Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOKINE COMPOSITION AND METHOD
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation-in-Part of prior application Serial No.
09/368,834, filed on August 5, 1999, which is a continuation of prior
application
Serial No. 08/908,212, filed on August 7, 1997, now U.S. Patent 5,989,857,
which is a
continuation of US patent application filed May 10, 1996 and assigned Serial
No.
08/644,399 for POLYPEPTIDE COMPOSITIONS AND METHODS, the entire
disclosure of which is incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to the treatment and prevention of viral
infections, including HIV infections.
BACKGROUND OF THE INVENTION
The lack of an effective vaccine and the increase in antiretroviral drug
treatment failures has led the HIV research community to continue the search
for
novel approaches to treat HIV infection. HIV can be inhibited at a number of
different steps in its lifecycle within the cell or, alternatively, vaccines
and immune
based therapies can eliminate HIV-infected cells directly.
The HIV lifecycle involves binding of the virus to specific cell receptors.
These receptors include CD4 and the recently discovered co-receptors called
chemokine receptors. Following receptor binding the virus is internalized into
the cell
and the viral RNA is converted into DNA by a process called reverse
transcription.
Reverse transcription requires an enzyme called reverse transcriptase, a
common
target for antiretroviral drugs. Following reverse transcription, the viral
DNA is
transported to the nucleus of the cell where it integrates info the host's
chromosome
by way of a process called integration; a process that requires the enzyme
integrase.
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Following integration into the host chromosome, the integrated DNA serves as a
template for transcription of viral gene products required for replication or
fox
packaging into new progeny virus. These viral mRNAs code for enzymatic or
structural proteins, some of which require cleavage by specific proteases to
produce
infectious viral particles. The new, much publicized HIV drugs called protease
inhibitors, inhibit this cleavage step resulting in the production of non-
infectious viral
particles.
In turn, anti-HIV compounds have been directed against HIV entry (entry
inhibitors), HIV fusion (fusion inhibitors), reverse transcription (nucleoside
and non-
nucleoside reverse transcriptase inhibitors), HIV integration (integrase
inhibitors),
HIV transcription inhibitors, and the aforementioned protease inhibitors.
Inhibition of
HIV at these different sites results in a specific pattern of HIV gene
expression that
requires sophisticated molecular techniques to decipher.
For instance, the (NIAm) categorizes anti-HIV compounds as having either
viral targets or cellular targets. Examples of those having viral targets
include Gag
proteins and precursors (e.g., capsid structural protein, matrix protein, RNA
binding
protein, and other Gag proteins,); viral enzymes (e.g., polymerase, protease
and
integrase); envelope proteins (e.g., surface glycoprotein and transmembrane
glycoprotein); accessory and regulatory proteins (e.g., Tat, Rev, Nef, Vif,
Vpr, Vpx
and Tev); and nucleic acids (e.g., HIV RNA).
Examples of anti-HIV compounds having cellular targets include cellular
receptors such as the immunoglobulin superfamily (e.g., CD4); and chemokines
(seven-transmembrane) receptor superfamily, examples of which include CXCR4
(also known as Eosin, LESTR, NPY3R), and CCRS (also known as CKR-5,
CIVIKRBS).
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Chemokines are a large family of low molecular weight, inducible, secreted,
proinflammitory cytokines which are pxoduced by various cell types. See, for
instance, Au-Yuong, et al., US Patent No. 5,955,303, which describes the
manner in
which chemokines have been divided into several subfamilies on the basis of
the
positions of their conserved cysteines. The CXC family includes interleukin-8
(IL-8),
growth regulatory gene, neutrophil-activating peptide-2, and platelet factor 4
(PF-4).
Although IL-8 and PF-4 are both polymorphonucleax chemoattractants,
angiogenesis
is stimulated by IL-8 and inhibited by PF-4. The CC family includes monocyte
chemoattractant protein-1 (MCP-1), RANTES (regulated on activation, normal T
cell-
expressed and secreted), macrophage inflammatory proteins (MIP-l.alpha., MIP-
l.beta.), and eotaxin. MCP-1 is secreted by numerous cell types including
endothelial,
epithelial, and hematopoietic cells, and is a chemoattractant for monocytes
and
CD45R0+lymphocytes (Proost, P. (1996) Int J. Clin. Lab. Res. 26: 211-223;
Raport,
C. J. (1996) J. Biol. Chem. 271: 17161-17166).
Cells respond to chemokines through G-protein-coupled receptors. These
receptors are seven trmsmembrane molecules which transduce their signal
through
heterotrirneric GTP-binding proteins. Stimulation of the GTP-binding protein
complex by activated receptor leads to the exchange of guanosine diphosphate
for
guanosine triphosphate and regulates the activity of effector molecules. There
are
distinct classes of each of the subunits which differ in activity and
specificity and can
elicit inhibitory or stimulatory responses.
Chemokine receptors play a major role in the mobilization and activation of
cells of the immune system. The effects of receptor stimulation are dependent
on the
cell type and include chemotaxis, proliferation, differentiation, and
production of
cytokines. Chemokine stimulation produces changes in vasculax endothelium,
chemotaxis to sites of inflammation, and activates the effector functions of
cells
(Taub, D. D. (1996) Cytokine Growth Factor Rev. 7: 355-376).
The chemokine receptors display a range of sequence diversity and ligand
promiscuity. The known chemokine receptor protein sequence identities range
from
22 to 40%, and certain receptors can respond to multiple ligands. Although
mainly
expressed in immune cells, viral homologues are expressed by human
cytomegalovirus and Herpesvirus saimiri. The chemokine receptor known as the
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Daffy blood group antigen binds both CC and CXC family chemokines and serves
as
the receptor on erythrocytes for the malarial parasite Plasmodium vivax.
Members of
the chemokine receptor family axe used as co-receptors with CD4 for HIV-1
entry
into target cells. Several receptoxs have recently been cloned.
See also, US Patent No. 5,994,515 (Hoxie) which describes the manner in
which the human immunodeficiency viruses HIV-1 and HIV-2 and the closely
related
simian immunodeficiency viruses (SIV), all use the CD4 molecule as a receptor
during infection. Other cellular molecules have long been suspected to form an
essential component of the cellular HIV-1 receptor; however, the nature of
such
cellular molecules was not lmown until the discovery of Eosin (Feng et al.,
1996,
Science 272:872-876).
Recently, two molecules, Eosin, which is now known as CXCR4 (also known
as Lestr, LCR-1, and HUMSTR) and CCRS, which are melnbers of the chemokine
receptor family of proteins, have been shown to function with CD4 as
coreceptors for
HIV-1 isolates that are tropic for T-cell lines or macrophages, respectively).
Results to
date indicate that the use of chemokine receptors is a general property of all
human
and nonhuman lentiviruses.
CXCR4 is a cellular protein which in conjunction with CD4, forms a
functional cellular receptor for entry of certain strains of HIV into cells.
This protein
is a member of a family of molecules that bind chemolcines which are involved
in the
trafficking of T cells and phagocytic cells to areas of inflammation (Power
and Wells,
1996, Txends Phaxmacol. Sci. 17:209-213).
CXCR4 fulfills the requirements of an HIV receptor co-factor. It renders a
number of marine, feline, simian, quail, and hamster cell lines, as well as
human cell
lines, which cells are normally resistant to HIV-1 entry, fully permissive for
HIV-1
env mediated syncytia formation. In addition, the T cell tropic HIV strain HIV-
1 IIIB,
is capable of infecting both marine and feline cells which co-express human
CD4 and
CXCR4. However, the macrophage tropic strain Ba-L, is not capable of infecting
cells
which co-express both CXCR4 and CD4. These results suggest that CXCR4 can
serve
as a co-factor for T-tropic, but not M-tropic, HIV-1 strains (Feng et aL,
1996, supra).
Moreover, the finding that change from M to T-tropic viruses over time in
infected
individuals correlates with disease progression suggests that the ability of
the viral
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envelope to interact with CXCR4 represents an important feature in the
pathogenesis
of immunodeficiency and the development of full blown AIDS.
Current anti-HIV therapy includes the use of compounds which inhibit various
aspects of HIV replication in a cell such as inhibition of replication andlor
transcription of viral nucleic acid and inhibition of protein processing.
While these
therapies, particularly when used in combination with one another, are
effective, they
are frequently short-lived in that viral strains rapidly develop that are
resistant to one
or more of the compounds used. There therefore remains an acute need to
develop
additional therapies and strategies for preventing HIV infection in humans.
On a separate subject, a previous patent issued to the present assignee, US
Patent No. 5,989,857, describes, inter alia, a method of preparing a bioactive
polypeptide in a stable, inactivated form, the method comprising the step of
treating
the polypeptide with ozonated water in order to oxidize and/or stabilize the
cysteine
residues, and in tum, prevent the formation of disulfide bridges necessary for
bioactivity. The method can involve the use of ozonated water to both oxidize
the
disulfide bridges in a bioactive polypeptide, and to then stabilize the
resultant cysteine
residues. Optionally, and preferably, the method can involve the use of
ozonated
water to stabilize the cysteine residues, and thereby prevent the formation of
disulfide
bridges, in a polypeptide produced by recombinant means in a manner that
allows the
polypeptide to be recovered with the disulfide bridges tmformed.
What are clearly needed are improved methods and compositions for the
treatment and prevention of HIV.
