catalyzed photo-oxidation of HZO by'Oz through determination of the
source of oxygen found in the HZOZ. Contents of'60f 80 in HZOZ were measured
by
modification of a standard HZOz detection method (Han et al., Anal. Biochem.,
234,
107 (1996)). Briefly, this method involves reduction with tris carboxyethyl
phosphine (TCEP), followed by mass-spectral (MS) analysis of the corresponding
phosphine oxides (Figure 12).
These experiments revealed that UV-irradiation of antibodies, in the presence
of oxygen, leads to oxygen incorporation from water into H202 (Figs. 12A and
12B).
The relative abundance of the'60/'80 ratio observed in the MS of the phosphine
oxide after irradiation of sheep polyIgG under conditions of saturating'GOZ
concentration in a solution of HZ'80 (98 %'g0) phosphate buffer (PB) is 2.2 ~
0.2:1
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(Figure 12A). When the converse experiment is performed, with an ' 80 enriched
molecular oxygen mixture (90 %'$O) in HZ'60 PB, the reverse ratio (1:2.0 +
0.2) is
observed (Figure 12B). These values of the ratios 'exhibit good
reproducibility (+ 10
%, n = 10) and are found for all antibodies studied.
The following control experiments were performed. First, under conditions
of'~Oz and HZ'60, irradiation of polyIgG (horse) generated Hz'602 (Figure
12C).
There is no incorporation of'$O when HZ'6O2 (400 ~,M in PB, pH 7.0) itself is
irradiated for 4 hours in HZ'80. This result alleviates concerns that'$O
incorporation
into HZOz may be occurnng via either an acid-catalyzed exchange with water or
by a
mechanism that involves homolytic cleavage of Hz'6Oz and recombination with
H'80' from water. To check the possibility that antibodies may catalyze both
the
production of HZ'602 and its acid-catalyzed exchange with H2'80, the isotopic
exchange of HZ'602 (200 ~M) in Hz'~OZ (98 %'80) PB in the presence of sheep
polyIgG (6.7 ~,M) after LTV-irradiation under an inert atmosphere was
determined.
Only a trace of incorporation of'80 into HZ'6O2 was observed (Figure 12D).
Isotope experiments were also performed with (3-galactosidase, the most
efficient non-immunoglobulin protein at generating HZOz, as well as 3-
methylindole.
In both cases, photo-oxidation led to negligible'80 incorporation into the
HZOZ
(Figures 12E and 12F), illustrating the view that the indole ring itself and
tryptophan
residues in this protein are behaving simply as reductants of'02.
This view is further supported because irradiation of 3-methylindole
generates H20z that does not include oxygen incorporation from HZ'$O. The same
experiment performed with tryptophan does give rise to exchange with a ratio
'60/'80 1.2:1. This result is thought to be due to the ammonium functionality
acting
as an intramolecular general acid, protonating the internal oxygen of a
diastereomeric mixture of 3'-hydroperoxides (inset Below). It should be noted
that
while this is interesting from a chemical point of view, it cannot account for
the
catalytic production of HZOZ by antibodies both because it is a stoichiometric
process
and Trp residues in proteins do not possess a free ammonium group.
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g2
H
Hz~ ~S ~ S+
+ ( '~ , N-H
8 O~H.
)+
z
The chemical mechanism. All antibodies studied can catalyze the oxidation
of water by singlet oxygen. The thermodynamic balance between reactants and
products for the oxidation of Hz0 by'OZ (heat of reaction, OH~ _ + 28.1
kcal/mol)
(D.R. Lide, in Hanbook of Chemistry and Ph, sics, 73rd ed. (CRC, 1992)),
demands a
stoichiometry in which more than one molecule of'OZ would have to participate
per
molecule of oxidized water during its conversion into two molecules of HZOZ.
This
stoichiometry assumes that no further light energy before that involved in the
production of singlet from triplet oxygen is participating in the process.
Qualitative
chemical reasoning on hypothetical mechanistic pathways, together with
thermodynamic considerations, makes the likely overall stoichiometries as in
either
equations 1b or c (all energetics are calculated from gas phase experimental
heats of
formation and are reported in kcal/mol):
'OZ + 2H20 --~ 2H202 ; VH~° = 28.1 (la)
2'0z + 2H20 --~ 2H202 + 302 ; OHr = 5.6 (1b)
3 'OZ + 2H20 --~ 2H202 + 2 302 ; ~Hr = -16.9 (1 c)
A recent report of a transition metal-catalyzed conversion of'Oz and water
into hydrogen peroxide, via a tellurium-mediated redox process (betty and
Gibson,
J. Am. Chem. Soc., 112, 4086 (1990)), provides experimental evidence for a
process
in which'OZ and HZO can be converted into H202 and, hence that the energetic
demands of this process can be overcome. It is thought that the mechanism for
the
antibody-mediated photo-oxidation process involves the addition of a molecule
water to a molecule of'Oz to form dihydrogen trioxide as the first
intermediate on
the way to HZO2. The antibody's function as a catalyst would have to be the
supply
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of a specific molecular environment that would stabilize the critical
intermediate
relative to its reversible formation and, or, would accelerate the consumption
of the
intermediate by channeling its conversion to HZO2. An essential feature of
such an
environment might consist of a special constellation of organized water
molecules at
an active site conditioned by an antibody-specific surrounding.