SUMMARY OF THE INVENTION
The present invention provides a composition and method for preventing HIV
infection of mammalian cells. One aspect of the invention relates to an anti-
immunodeficiency virus immunokine capable of binding to a cellular protein in
a
manner that prevents HIV infection of that cell. In another aspect, the
immunodef ciency virus is selected from the group consisting of HIV-1,HTV-2
and
SIV. In another aspect, the invention relates to the identification of a
biologic
anticholinergic agent capable of binding to a cellular protein in a manner
that prevents
HIV infection of that cell. In yet another aspect the invention relates to an
anti-
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immunodeficiency virus immunokine derived from a biologic anticholinergic
agent
which can be administered isz vivo for the treatment of HIV infection. The
immunodeficiency virus can be selected from the group consisting of
Lentiviruses
(HIV-1, HTV-2, SIV, EIAV, BTV, FIV and FeLV).
Compositions of this invention can include either an "active bioactive
polypeptide", such as native cobratoxin, and/or an "inactivated bioactive
polypeptide", such as cobratoxin in which one or more of the native disulfide
bridges
have been prevented from forming. While not presently preferred for iya vivo
applications, it appears that the active polypeptides exhibit the desired
antiviral
activity, and in turn, can be used for ifZ vitro (e.g., diagnostic)
applications. The term
"immunol~ine" will generally be used to refer to an inactivated bioactive
polypeptide,
whether inactivated by chemical, genetic, and/or synthetic means as described
herein,
with the proviso that a corresponding active bioactive polypeptides can be
included
where applicable (e.g., for in vitro use).
A composition of this invention is useful in preventing infection of a cell,
both
with in terms of treating existing HIV spread within an infected individual as
well as
preventing initial HIV infection of that individual. As such, the composition
can be
useful in limiting the spread of virus from one cell to another in an infected
host and,
if present, (i.e. circulating within a host) prior to exposure (but not
productive
infection) of a cell.
' Proteins such as those from venoms, as described herein, have long been
recognized for their ability to bind to specific receptors on the surface of
human cells.
These neurospecific proteins bind to such common receptors as the
acetylcholine
receptor for example. Significantly less well lmown than the interactions
between
venom proteins and human cells is the ability of these venoms to cause cells
to
migrate toward or in response to the venom proteins. This cellular activity is
called
chemotaxis and, until the characterization of these venom proteins by the
present
Applicants, this property has only been attributed to compounds called
chemokines
produced in immune cells. For these reasons, we will heretofore refer to our
venom
proteins as "immunokines".
In yet another aspect of the invention, the protein to which the immunokine of
the invention binds is one or more of a chemokine receptor protein,
preferably, an
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HIV receptor protein and/or a cellular cofactor for a cellular HIV receptor
protein.
More preferably, the protein to which the immunokine of the invention binds is
selected from the group consisting of CD4, CXCR4 and CCRS; and most
preferably,
the protein to which the immunokine binds is CD4/CXCR4 and/or CD4/CCR%
complexes.
In another aspect of the invention, the immunokine is most preferably selected
from the group consisting of post-synaptic alpha-neurotoxins (Group II) and
anticholinergic peptides.
The invention also relates to an isolated DNA encoding an immunokine
capable of binding to a cellular protein in the manner described herein.
The invention also relates to a method of inhibiting infection of a cell by
HIV
comprising adding to the cell an anti-immunodeficiency virus immtznokine
capable of
binding to a cellular protein on the cell, wherein upon binding of the
immunokine to
the cellular protein infection of the cell by HIV is inhibited.
Also included in the invention is a method of treating HIV infection in a
human comprising administering to the human an anti-immunodeficiency virus
immunokine capable of binding to a cellular protein on a cell, wherein upon
binding
of the iixnnunokine to the cellular protein, infection of the cell by HIV is
inhibited,
thereby treating the HIV infection in the human.
The invention further includes a method of obtaining an anti-
immunodeficiency virus immunokine capable of binding to a cellular protein on
a
cell, in one embodiment the method comprising an oxidative process for the
chemical
production of immunokine by combining ozone with the protein of interest,
e.g., a
native or synthetic neurotoxin.
Also included in the invention is a method of identifying a target cell for
immunodeficiency virus infection, the method comprising adding to a population
of
cells native or synthetic active bioactive polypeptide (e.g., alpha-
cobratoxin) or an
anti-immmodeficiency virus immwokine capable of binding to a cellular protein
on a
cell, wherein binding of the immunokine to a cell in the population is an
indication
that the cell is an immunodeficiency virus target cell.
In addition, there is provided a method of identifying a candidate anti
irnmunodeficiency virus compound. This method comprises isolating a test
compound
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capable of binding to an active bioactive polypeptide such as alpha-cobratoxin
or an
anti-immunodeficiency virus immunokine, which immunokine binds to a cellular
protein, and assessing the ability of the test compound to inhibit infection
of a cell by
an immunodeficiency virus in an antiviral assay, wherein inhibition of
infection of the
cell by the immunodeficiency virus in the presence of the test compound is an
indication that the test compound is an anti-immunodeficiency virus compound.
DETAILED DESCRIPTION
In one preferred embodiment, the invention relates to an antiviral,
anticholinergic protein and immunokine which binds to one or more cellular
proteins
essential for entry of a virus into a cell expressing that protein The
immunolcine of
the invention is an antiviral immunokine in that it is an immunokine which
binds to
one or more cellular proteins that are essential for virus entry into the cell
in which the
cellular protein is expressed. By binding to the cellular protein, the
irrnnunokine of the
invention inhibits entry of the virus into the cell and is therefore termed an
antiviral
immunokine despite the fact that it does not bind to a viral protein, but
rather, binds to
a cellular protein. The invention further relates to an antiviral immunokine
which
binds to one or more cellular proteins essential for entry of a virus into a
cell
expressing that protein.
The virus against which the antiviral immunokine is directed is an
inununodeficiency virus, that is, a virus which causes an immunodeficiency
disease.
Thus, the immunokine of the invention is termed an anti-immunodeficiency virus
immunokine. Such immunodeficiency virus should be construed to include any
strain
of HIV or SIV, as well as other lentiviruses (FIV, FeLV, BIV, and EIAV).
By "HIV" as used herein, is meant any strain of a human immunodeficiency
virus belonging to the group of either HIV type 1 or HIV type 2. By "SIV" as
used
herein is meant any of five recognized strains of SIV (STVmac, SIVsnnn,
SIVagm,
SIV~nnd and SIVcpz) which are known to infect non-human primates.
Without intending to be bound by theory, it appears that both native alpha-
cobratoxin and an immunokine of the invention are each capable of binding to a
cellular protein required to form a functional cellular receptor for entry of
HIV into a
cell. In one preferred embodiment, the immunokine of the invention is an
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immunokine which binds to a cellular receptor and/or to a cellular co-factor
required
for entry of HIV into a cell. A "cellular co-factor" as used herein, is
defined as a
protein which is required, in association with a cellular receptor for HIV,
for entry of
HIV into cells.
According to the invention, the polypeptides (e.g., native or immunokine) of
the invention is useful in a method of inhibiting infection of a cell by HIV
as
described herein. Moreover, the imrnunolcine of the invention is useful in a
method of
screening compounds for anti-HIV activity as described herein. Additional uses
for
alpha-cobratoxin or an immunol~ine of the invention include the identification
of cells
in the body which are potential targets for viral infection. The immunokine is
thus
also useful for the isolation of such cells using flow cytometry technology or
other
cellular isolation techniques which are common in the art. The invention also
relates
to methods of use of the immunokine of the invention, which methods include
diagnostic and therapeutic uses.
By "antiviral activity" as used herein, is meant an immunokine which when
added to an immunodeficiency virus or to a cell to be infected with such a
virus,
mediates a reduction in the ability of the virus to infect and/or replicate in
the cell
compared with the ability of virus to infect and/or replicate in the cell in
the absence
of the immunokine. Examples of assays for antiviral activity are described in
detail in
the experimental detail section and include, but are not limited to, reverse
transcriptase assays, immunofluorescence assays, assays for formation of
syncytia,
antigen capture assays and the lilce.
Immunolcine Pre aration
A composition of this invention can be prepared in any suitable manner. For
instance, native cobratoxin can be obtained arid used in its native (e.g.,
unmodified)
form, and is shown to inhibit HIV infection of cells (PMNC) with a similar
efficacy to
the corresponding alpha-innnunokine described herein. Toxins themselves can be
chemically modified (e.g., using ozone, performic acid, iodoacetamide etc.),
and other
cobratoxin homologues (see Group II) can be prepared. Toxin modifications
include
site-directed mutants (mono and poly-substituted mutants such as tryptophan,
tyrosine, lysine and arginine), chimeras and other homologous peptide
fragments
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produced from the parent protein through genetic engineering or synthetic
peptide
production.
An inactivated bioactive polypeptide (e.g., immunokine) of this invention can
be prepared using any suitable means. As described herein, the immunokine can
be
5 chemically produced in an oxidative process in combination with the protein
of
interest, e.g., a neurotoxin. The use of ozone treatment to prepare the
immunokine is
particularly preferred, e.g., in view of the simplicity of manufacture, the
modest
facility requirements and self sterilizing nature of the production procedure.
Under
controlled conditions, ozone specifically modifies certain amino-acids such as
10 methionine, cysteine and tryptophan to methionine sulphone, cysteic acid
and
kynurenine respectively. Cobratoxin has no methionine, ten (10) cysteine and
one (1)
tryptophan residues.
Other procedures can be used as well, though these with each such procedure
providing a product that varies in its relative potencies when compared to
immunokine produced with ozone. Those procedures include the use of hydrogen
peroxide, perfonnic acid, carboxyamidomethylation, iodoacetamide, iodoacetic
acid
and Oxone (faro's Acid) but includes any chemical agent that acts as an
oxidizer or
alkylator that can render proteins like cobratoxin atoxic and suitable for
administration to a host. The circumstances where a difference procedure would
be
employed would be if the resultant product demonstrated better therapeutic
activity in
other applications, for example superior imrnuno-modulatory, anti-tumor or
anti-viral
activity, but they emphasize the importance of breaking the disulphide bonds
with a
concomitant conformational reorganization similar to that during disulphide
oxidation. The requirement for scission of all the disulphide bonds for
optimal
function has not yet been fully investigated but sufficient bonds must be
broken to
render a protein like alpha-cobratoxin safe for administration to a host.
Applicant's paxent application (now US Patent No 5,989,857) described, inter
alia, a method that involved bubbling ozone through a solution (lOmg/ml) of
cobratoxin in water. This approach could be used, for instance to produce 12
gram
batches with a concentrate that could be diluted to any desired concentration.
This
approach typically involved a 6-8 hour process requiring close monitoring to
determine the optimal endpoint. The endpoint of the reaction was typically
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determined by toxicity studies in mice. Ozonation was determined to be
complete
when mice survived a 1 mg (0.1 cc) inj ection. It was determined by this
technique,
however, that excess ozone could adversely effect the quality~of the final
drug
product.
In a presently preferred embodiment (for the ozonation of neurotoxins to make
therapeutic molecules) the present invention now uses only enough ozone to
render
the toxin atoxic (breaking the disulphide bonds) while minimizing damage to
other
sensitive sites of oxidation. Secondly, it is now preferred to ozonate
physiological
saline (0.9% NaCl) such that it contains a known, preferred amount of ozone
which is
then added to solubilized toxin in the 0.9% NaCI. The oxidation is
stoichiometic as
described below.
Those skilled in the art will be able to determine a stoichiometric approach,
given the present description, as exemplified by the use of cobratoxin as
follows:
In theory, 1 ug/ml of Ozone contains 0.02083 umoles/ml = 1 u~/ml Ozone
48 MW
Likewise, 1 ug of toxin contains 0.0001276 umoles of toxin = 1 a ml toxin
7831 MW
If one multiplies by ten to account for the sulphurs (half cystines) to be
oxidized by the ozone (i.e., 0.000127 umoles x 10) one obtains the value of
0.00127
umoles of sulphur molecules (half cystines).
Experimental models were used to confirm these assumptions. Varying
amounts of toxin (25mg-1230mg dissolved in 10 ml solution) were brought up to
a
final volume of 1 liter using ozonated saline, as described above. In this
model,
samples containing 25 mg/1 to 300 mg/1 toxin were not toxic in mice while the
610mg
and 1230mg samples killed mice. Additional experiments used 19.1 ug/ml ozone
dissolved in 0.9% saline in which samples containing 600 mg/1 and 700 mg/1 of
toxin
were oxidized. The 600mg/1 samples were not toxic in mice and the 700mg/1
samples
killed mice, thus defining the range of use.
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In one aspect, the present invention provides a method of preparing a
parenteral composition comprising an immunokine (e.g., an imrnunokine), the
method
comprising the steps of
a) identifying a polypeptide having a biological activity dependent on the
presence of one or more disulfide bridges in its tertiary structure,
b) preparing a cDNA strand encoding the polypeptide,
c)' expressing the cDNA under conditions in which the polypeptide is
recovered in an inactive form due to the failure to form one or more disulfide
bridges,
and
d) recovering the inactive polypeptide and formulating it into a
composition suitable for parenteral administration to a host.
In another aspect, the invention provides a composition comprising an
immunokine that has been rendered inactive by virtue of the failure to form
one or
more of its disulfide bridges. In a related aspect, the invention provides a
composition
for in vivo administration comprising a bioactive immunokine that has been
inactivated in the manner described herein.
The method can be used to prepare immunokines from, or based upon, a
variety of natural compounds, including "Group I neurotoxins" (namely, toxins
affecting the presynaptic neurojunction), Group II neurotoxins (namely those
affecting the postsynaptic neurojunction), and Group III neurotoxins (those
affecting
ion channels). cDNA sequences fox such polypeptides are generally known, or
can be
determined using conventional techniques.
The cDNA can be expressed using any suitable expression system, under
conditions in which the product can be recovered with one or more disulfide
bridges
unformed. Suitable expression systems include heterologous host systems such
as
bacteria, yeast or higher eucaryotic cell lines. Examples of useful systems
are
described, for instance, in "Foreign Gene Expression in Yeast: a Review",
Romanos,
et al., Yeast, 8:423-488 (1992). See also, "Yeast Systems for the Commercial
Production of Heterologous Proteins", Buckholz, et al., Bio/Technology 9:1067-
1072
(1991), the disclosures of both Romanos et al. and Buckholz et al. being
incorporated
herein by reference.
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These articles are generally directed at the more common goal of affirmatively
achieving posttranslational processing and extracellular secretion. Under such
conditions, the formation of appropriate disulfide linkages would be included
as a
necessary step. Given the present description, however, these articles, and
the
techniques described therein, will be of considerable use to those skilled in
the art in
achieving the recovery of the unfolded product, e.g., by intracellular
expression in
yeast.
Preferably, the cDNA is expressed using a microbial expression system, such
as Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. From a
safety and
environmental perspective it is preferable that the cDNA is expressed in a
microbial
expression system under conditions in which the product is cytoplasmically
produced,
as opposed to extracellularly secreted. In an exemplary embodiment, the
immunokine
is expressed using a microbial expression system, under conditions in which
the
leader sequence of naturally-occurring cDNA is removed and replaced with only
the
initiation codon.
Tm_m__unokines of the present invention are generally stable under suitable
conditions of storage and use in which the disulfide bonds are prevented from
spontaneously reforming, or are allowed to reform in a manner that precludes
the
undesirable activity of the immunokine. Optionally, and preferably, once the
inactive
polypeptide has been recovered, it is treated by suitable means to ensure that
the
cysteine residues do not spontaneously reform to form disulfide bridges. An
example
of a preferred treatment means is the use of ozone treatment as described
herein.
In another optional, and alternative, embodiment a immunokine such as
neurotoxin is produced in an inactive form using the Pichia expression system
described herein. To the best of Applicants knowledge, the prior art fails to
teach or
suggest the preparation of a toxin in inactive form by the route of
cytoplasmic
expression in yeast.
The method and composition of the present invention provide a unique and
valuable tool for the synthesis and recovery of bioactive irninunokines in a
manner
capable of diminishing undesirable activity, yet retaining other useful
properties of the
immunokine (such as immunogenicity and antiviral activity).
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As used herein, the following words (and inflections thereof) and terms will
have the meanings ascribed to them below:
"bioactive" will refer to a polypeptide capable of eliciting at least one
biological response when administered if? vivo.
"polypeptide" will refer to any biomolecule that is made up, at least in part,
of
a chain of amino acid residues linked by peptide bonds.
"inactive" will refer to a polypeptide that is provided in a form in which at
least one form of its bioactive responses is substantially terminated or
decreased to a
desired extent.
"neurotoxin" will refer to a bioactive polypeptide wherein at least one
activity
(e.g., binding to the acetylcholine receptor) produces a toxic effect on the
nervous
system of a mammalian host.
The method of the present invention involves an initial step of identifying a
bioactive immunokine having a tertiary structure in which bioactivity is
dependent, at
least in part, on the formation of one or more disulfide bridges between
cysteine
residues. Typically, the immunokine will be one that is naturally secreted in
the
course of its synthesis, since it is the secretion process that will provide
the necessary
posttranslational steps, including disulfide bond formation. Preferably, the
immunokine is one that is stable when recovered and that retains other
desirable
properties in the unfolded state, such as immunogenicity and/or antiviral,
anti-tumor
or wound healing activity.
The amino acid sequence and tertiary structure of a number of bioactive
polypeptides is known. Suitable immunokines include those in which one or more
disulfide bridges are known to form in the natural configuration, and in which
such
bridges) are necessary for the bioactivity of the inununokine. Such bridges
can be of
either an intramolecular (i.e., within a single polypeptide) nature and/or an
intermolecular (e.g., between discrete subunits) nature.
Secreted or cell-surface proteins often form additional covalent intrachain
bonds. For example, the formation of disulfide bonds between the two -SH
groups of
neighboring cysteine residues in a folded polypeptide chain often serves to
stabilize
the three-dimensional structure of the extracellular proteins. Protein
hormones such
as oxytocin, arginine vasopressin, insulin, growth hormone and calcitonin, all
contain
CA 02404078 2002-09-23
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disulfide bonds. Enzymes such as ribonuclease, lysozyme, chymotrypsin,
trypsin,
elastase and papain also have their tertiary structure stabilized by disulfide
bonds.
Besides the bioactive proteins listed above, there are numerous other proteins
that
contain disulfide bonds, such as the innnunoglobulins (IgA, IgD, IgE, IgM),
5 fibronectin, MHC (major histocompatible complex) molecules and procollagen.
Many polypetides from animal venoms also contain disulfide bonds.
In a preferred embodiment, the method of the present invention is used to
prepare inactivated forms of neurotoxins, and more preferably neurotoxins from
amongst the four groups provided below. As described above, those in Group I
IO typically affect the presynaptic neurojunction, those in Group II typically
affect the
postsynaptic neurojunction, and those in Group III typically affect ion
channels.
Lastly, there axe also included toxins known only to have a toxic affect by
causing
membrane damage.