While Hz03 has not yet been detected in biological systems, its chemistry ifa
vivo has been a source of considerable speculation and its in vitro properties
have
been the subject of numerous experimental and theoretical treatments (C. Deby,
La
Recherche, 228, 378 (1991); Sawyer, in Oxy~en Chemistry (Oxford University
Press, Oxford, 1991); Cerkovnik and Plesnicar, J. Am. Chem. Soc., 115, 12169
(1993); Vincent and Hillier, J. Phys. Chem., 99, 3109 (1995); Plesnicar et
al., Chem.
Eur. J., 6, 809 (2000); Corey et al., J. Am. Chem. Soc., 108, 2472 (1986);
Koller and
Plesnicar, J. Am. Chem. Soc., 118, 2470 (1996); Cacace et al., Science, 285,
81
(1999)). Plesnicar and co-workers have shown that H203, reductively generated
from ozone, decomposes into HZO and'Oz (Koller and Plesnicar, J. Am. Chem.
Soc.,
118, 2470 (1996)). Applying the principle of microscopic reversibility, it was
surmised that the reverse reaction should also be catalyzed by one or more
molecules
of water. To delineate plausible reaction routes and energetics of such a
process,
first principles quantum chemical (QC) methods were used (B3LYP Density
Functional Theory) as described herein. The results are illustrated in
equations 2a-c
(all energetics are in kcal/mol):
H20 +'0Z .-~ .1s --i HzO3 (2a)
0.0 69.5 1-5.5
2H20 +'0Z -~ TS --~ HZO3 + H20 (2b)
0.0 31.5 15.5
3H20 +'0Z --~ TS --~ H203 + 2H20 (2c)
0.0 15.5 15.5
The direct reaction of water and'OZ to give H203 is quite unfavorable, with
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an activation barrier of 70 kcal/mol (Eqn. 2a). However, with the addition of
a
second or third water molecule a concerted process is found that decreases the
activation barrier to 31.5 and 15.5 kcal/mol respectively. Indeed these
additional
waters do play the role of a catalyst (in eqn. 2b the H of the 2nd water goes
to the
product HOOOH, simultaneous with the H of the 1 st water replacing it). These
barriers are small compared with the first HO bond energy of water (119
kcal/mol)
and the bond energy of'OZ (96 kcal/mol). Note that the reverse reaction in
eqn. 2b
and eqn. 2c has a barrier of only 15.5 or 0 kcal/mol respectively, suggesting
that
Hz03 is not stable in bulk water or water rich systems. Thus, the best site
within the
antibody structure for producing and utilizing H203 is expected to be one in
which
there are localized waters and water dimers next to hydrophobic regions
without
such waters.
The'~O/'80 ratio in the phosphine oxide derived from the antibody-catalyzed
photo-oxidation of water poses a significant constraint to the selection of
reaction
paths by which this primary intermediate H203 would to convert to the final
product
H202. The ratio is primarily determined by the number of'Oz molecules that
chemically participate in the production of two moles of H202 from two moles
of
HZO as well as by mechanistic details of this process. A ratio of 2.2:1 would
coincide exactly with the value predicted for certain mechanisms in which two
molecules of'OZ and two molecules of HZO are transformed into two molecules of
H20z and one molecule of molecular oxygen (which would have to be 30z for
thermodynamic reasons). An example of such a mechanism is an SN2-type
disproportionation of two molecules of H203 into H204 and H202, followed by
the
decomposition of the former into H202 and 30z. The complex problem of
defining,
theoretically feasible reaction pathways for the conversion of H203 into H202
with or
without the participation of'02 has been tackled in a systematic way using
quantum
chemical methods (B3LYP Density Functional Theory). These studies show
extensive docking calculations of H203 and the transition states for its
formation and
conversion into HZOZ to a number of proteins. Indeed there are unique sites of
stabilizing these species in a region of antibodies (and the a(3-T cell
receptor) in a
region with isolated waters and next to hydrophobic regions. This extended
study
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revealed the potential existence of a whole spectrum of theoretically feasible
chemical pathways for the H203 to H20z conversion.
Structural studies of xenon binding to antibodies. Given the conserved
ability of antibodies, regardless of origin or antigen specificity, or of the
a~3- TCR to
mediate this reaction, X-ray structural studies were instigated to search for
a possible
conserved reaction site within these immunoglobulin fold proteins. A key
constraint
for any potential locus is that molecular oxygen (either'OZ or triplet with a
potential
sensitizing residue in proximity, preferably tryptophan) and water must be
able to
co-localize, and the transition-states and intermediates along the pathway
must be
stabilized either within the site or in close proximity.