Neurotoxins Membrane-damaging toxins
15
Grou I Group II Group III Toxins
notexin a-conotoxin dendrotoxins
myotoxins
13-bungarotoxin a-cobrotoxin scorpion toxins
cardiotoxins
crotoxin erabutoxin ~.-conotoxins
mellitin
taipoxin a,-cobratoxin sea anemone toxins
phospholipases
textilotoxin a,-bungarotoxin
a-latrotoxin
The method involves a further step of preparing or isolating a corresponding
gene (e.g., a cDNA strand) encoding the polypeptide. Using the primary amino
acid
sequence discussed above, and in view of the present teaching, those skilled
in the art
will appreciate the manner in which such polypeptides can be synthesized using
genetic engineering techniques. Generally, and preferably, one or more of the
native
control (e.g., leader) sequences of the desired cDNA are removed and replaced
with
one or more corresponding sequences in order to facilitate the desired
expression.
Immunokine components from animal venoms, for instance, can be obtained
from the animals themselves or from other sources, or they can be created in
the
CA 02404078 2002-09-23
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16
laboratory using conventional protein engineering techniques. In the former
approach, animals are induced by mechanical or electrical stimuli to release
venom
from their glands, which travels through a venom canal and out the fang or
stinger.
The venom is collected and various constituents of the venom are purified by
conventional chromatographic techniques.
In the latter approach, constituents from the venom are synthesized by cloning
the genes encoding the various ixnmunokine elements and expressing these genes
in
heterologous host systems such as bacteria, yeast or higher eucaryotic cell
lines.
Yeast expression systems are presently preferred, since they tend to provide
an
optimal combination of such properties as yield and adaptability to human use
products.
Expressed products are then purified from any other contaminating host
polypeptides by means of chromatographic techniques similar to those used to
isolate
the polypeptides directly from the venom.
There are significant advantages to the use of host systems other than the
venomous animals to obtain the venom components. The danger to human lives in
obtaining the venom from the animal is eliminated. There will no longer be a
need
for the costly animal husbandry required to maintain venomous animals for
venom
extraction. The quantities of materials that can be obtained from the genetic
engineering approach can be one or more orders of magnitude greater than the
quantities that can be derived from the venom itself. Moreover, once the
genes) is
cloned and expressed, it can be used to provide a continual, reproducible
source in the
form of a bacterial, yeast or higher eucaryotic cell line seed culture.
Seed cultures can be stored and transported in the frozen state, lyophilized,
or,
in some cases, plated on media. Also, the use of genetic engineering tools
will enable
those skilled in the art to manipulate the genes for the purpose of altering
the
polypeptide product in any fashion feasible. Using the method of the present
invention, in combination with available tools for protein engineering (e.g.,
site-
directed mutagenesis), those skilled will be able to prepare a bioactive
polypeptide
having any desired level of toxicity, whether non-toxic, or of diminished,
equal or
greater toxicity than the native form.
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17
The method of the invention provides a further step of expressing the cDNA
under conditions in which the polypeptide is recovered in an inactive form due
to the
failure to form one or more disulfide bridges. As described in greater detail
below,
this step involves the avoidance of posttranslational processes that would
otherwise
serve to form such lintcages.
Optionally, and preferably, the method provides a further step of treating the
immunokines in order to retain the cysteine residues and prevent the
spontaneous
formation of disulfide bonds. A preferred treatment includes ozone treatment,
in the
manner described herein. Ozonation affects the cysteine residues by converting
the
IO pendent sulfhydryl (-SH) groups to corresponding -S03X groups, which,
unlike the
sulfhydryl groups, are unable to form a disulfide bridge. Such treatment is
not
necessary, however, for those inactivate polypeptides that are found to not
spontaneously reform, and that provide the desired activity. Ozonation is
preferred
for polypeptides such as neurotoxins, where Applicant has shown that upon
cleavage
and ozonation of the sulfliydryl groups, native neurotoxins are both stable
and active.
The invention further provides a bioactive polypeptide that has been rendered
inactive by virtue of the failure to form one or more disulfide bridges. Such
polypeptides can be stably stored and used under conditions in which disulfide
bonds
are prevented from spontaneously reforming.
In yet another aspect, the invention provides a method of administering a
bioactive polypeptide to a host, comprising the step of providing the
polypeptide in an
inactive form and within a suitable composition, and administering the
composition to
a host. In a related aspect, the invention provides a host having administered
such a
polypeptide. Compositions of the present invention can be used for a variety
of
purposes. Compositions are particularly useful in situations calling for a
polypeptide
in a form that is as close to native as possible, yet without an unwanted
bioactivity.
Poplypeptides such as the preferred neurotoxins and immunokines can be
prepared using genetic engineering techniques within the skill of those in the
art,
given the present desription. See, for instance, (Fiordalisi et al., (1996)
Toxicon 34, 2,
213-224, I~rajewski et al (1999) "Recombinant ml-toxin" presented at the 29th
Annual Meeting of the Society for Neuroscience) and (Smith et al., (I997)
Biochemistry, 36, no. 25, 7690-7996 . As the native cobratoxin gene is
available, a
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18
number of bioengineered variants can be prepared which replace the residues
required
for disulphide bond formation with other residues. As these amino acid
substitutions
must be expressed ih vivo, the availability of modifications are typically
limited to the
use of native residues (the standard 20 naturally occurring amino acids) and
the host
to be employed for expression. In the host, the codon usage will be important
in
ensuring efficient and maximal expression of the novel protein. Theoretically
any
amino acid can be substituted for cysteine but as this is a more costly
approach to
generating immunokine variants relative to synthetic peptide techniques
certain
residues have been selected which best reproduce the protein characteristics
resulting
from chemical exposure.
It is preferred to make what are considered to be conservative substitutions,
e.g., to limit the cysteine replacement to the following residues; methionine
(M),
glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (I~, serine
(S), glycine
(G) and alanine (A). Methionine incorporation can be considered to be the more
conservative substitution by replacing one sulphur-containing residue for
another.
Unlike cysteine, methionine cannot form disulphide bonds. Methionine also
reacts
readily with ozone to produce the sulfone derivative, therefore the purified
product
can be exposed to ozone or other chemical agents to confer upon the protein
other
desirable properties (i.e. low immunogenicity). Also the presence of
methionine also
allows for the cleavage of the protein into fragments employing cyanogen
bromide.
Cleavage of the native cobratoxin and immunokine protein can be achieved
with serine proteases (i.e. trypsin) but at sites containing positive
residues. This
permits also the evaluation and production of smaller peptide fragments for
biological
activity. The conversion of cysteine to cysteic acid also permits the
substitution by
other acidic residues such as E, D, Q, N and S. The substitution of E and D
for
cysteine is estimated to produce a protein with a pI similar to that of alpha-
immunokine (pI = 4.5). The substitution of cysteine with the residues glycine
and
alanne would represent standard "neutral" substitutions. A suitable method for
creating these genes has been described previously (Smith et al., (1997)). The
codon
usage of the DNA fragments is optimized for use in commercially used bacterial
and
yeast expression systems Esc7aef°iclaia coli and Piclaia pasto~is
respectively.
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19
Given the advances in technology in cloning DNA encoding proteins
comprising antibodies, the invention also includes DNA which encodes the
immunokine of the invention, or a portion of such immunokine. The nucleic acid
encoding the immunoltine may be cloned and sequenced using technology which is
S available iri the art, and is described, for example, in Wright et al.
(1992, Critical Rev.
in Immunol. 12(3,4):125-168) and the references cited therein. Further, the
imtnunokine of the invention may be "humanized" using the technology described
in
Wright et al., (supra) and in the references cited therein.
For example, to generate a phage immunokine library, a cDNA library is first
obtained from mRNA which is isolated from cells, e.g., the hybridoma, which
express
the desired protein to be expressed on the phage surface, e.g., the desired
immunoltine. cDNA copies of the mRNA are produced using reverse transcriptase.
cDNA which specifies immunoglobulin fragments are obtained by PCR and the
resulting DNA is cloned into a suitable bacteriophage vector to generate a
1S bacteriophage DNA library comprising DNA specifying immunoglobulin genes.
The
procedures for malting a bacteriophage library comprising heterologous DNA are
well
known in the art and are described, for example, in Sambroolt et al. (1989,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).
Bacteriophage which encode the desired immunokine, e.g., an immunokine,
may be engineered. such that the protein is displayed on the surface thereof
in such a
manner that it is available for binding to its corresponding binding protein,
e.g., the
antigen against which the immunokine is directed. Thus, when bacteriophage
which
express a specific immunokine are incubated in the presence of a cell which
expresses
the corresponding antigen, the bacteriophage will bind to the cell.
Bacteriophage
2S which do not express the immunokine will not bind to the cell. Such panning
techniques are well known in the art and are described for example, in Wright
et al.,
(supra).
By the term "synthetic immunoltine" as used herein, is meant an immunokine
which is generated using recombinant DNA technology, such as, for example, an
immunokine expressed by a bacteriophage as described herein. The term should
also
be construed to mean an immunokine which has been generated by the synthesis
of a
DNA molecule encoding the immunokine and which DNA molecule expresses an
CA 02404078 2002-09-23
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immunokine protein, or an amino acid sequence specifying the immunokine,
wherein
the DNA or amino acid sequence has been obtained using synthetic DNA or amino
acid sequence technology which is available and well known in the art.
The invention thus includes a DNA encoding the immunokine of the invention
5 or a portion of the irnmunol~ine of the invention. To isolate DNA encoding
an
immunokine, for example, DNA is extracted from immunokine expressing phage
obtained according to the methods of the invention. Such extraction
teclv~iques are
well known in the art and are described, for example, in Sambrook et al.
(supra).
An "isolated DNA", as used herein, refers to a DNA sequence, segment, or
10 fragment which has been purified from the sequences which flank it in a
naturally
occurnng state, e.g., a DNA fragment which has been removed from the sequences
which are normally adjacent to the fragment, e.g., the sequences adjacent to
the
fragment in a genome in which it naturally occurs. The term also applies to
DNA
which has been substmtially purified from other components which naturally
15 accompany the DNA, e.g., RNA or DNA or proteins which naturally accompany
it in
the cell.
The invention should also be construed to include DNAs which are
substantially homologous to the DNA isolated according to the method of the
invention. Preferably, DNA which is substantially homologous is about 50%
20 homologous, more preferably about 70% homologous, even more preferably
about
80% homologous and most preferably about 90% homologous to DNA obtained using
the method of the invention.
"Homologous" as used herein, refers to the subunit sequence similarity
between two polymeric molecules, e.g., between two nucleic acid molecules,
e.g., two
DNA molecules or two RNA molecules, or between two polypeptide molecules.
When a subunit position in both of the two molecules is occupied by the same
monomeric subunit, e.g., if a position in each of two DNA molecules is
occupied by
adeune, then they are homologous at that position. The homology between two
sequences is a direct function of the number of matching or homologous
positions,
e.g., if half (e.g., five positions in a polymer ten subunits in length) of
the positions in
two compound sequences are homologous then the two sequences are 50%
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21
homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous,
the
two sequences share 90% homology.
To obtain a substantially pure preparation of a protein comprising, for
example, an immunokine, generated using the methods of the invention, the
protein
may be extracted from the surface of the phage on which it is expressed. The
procedures for such extraction are well known to those in the art of protein
purification. Alternatively, a substantially pure preparation of a protein
comprising,
for example, an immunokine, may be obtained by cloning an isolated DNA
encoding
the immunolcine into an expression vector and expressing the protein
therefrom.
Protein so expressed may be obtained using ordinary protein purification
procedures
well known in the art.
An inactivated bioactive polypeptide of this invention can also be provided by
synthetic means, e.g., solid phase synthesis (also known as combinatorial
chemistry).
For instance, current technology permits the production of polypeptides such
as
neurotoxins through peptide synthesis. Many smaller neurotoxins (from conus
snails,
bee venom and scorpion venom) are routinely produced by synthetic peptide
methodology (Hopkins et al., (1995) J. Biol. Chem., 270, no. 38, 22361-22367,
Ashcom and Stiles, (1997) Biochem. T. 328, 245-250, Granier et al., (1978)
Eur. J.
Biochem, 82, 293-299 and Sabatier et al., (1994) Int. J. Pept. Protein Res.,
43, 486-
495) and some are available from commercial organizations. The above
references
also describe the synthesis of such peptides incorporating mutant residues
(Hopkins et
al. (1995) and Sabatier et al (1994)).
Current techniques in peptide chemistry allow for proteins in excess of 80
amino acids can be reliably produced using automated Fmoc solid phase
synthesis
(ABI 433A Peptide Synthesizer, Perkin Elmer - see www.perkin-elmer.com). Non
native amino acids (acetamidomethyl cysteine, carboxyamidomethyl cysteine,
cysteic
acid, kynurenine and methionine sulphone) are acquired from Advanced Chemtech
(Louisville, Kentucky) or Quchem (Belfast, Ireland). Other oxidized or
alkylated
amino acid variants are available from these agents. The generation of alpha-
immunol~ine is achieved by substituting primarily the cysteine residues (from
1 pair to
all 5 disulphide couples) with those residues described above to mimic the
effects of
ozone and other chemical modifications. Furthermore the substitution of other
native
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22
and non-native residues for cysteine can be investigated in an attempt to
identify
immunokine variants with improved biological activity. Also peptide fragments
from
within the cobratoxin sequence can be created (analogous to Hinmann et al.,
(1999),
T_mmunoparmaCOl. Tznmunotoxicol, ZI (3), 483-506) and examined for receptor
binding activity.
Inactivated bioactive polypeptides of this invention can be formulated and
delivered in any suitable manner. For instance, for use in treating existing
HIV
infections, an immunokine will typically be provided in a substantially pure
and
sterile form, and in a vehicle adapted for delivery. As used herein, the term
"substantially pure" describes a compound, e.g., a protein or polypeptide
which has
been separated from components which naturally accompany it. Typically, a
compound is substantially pure when at least 10%, more preferably at least
20%,
more preferably at least 50%, more preferably at least 60%, more preferably at
least
75%, more preferably at least 90%, and most preferably at least 99% of the
total
material (by volume, by wet or dry weight, or by mole percent or mole
fraction) in a
sample is the compound of interest. Purity can be measured by any appropriate
method, e.g., in the case of polypeptides by column chromatography, gel
electrophoresis or HPLC analysis. A compound, e.g., a protein, is also
substantially
purified when it is essentially free of naturally associated components or
when it is
separated from the native contaminants which accompany it in its natural
state.
To inhibit infection of cells by HIV in vitro, cells are treated with the
immunokine of the invention, or a derivative thereof, either prior to or
concurrently
with the addition of virus. Inhibition of infection of the cells by the
immunokine of
the invention is assessed by measuring the replication of virus in the cells,
by
identifying the presence of viral nucleic acids and/or proteins in the cells,
for
example, by perfornling PCR, Southern, Northern or Western blotting analyses,
reverse transcriptase (RT) assays, or by immunofluorescence or other viral
protein
detection procedures. The amount of immunokine and virus to be added to the
cells
will be apparent to one skilled in the art from the teaching provided herein.
To inhibit infection of cells by HIV ire vivo, the immunokine of the
invention,
or a derivative thereof, is administered to a human subject who is either at
risk of
acquiring HIV infection, or who is already infected with HIV. Prior to
administration,
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23
the immunokine, or a derivative thereof, is suspended in a pharmaceutically
acceptable formulation such as a saline solution or other physiologically
acceptable
solution which is suitable for the chosen route of administration and which
will be
readily apparent to those skilled in the art of immunokine preparation and
administration. The dose of imrnunokine to be used may vary dependent upon any
number of factors including the age of the individual, the route of
administration and
the extent of HIV infection in the individual. The immunokine is prepared for
administration by being suspended or dissolved in a pharmaceutically
acceptable
earner such as saline, salts solution or other formulations apparent to those
skilled in
such administration.
Typically, the imrnunokine is administered in a range of 0.1 microgram to 1 g
of protein per dose. Approximately 1-10 doses are administered to the
individual at
intervals ranging from once per day to once every few years. The immunokine
rnay be
administered by any number of routes including, but not limited to,
subcutaneous,
intramuscular, oral, intravenous, intradermal, intranasal or intravaginal
routes of
administration. The immunokine of the invention may be achninistered to the
patient
in a sustained release formulation using a biodegradable biocompatible
polymer, or by
on-site delivery using micelles, gels and liposomes, or rectally (e.g., by
suppository or
enema). The appropriate pharmaceutically acceptable carrier will be evident to
those
skilled in the art and will depend in large part upon the route of
administration.
The immunokine (including the corresponding active bioactive polypeptide) of
the invention may also be used in a method of screening compounds for anti-HIV
activity. A test compound is first screened for the ability to bind to the
immunokine of
the invention. Compounds which bind to the immunol~ine are likely to share
structural
and perhaps biological activities with CXCR4 and thus, may serve as
competitive
inhibitors for inhibition of the interaction of HIV envelope protein with CD4
and/or
CXCR4 plus CD4. An immunokine-binding compound is further tested for antiviral
activity by treating cells with the compound either prior to or concurrently
with the
addition of virus to the cells. Alternatively, the virus and the compound may
be mixed
together prior to the addition of the mixture to the cells. The ability of the
compound
to affect virus infection is assessed by measuring virus replication in the
cells using
any one of the known techniques, such as a RT assay, immunofluorescence assays
CA 02404078 2002-09-23
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24
and other assays known in the art useful for detection of viral proteins or
nucleic acids
in cells. Generation of newly replicated virus may also be measured using
known
virus assays such as those which are described herein.
The immunokine of the invention may also be used in competition assays to
screen for compounds that bind to CXCR4 arid which therefore prevent binding
of the
immunokine to CXCR4. Such compounds, once identified, may be examined further
to determine whether or not they prevent entry of virus into cells. Compounds
which
prevent entry of virus into cells are useful as anti-viral compounds.
Additional uses for the immunokine of the invention include the identification
of cells in the body which are potential targets for infection by an
immunodeficiency
virus.
By the term "target cell for immunodeficiency virus infection" as used herein,
is meant a cell which expresses receptor proteins) for an immunodeficiency
virus and
which cell is therefore capable of being infected by an immunodeficiency
virus.
Cells which axe potential targets for HIV infection may be identified by
virtue
of the presence of CXCR4 on their surface. The immunokine of the invention
facilitates identification of these cells as follows: The immunokine of the
invention is
first combined with an identifiable marker, such as an immunofluorescent or
radioactive marker. Cells which are obtained from a human subject are then
reacted
with the tagged immunokine. Binding of the immunokine to cells is an
indication that
such cells are potential targets for HIV infection. The identification of
cells which
may be infected with HIV is important for the design of therapies for the
prevention
of HIV infection. Fox example, CXCR4 is differentially expressed and regulated
on
human T lymphocytes (Bleul et al., 1997, Proc. Natl. Aced. Sci. USA 94:1925-
1930).
Further, reactivity of immune cells to MAb 1265 is high on naive cells and low
on
memory cells and thus, the pattern of expression of CXCR4 and its utilization
by
viruses may contribute to immune dysfunction. CXCR4 has also been detected,
using
the immunokine of the invention, on some endothelial cells (in atherosclerotic
plaques), platelets and some hematopoietic precursor cells. In the case of
individuals
who are infected with HIV, the identification of target cells provides an
immune
profile of these individuals which provides useful information regarding the
progress
of their infection.