There is strong evidence to support the notion that Xe and OZ co-localize in
the same cavities within proteins (Tilson et al., J. Mol. Biol., 199, 195
(1988);
Schoenborn et al., Nature, 207, 28 (1965)). Accordingly, xenon gas was used as
a
heavy atom tracer to locate cavities within the murine monoclonal antibody 4C6
that
may be accessible to molecular oxygen (Li et al., J. Am. Chem. Soc., 117, 3308
(1995)).
Three xenon sites were identified (Figure 13A), and all occupy hydrophobic
cavities as observed in other Xe-binding sites in proteins (Scott and Gibson,
Biochemistry, 36, 11909 (1997); Prange et al., PROTEINS: Struct.. Funct. and
Genet., 30, 61 (1998)). Superposition of the refined native and Xe-derivatized
structures shows that, aside from addition of xenon, there is little
discernible change
in the protein backbone or side chain conformation or in the location of bound
water
molecules.
The xenon I binding site (Xe 1 site) has been analyzed here in more detail
because it is conserved in all antibodies and the a~i TCR (Figure 13B). Xel is
in the
middle of a highly conserved region between the ~3-sheets of VL, 7 A from an
invariant Trp. The Xel site is sandwiched between the two [3-sheets that
comprise
the immunoglobulin fold of the VL, approximately 5 A from the outside
molecular
surface. Xenon site two (Xe2) sits at the base of the antigen binding pocket
directly
above several highly conserved residues that form the structurally conserved
interface between the heavy and light chains of an antibody (Figure 13A). The
CA 02422586 2003-03-17
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-68-
residues in the VL VH interface are primarily hydrophobic and include
conserved
aromatic side chains, such as TrpH'o9.
The contacting side chains for Xel in Fab 4C6 are AIaL'9, IleL2', LeuL'3, and
IIeL'S, which are highly conserved aliphatic side chains in all antibodies
(Kabat et al.,
Sequences of Proteins of Immunolo~cal Interest (LTS Department of Health and
Human Services, Public Health Service, NIH, ed. 5th, 1991)). Additionally,
only
slight structural variation was observed in this region in all antibodies
surveyed.
Notably, several other highly conserved and invariant residues are in the
immediate
vicinity of this xenon site, including Trp''35, Phe''6z, TyrLS6,
LeuL'°4, and the
disulfide-bridge between CysLZ3 and CysLSS. Trp''35 stacks against the
disulfide-bridge and is only 7 A from the xenon atom. In this structural
context,
Trpr.3s may be a putative molecular oxygen sensitizer, since it is the closest
Trp to
Xel. Comparison with the 2C a(3 TCR structure and all available TCR sequences
shows that this Xel hydrophobic pocket is also highly conserved in TCRs
(Figure
SB) (Garcia, Science, 274, 209 (1996)).
Human biz-microglobulin, which does not generate HZO2, does not have the
same detailed structural characteristics that define the antibody Xel binding
pocket,
despite its overall immunoglobulin fold. Also, (3z-microglobulin does not
contain
the conserved Trp residue that occurs there in both antibodies and TCRs. If
TrpL3s
(antibodies) or Trp"3a (TCR) is the oxygen sensitizer, the lack of a
corresponding Trp
in [32-microglobulin may relate to the finding that it does not catalyze the
oxidation
of water.
Thus, the xenon experiments have identified at least one site that is both
accessible to molecular oxygen and is in a conserved region (VL) in close
proximity
to an invariant Trp; an equivalent conserved site is also possible in the fold
of VH.
The structure and sequence around the Xel site is almost exactly reproduced in
the
VH domain by the pseudo two-fold rotation axis that relates VL to VH. Although
a
xenon binding-site was not located in this domain, it is thought that
molecular
oxygen can still access the corresponding cavity in VH. The proposed heavy
chain
xenon site may not have been found because the crystals were pressurized for
only
two minutes, which may have been insufficient time to establish full
equilibrium, or
CA 02422586 2003-03-17
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simply because xenon is too large compared to oxygen for the corresponding
cavity
on the VH side, or due to crystal packing. In other antibody experiments, Xe
binding
sites were found in only one of the two molecules of the asymmetric unit that
suggests that crystal packing can modulate access of Xe in crystals. Analysis
of the
sequence and structure around these sites shows that they are highly conserved
in
both antibodies and TCRs thus providing a possible understanding of why the
Ig-fold in antibodies and the TCR can be involved in this unusual chemistry.
Antibodies are unique among proteins in their ability to catalytically convert
'OZ into HZO2. It is thought that this process participates in killing by
event-related
production of HZOz. Alternatively, antibodies can fulfill the function of
defending an
organism against'Oz. This would require the further processing of hydrogen
peroxide into water and triplet oxygen by catalase.
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All publications, patents and patent applications are incorporated herein by
reference. While in the foregoing specification this invention has been
described in
relation to certain preferred embodiments thereof, and many details have been
set
forth for purposes of illustration, it will be apparent to those skilled in
the art that the
invention is susceptible to additional embodiments and that certain of the
details
described herein may be varied considerably without departing from the basic
principles of the invention.