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In addition to the aforementioned uses for the immunokine of the invention,
the immunokine is useful for the detection of CXCR4 on a variety of cell types
on
which CXCR4 may be expressed. For example, CXCR4 is expressed on human
neurons (Hesselgesser et al., 1997, Current Biology 7:112-121), including
cells in the
5 human brain.
EXAMPLES
Example 1
Isolation of Gland Tissue for RNA Extraction
The following protocol was used to clone the gene encoding a-cobratoxin
10 from the venom of Naj a naj a siamensis.
(al Recovery of Venom
Naj a naj a siamensis snakes were obtained from Siam Farms, Bangkok,
Thailand. Animals were shipped to and housed at Ventoxin, Inc., Frederick, MD.
USA. The venom glands from N. n, siamensis animals were surgically removed and
15 used to prepare mRNA for generating a cDNA library. Snakes were placed on a
schedule for milking (venom extraction). They were milked on day 1 and eight
days
Iater milked a second time. On the 2nd or 3rd day, they were anesthetized with
sodium pentobarbital and their glands removed (Vandenplas et al., 1985). Gland
tissue was quickly cut into small pieces and immediately frozen in liquid
nitrogen.
20 Samples were kept at -70°C until use.
(b) RNA Isolation
Total RNA was isolated from gland tissue by using a standard
guanidiniumlhot phenol method (Feramisco et al., 1982). Frozen gland tissues
(5 g)
were placed in a polytron mixer and 10 ml of Solution A (guanidinium
isothiocyanate
25 mixture) was added to the tissue. Solution A was prepared by resuspending
100 g of
guanidinium isothiocyanate in 100 ml of deionized water, 10.6 ml of 1 M Tris-
Cl (pH
7.6), and 10.6 ml of 0.2 M disodium ethylene diamine tetraacetate (EDTA). It
was
stirred overnight at room temperature.
The solution was then warmed while stirnng to 60-70°C for 10 min to
assist
dissolution. Any insoluble material remaining was removed by centrifugation at
3000g for 10 min at 20°C. To the guanidinium isothiocyanate solution,
was added
CA 02404078 2002-09-23
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26
21.2 ml of 20% sodium lauryl sarkosinate and 2.1 ml of 13-mercaptoethanol to
the
supernatant and the volume was brought to 212 ml with water. The final
solution was
filtered through a disposable Nalgene filter and stored at 4°C in a
tightly sealed,
broom glass bottle.
The glands were mixed in the polytron mixer at 4°C until most of the
tissue
had been disrupted (about 3-5 min.). The gland solution was placed in a 50 ml
polypropylene centrifuge tube and 20 ml more of the guanidinium isothiocyanate
mixture was added. The mixture was brought to 60°C and passed through a
syringe
fitted with an 18 gauge needle. This shearing technique was repeated 2 to 3
times or
until the viscosity of the suspension was reduced. An equal volume of ultra
pure
liquid phenol preheated to 60°C was added to the tissue suspension and
this was again
passed through the syringe 2 to 3 times.
At this point, 0.5 volume of Solution B (0.1 M sodium acetate (pH 5.2), 0.01
M Tris-Cl (pH 7.4), 0.001 M. EDTA) was added to the emulsion and mixed. An
equal volume of chloroform/isoamyl alcohol (24/1 v/v) was added and the
mixture
shaken vigorously for 15 min. wlule maintaining the temperature at
60°C. The
mixture was cooled on ice and centrifuged at 2000g for 15 min. at 4°C.
The aqueous
phase, containing the RNA, was recovered a~.id reextracted with
phenol/chloroform.
To the aqueous phase was added 2 volumes of absolute ethanol and the mixture
was
stored at -20°C overnight. All glassware used in extracting and
worl~ing with RNA
had been baked at 250°C for at least 4 h. Sterile, disposable
polypropylene
plasticware is essentially free of RNase and can be used for the preparation
and
storage of RNA without pretreatment.
The RNA was recovered by centrifugation was dissolved in 30 ml of Solution
C (0.1 M Tris-Cl, pH 7.4, 0.05 M NaCI, 0.01 M EDTA, 0.2% (v/v) sodium dodecyl
sulfate (SDS)). Proteinase K was added to a final concentration of 200 ~,g/rnI
and
incubated at 37°C for 2 h. The solution was then heated to 60°C
and 0.5 volume of
phenol, preheated to 60°C, was added and mixed vigorously with the RNA-
containing
solution. Chloroform (0.5 volume) was added to the solution and again mixed
vigorously at 60°C for 10 min. The solution was cooled on ice for 10
min. and then
centrifuged at 2000g for 15 min.
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27
The aqueous phase was recovered and re-extracted one more time with
phenol/chloroform at 60°C. The aqueous phase was recovered and
reextracted twice
with chloroform at room temperature. To the aqueous phase was added 2 volumes
of
absolute ethanol and put at -20°C overnight. The nucleic acids were
precipitated by
centrifugation and the pellet rinsed with 70% cold ethanol. RNA was stored at -
70°C
in 70% ethanol until used. When the RNA was ready to be used, it was
centrifuged,
dried and resuspended in Rnase-free sterile water.
(c) mRNA Purification
Poly(A)+ RNA was enriched by passage over an oligo(dT)-cellulose colmnn
using a conventional method (Aviv and Leder, 1972). Conunercial oligo(dT)-
cellulose was equilibrated with sterile, RNase-free Solution D (0.02 M Tris-
Cl, pH
7.6, 0.5 M NaCl. 0.001 M EDTA and 0.1 % (v/v) SDS). A 1.0-ml bed-volume of
equilibrated matrix was poured into either an Rnase-free disposable
polypropylene
colurrul or siliconized RNase-free pasteur pipette. The matrix was washed with
3
IS column volumes of (1) Rnase-free sterile water; (2) 0.1 M NaOH containing
0.005 M
EDTA; and (3) sterile water. The column effluent should have a pH less than 8.
The
column was then washed with 5 volumes of sterile Solution D.
The RNA isolated as described above was heated to 65°C for 5 min
and a 2X
concentration of an equal volume of Solution D was added to the RNA solution.
The
sample was cooled to room temperature and loaded onto the oligo(dT)-cellulose
column. The flow-through from the column was heated to 65°C, cooled to
room
temperature, and reapplied to the column. The column was washed with 10
volumes
of Solution D followed by 4 column-volumes of Solution D containing 0.1 M
NaCI.
The poly(A)+ RNA was then eluted with 2-3 column volmnes of sterile Solution E
(0.01 M Tris-Cl, pH 7.5, O.OOIM EDTA and 0.05% (v/v SDS).
Typically, NaCI was added to the mRNA to obtain a salt concentration of 0.5
M, and the mRNA was repurified on a second passage over the oligo(dT)-
cellulose
column using the same procedures as described for the initial column run.
Sodium
acetate (NaOAc) (3M, pH 5.2) was then added to the mRNA from the second column
run to obtain a final concentration of 0.3 M NaOAc. Cold absolute ethanol (2.5
volumes) was added to the mRNA solution and the solution was placed at -
20°C
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overnight. The N. n. siamensis mRNA was then centrifuged at 12,000g, the
pellet
washed with cold 70% ethanol, and stored in 70% ethanol at -70°C until
used. The
yield of mRNA from 5 g of gland tissue was 16 ~,g.
(d) Construction of a N. n. siamensis cDNA Library.
Complementary DNA (cDNA) was prepared from 5 p.g of N. n siamensis
mRNA (Guber and Hoffinan, 1983) using commercially-available cDNA synthesis
kits. A variety of sources provide cDNA synthesis kits that are useful for
such
purposes. In this particular case, cDNA synthesis kit, EcoR I/Not I adaptors,
T7
sequencing kit, Deaza T7 sequencing mixes, and restriction enzymes were
obtained
from Pharmacia (Piscataway, NJ).
A lambda ZAP II /EcoR I CIAP treated vector kit and Gigapack II Gold
packaging extract were obtained (Stratagene, LaJolla, CA), as was a "GeneAmp
PCR
reagent kit" (Perlcin-Elmer Cetus, Norwalk, CT). Oligonucleotides used for
screening
cDNA libraries and as primers for polymerase chain reactions (PCR) and
dideoxynucleotide sequencing were synthesized on a Biosearch 8700 DNA
synthesizer by 13-cyanoethyl phosphoramidite chemistry and purified on Oligo-
Pak
columns (MilliGen/ Biosearch, Burlington, MA).
A protocol for the cDNA synthesis is provided in "You-Prime cDNA
Synthesis I~it Instructions", Pharmacia LKB Biotechnology, the disclosure of
which is
incorporated herein by reference. (See, in particular, pages 12, 13, 18, 19
and 29 and
Procedures A, B and D thereof for the prototypical procedure.) Using procedure
B,
hemiphosphorylated adaptors contaiung Not I and EcoR I restriction enzyme
sites
were ligated to the termini of the synthesized, double-stranded cDNA prepared
in
Procedure A. After purification of the cDNAs (Procedure D), the N. n.
siamensis
cDNA were inserted into EcoR I-predigested, phosphatased Lambda ZAP IT arms
and
packaged into viable phage particles by using packaging extracts. The latter
was
accomplished using a commercially available kit from Stratagene (LaJolla, CA)
(Catalog #236211, "Predigested Lambda ZAP II/EcoRl Cloning Kit").
N. n. siamensis cDNA was ligated to Lambda ZAP II arms using the
procedure on page 3 of the Strategene instructions (substituting the test
insert for the
N.n. siamensis cDNA). The Iigated sample was then packaged into viable phage
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particles using a "Gigapack Gold" packaging extract from Strategene (product
insert,
page 4). The recombinant bacteriophage was used to infect E. coli host strain,
XLl-
Blue, which generated the primary cDNA library. The primary library contained
approximately 1.35 X 105 pful~.g mRNA.
(e) Isolation of a-cobratoxin cDNA from the cDNA Libra and
Subcloning of cDNA Inserts from Lambda ZAP II Clones
Approximately 100,000 plaques from an amplified cDNA library were
analyzed for sequences encoding a-cobratoxin using a degenerate
oligonucleotide
probe prepared from the known amino acid sequences of a-cobratoxin. The probe
(LAS 1) was prepared as follows:
5' - GGI CAI GTI TGT/C TAT/C ACI AAA/G ACI TGG TGT/C GAI GCI TTI TG -
3'
The oligonucleotide probe above was end-labeled on the 5' end using
[32P~ATP and T4 polynucleotide kinase using standard protocols (Sambrook et
al.
1989). The library was screened for the presence of alpha-cobratoxin cDNA on
nitrocellulose filters according to standard procedures (Sambrook et al.,
1989). Filters
were prehybridized for 4 h at 42C in 6X SSC (90 mM sodium citrate containing
0.9
M NaCI, pH 7.0), containing 1X Denhardt's and 100 mg/ml sonicated and
denatured
salmon sperm DNA. Filters were then hybridized in 4X SSC, pH 7.0, containing
1X
Denhardt solution (SOX = 5 g ficoll, 5 g polyvinylpyrrolidone, 5 g bovine
serum
albumin/500 ml water) and the radiolabelled oligonucleotide probe for 16 h at
42C.
Successive washes were performed in 2X SSC, pH 7.0, at 37C for 30 min
before autoradiography for 16 h at -70C using X-AR film with intensifying
screens.
Double-stranded cDNA inserted into the multiple cloning site (MCS) of
pBluescript
SIB- contained within lambda ZAP II, ware removed as phagemids by an in vivo
excision process designed by Stratagene (LaJolla, CA) (see Stratagen insert,
page 7,
"In Vivo Excision Protocol"). Colonies from the ih vivo excision were selected
by
ampicillin resistance, propagated, and the phagemids were isolated by alkaline
extraction (see pp. 368-369, "Analysis Lysis Method"). The size of the inserts
from
the recombinant phagemids were measured on agarose gel electrophoresis after
digestion with the restriction enzyme, EcoR I.
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Characterization of the Alpha-Cobration cDNA by Asymmetric PCR
and DNA Sequencing
The template for asymmetric PCR was double-stranded pBluescript SK-
containing cDNA inserts of approximately 400 bp. Oligonucleotides designated
as
5 LAS 2 (5' GAGTTAGCTCACTCATTAGGC 3') and LAS 3 (5' ATT-
TTCATTCGCCATTCAGGC 3') were used as primers in asymmetric PCR (see "T7
Sequencing Kit Instructions", Pharmacia LKB Biotechnology"). Sanger
dideoxynucleotide sequencing employed T7 DNA polymerase according to the
manufacturer's protocol accompanying the T7 Sequencing (TM) Kit of Pharmacia
10 LKB Biotechnology. N. h siay~eyasis cDNA template, and the primers (LAS 4
and
LAS 5) were as described below. Single stranded DNA was used as a template.
Programs for sequence analysis from Intelligenetics,Inc. (Mountain View, CA),
including GENED, SEQ, and IF1IVD, were used on a VAX from Digital Equipment
Corp. (Maynard, MA). One of the cDNAs sequences encoded alpha-cobratoxin
15 (identified as Naj a naj a kaouthia cDNA library clone "NNK III 6.2"). The
alpha-
cobratoxin cDNA was an incomplete gene in that the leader sequence coding for
the
snake signal sequence was incomplete (-1 to -20) lacking an in initiation
codon
(ATG). For purposes of expression, this was immaterial, since the leader
sequence
was replaced with a functional start codon and restriction enzyme site (as
described
20 herein with reference to expression of cDNA in yeast).
The gene encoding alpha-cobratoxin could also have been prepared using the
genetic coding sequence for the known amino acid sequence of the protein, and
synthetically constructing a suitable gene using automated biochemical
techniques.
The PCR-derived DNA was resuspended in TE buffer (20 mM tris-CL, 1mM
25 EDTA, pH 7.5) and cleaved with the restriction enzyme, EcoR I (see Gibco
product
insert for EcoR I catalog #15202-013, restriction enzyme assay for EcoR I).
The
yeast DNA vector (pHILD4) was also taken, resuspended in TE buffer and cleaved
with EcoR I.
The vector DNA was cleaved in the same manner as the PCR-derived DNA
30 (see Gibco instructions). After digestion with EcoR I, the PCR-derived DNA
and
yeast vector DNA was purified by the addition of an equal volume of
phenol/chloroform (50/50 v/v), vortexing, and centrifugation in a microfuge
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(12,000g). A second chloroform extraction was performed (equal volume of CHCI3
and sample), vortexing, centrifugation and ethanol precipitation. Ethanol
precipitation was performed by adding sodium chloride to the sample (0.2 M
final
concentration) and 2.5 volumes of cold ethanol. After mixing, the sample was
placed
on dry ice for 15 min, then centrifuged at 4C in a microfuge (12,000g) for 15
min.
The DNA pellet was dried under vacuum.
Both of the EcoR I-treated DNAs were resuspended in TE buffer and
covalently joined together using T4 DNA Ligase (see insert materials, Gibco
BRL,
Cat. # 5224SC, T4 DNA Ligase). The ligated DNA was used to transform competent
E. coli cells (see Enclosure 10 for transformation conditions). Transformants
growing
on TB agar (Terrific Broth + agar) containing ampicillin were isolated and the
recombinant DNA analyzed by restriction enzyme analysis.
Optionally, the DNA can be purified from E. coli cells, e.g., in the manner
described in "Wizards Maxipreps DNA Purification System", Promega. Recombinant
DNA from clones harboring the a-cobratoxin gene/pHILD4 construct was used for
integration into the yeast, Pichia pastoris.
(g) Clonin~and Cytoplasmic Expression
Expression of the alpha-cobratoxin gene in the vector, pHILD4 yields a
cytoplasmic product that lacks posttranslational modifications, including
disulfide
bond formation.
Suitable techniques for cloning and expressing genes into Pichia pastoris have
been developed by the Phillips Petroleum Company and compiled in "Pichia
Expression Kit - A Manual of Methods for Expression of Recombinant Proteins in
Pichia pastoris", which was prepared by Invitrogen and accompanies their
expression
kit having catalog # K1710-OI.
The gene encoding alpha-cobratoxin from amino acids +1 to +~1 can be
removed from the cDNA by using the following polymerase chain reaction
primers:
(a) 5' sense primer (LAS 4) = 5'-GGATCC GAATTC ACG atg [ATA AGA
ACA]-3' (36 mer) and
(b) 3' antisense primer (LAS 5) = 5'-CCTAGG GAATTC TTA TCA [AGG a
TGG]-3' (36-mer).
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Recombinant DNA prepared as described herein was treated with Sst I
restriction enzyme under the same reaction conditions as described above with
respect
to EcoR I, except using reaction buffer No. 2 described in the above-captioned
Gibco
EcoR I product insert. The restricted DNA is purified by the addition of an
equal
volume of phenol/chloroform (50/50 v/v), vortexing, and centrifugation in a
microfuge (12,000g).
A second chloroform extraction was performed (equal volume of CHCI3 and
sample), vortexing, centrifugation and ethanol precipitation. Ethanol
precipitation
was performed by adding sodium chloride to the sample (0.2 M final
concentration)
and 2.5 volumes of cold ethanol. After mixing, the sample was placed on dry
ice for
min, then centrifuged at 4°C in a microfuge (12,000g) for 15 min. The
DNA pellet
was dried under vacuum and resuspended in TE buffer.
The DNA pellet is then integrated into the chromosome of Pichia pastoris
strain GS 115 using conventional proceduxes for integrating genes into Pichia
pastoris
15 (e.g., p. 29-38, "Growth of Pichia for Spheroplasting") and expressing the
integrated
genes (pp. 41-45, "Expression of Recombinant Pichia strains").
Example 2
Recovery and Yield
A fermentation of a cytoplasmically-expressing clone harboring the gene
encoding a.-cobratoxin can be performed in a 5 L New Brunswick BioFlo III
fermentor. The size of the fermentation can be scaled up or down depending on
the
requirement for product. For a 5 L batch, a frozen seed culture containing the
alpha-
cobratoxin construct is used to inoculate 10 ml of MGY media (see attached
media
recipe) in a test tube. After 18 to 20 hours growth at 30°C, 0.5 ml is
used to inoculate
50 ml of MGY in a 250 ml flask. After 36 to 38 hours of growth, the entire 50
ml is
used to inoculate the 5 L fermentor. The fermentation is performed in a basal
salt
medium with 26.7 ml 85% phosphoric acid, 0.93 g/L calcium sulfate-2H20, 18.2
g/L
potassium sulfate, 14.9 g/L magnesium sulfate, 4.13 g/L potassium hydroxide,
40 g/L
glycerol and 2 m/L of basal salts (PTM) are added. PTM basal salts consist of
2.0 g
cupric sulfate, 0.08 g sodium iodide, 3.0 g magnesium sulfate, 0.2 g sodium
molybdate, 0.02 g boric acid, 0.5 g cobalt chloride, 7.0 g zinc chloride, 22 g
ferrous
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sulfate, 0.2 g biotin and 1 ml sulfuric acid per liter. The fermentation
culture is fed
with a 50% solution of glycerol in deionized water, while the methanol feed
solution
is 100% methanol with 2 ml of PTM basal salts and 1 mg biotin per liter.
"Structol"
brand antifoamer can be used as antifoam control; the pH during the glycerol
phase is
maintained at pH 5.0 using 30% ammonium hydroxide; dissolved oxygen is
controlled above 25% saturation by supplementing with pure oxygen.
A standard fermentation procedure is followed which includes an initial batch
phase followed by a 4 hour glycerol fed-batch with a feed rate of 15m1/L/h of
a 50%
glycerol solution. At the completion of the glycerol fed-batch phase the
methanol
induction phase is started. The rate of methanol feeding is increased
gradually from
3.5 to 12 ml/L/h within 6 to ~ hours and maintained at 12 ml/L/h. Samples are
taken
during fermentation for measuring optical density at 600"m, cell dry weight
and SDS-
PAGE analysis.
Yeast cells are recovered from the fermentation by centrifugation. Cells are
1 S washed in breaking buffer (50 mM NaH2P04, 1 mM EDTA, 5 % glycerol, 1 %
PMSF,
pH 6.0), and resuspended in the same buffer prior to disruption in an APV
Matnon
Gaulin 30CD pilot scale homogenizer. Cell debris is removed by centrifugation
and a
PEI precipitation is performed on the cell extract in order to remove
endogenous
nucleic acids,. Polyethyleneimine (PEI) (10%) is added to the cell extract to
obtain a
final concentration of 0.4% PEI. The mixture is allowed to sit for 3 to S
hours at 4C
with stirring. The mixture is centrifuged at 27,000 x g for 15 min and the
supernatant
is dialyzed against 50 mM NaH2P04, pH 6.0 at 4C. The recombinant product is
purified by ion exchange (e.g., cationic exchange matrix) and molecular sieve
chromatography.
There have been a number of heterologous proteins produced using the Pichia
pastoris expression system. The levels of expression from intracellularly
expressed
proteins has ranged from 0.3 to 12 g/L depending on the protein expressed
(Biotechnology I 1, 905-910 (1993)). The level of expression is usually
dependent on
such factors as the genetic construct itself, cell copy number and
fermentation
optimization (e.g., cell density, optimal pH and dissolved oxygen
concentration).
Yields from an alpha-cobratoxin gene expressed intracellularly in Pichia
pastoris will
typically fall in the range stated above.
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Example 3
Ozonation
Ozone (03), a powerful oxidant, is used for water disinfection. In the course
of the present invention, ozone treatment is preferably used to treat the
recovered,
inactive polypeptide in order to render it incapable of spontaneous
reformation.
Optionally, ozonated pure water can be used to itself selectively brealc the
disulfide
bonds of a formed polypeptide in order to provide an inactive, denatured, and
stable
form thereof.
Ozone treatment can be used to quickly provide microbial sterilization and
disinfection, organic compound destruction, and conversion of iron or
manganese
salts to insoluble oxides which can be precipitated from the water. The major
reaction
byproducts are water, oxygen and carbon dioxide. For environmental and safety
concerns, unreacted or residual ozone should be monitored. A number of UV
spectrophotometric methods can be used to determine the level of ozone in
water or
physiological saline. Ozone has an absorption pear at 260 nrn whereas oxygen
does
not absorb at this wavelength. When ozone concentration was measured ice water
(1°C ~ 1°C) by three different colorimetric methods, the
absorbance coefficient in
ozone at 260 nm as Alcmlmg/L is 0.11.
A wavelength scan of ozonated water was determined at various dilutions.
Using the same ozonated water, the ozone concentration was determined by
Accuvac
method described below. Using this, or similar methods, it is possible to
calculate the
ozone content of the ozonated water in mg of 03/L.
A standard curve for the ozonated water was also prepared. From this curve
one can derive the absorbance coefficient of ozone in any given solution.
Table 1
below provides a representative relationship between absorbance coefficients
and
concentration for ozonated water.
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Absorbance coefficient (A) mil = (Absorbance at 260 nm) = (Concentration of
Ozone)
TART,R 1
Absorbance of OzonatedConcentration of OzoneAbsorbance Coefficient
water at 260 nm by of
Accuvac method mg/L Ozone at 260 nm
1.5717 13.48 0.11659
0.628 6.44 0.0975
.39822 2.908 0.1369
.25953 2.6792 0.0968
.19797 1.722 0.11496
.13605 1.28 0.1062
AVERAGE VALUE 0.11
Three different colorimetric methods ("Accuvac", "Alizarin" and "Indigo
5 Trisulphonate" methods) were used for the determination of ozone
concentration in
ice water (1°C ~ 1°C), and compared to absorbance at 260 nm.
Ozonated water was
prepared as described in above. Certain of these methods are used by the
W ternational Ozone Association Standardization Committee.
METHOD 1: ALIZARIN METHOD
10 The method is directly applicable in the range of 0.03 to 0.6 mg/L. A stock
solution of Alizarin violet 3R is made up as a 0.2 mM solution. Disperse
124.45 mg
of the dye into an aliquot of distilled water in a 1 liter volumetric flask.
Mix
magnetically overnight. Add 20 mg of analytical grade sodium
hexametaphosphate,
48.5 g of analytical grade ammonium chloride and 1.6 g of ammonia expressed as
15 NH3. Dilute with distilled water to 1 liter and stir overnight. A 10-fold
dilution of
this solution has an absorbance of 0.155cni 1.) 20 ml of the reagent solution
is
introduced into each of two 200 ml volumetric flasks. Fill one flask with
ozone free
water. Fill the other flask with the sample water by introducing the sample
below the
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36
surface of the dye solution to prevent ozone loss by degassing. When measured,
the
difference in absorbance at 548 nM is 2810 L/M/cm. This equates to the
expression:
mg/L 03 =Total volume (200rn1) x (change in absorption) = (Cell length (1cm) x
0.059 x volume of sampled water (180 rnl))
METHOD 2: INDIGO TRISULPHONATE METHOD
The method is directly applicable in the range of 0.01 to 0.1 mg/L of ozone in
water. A stock solution of indigo-trisulphonate is made up as a 1 mM solution
by
dispersing the dye into a solution of analytical grade phosphoric acid at a
concentration of 1 x 10-3 M. A 100-fold dilution of this solution has an
absorbance of
0.16 +/- 0.01/cm at 600 nm and should be discarded if the absorbance is lower
than
80% of the starting value. Normal stability lasts one month. As a diluted
reagent, 20
ml of the stock solution is diluted to 1 liter together with l Og of
analytical grade
NaH2PO4 and 7 ml concentrated analytical grade H3P04. (stability of the
diluted
solution: one week).
In use, 10 ml of diluted reagent solution is introduced into each of two 100
ml
volumetric flasks. Fill one flask with ozone free water (e.g. distilled
water). Fill the
other flask with the sample water by introducing the sample below the surface
of the
dye solution to prevent ozone loss by degassing. Measure the difference in
absorbance at 600 run between blank and sample with 5 or 10 cm cells. The
measurement is to be made as soon as possible but preferably within 4 hours.
The pH
value of the measured solution must be lower than 4.
The proportionality constant is 0.42 +/- 0.01 /cxn/mg/L ozone, which is equal
to a difference in absorbance of 20 L/Mlcm (Stoichiometry is considered as
1:1).
mg/L (03) _ (total volume (100 Ml) x Change in absorption) = (cell length (cm)
x
0.42 x Volume of sampled water (90 ml))
METHOD 3: ACCUVAC METHOD
As ozone reacts quantitatively with indigo trisulfonate (Blue indigo dye), the
color of the solution fades. Color intensity is inversely proportional to the
amount of
ozone present, is then measured at 600 nm with a spectrophotometer. The
reagent is
formulated to prevent interference from any chlorine residual which may be
present.
The method is directly applicable in the range of 0 to 0.25 mg/L.
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In use, gently collect at least 40m1 of sample in a SOmI beaker. Collect at
least
40m1 of ozone free water (Blank) in another beaker. Fill one W digo ozone
reagent
Accuvac ampule with the sample and one ampule with the blank. This is done by
immersing the ampule in the beaker which has the sample. Quickly invert the
ampules several times to mix. Take an aliquot of the samples and read at 600
nm in
spectrophotometer.
Read a blai~lc value as X at 600 nm. 0.125 mg/L 03 should have absorbance of
x/2 g/L of 03= (0.125 x O.D. of the blank value / 2) = (0.D. of the sample at
600 nm
x Dilution factor).
TABLE 2
METHOD OZONE NOTE
CONCENTRATION
(mg/L of water)
Accuvac 13.676
Alizarin 16.8
Indigo - Trisulphonate15.85
UV absorption at 260 15.45 (Abs/Alc",~r"~1)
nm 1.710/.11
Table 2 shows the ozone concentration, as determined by these various
methods, for aliquots of the same ozonated water. From the results in TABLE 2
it can
be seen that each method provides substantially the same concentration of
ozone.
Since all the four methods seem to be comparable to each other, the W
absorption
method is preferred since it is simple and inexpensive to perform.
Ozone was produced by a high voltage discharge using Tri Atomic Oxygen
Generator (Model No. 3, Serial No. 34 from modern Medical Technology Boca
Raton, Florida) The oxygen was passed through the generator to produce the
ozone.
Approximately 0.2% of ozone was produced in the equipment at the rate of
bubbling
used (about 200m1/min). However, for quantitation studies a sample was taken
with
each series of experiments.
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Absorption measurements were made in the Beckman DU 650 Spectrometer
using cm quartz cuvettes. A standard curve was obtained by serially diluting
the
ozonated water and measuring the absorbance at 260 nm. The standard curve was
also obtained by using a colorimetric method using commercially available
Accuvac
ampules (From Hach, P.O. Box 389, Loveland, CO 80539)
Saturated ozone water was prepared in the following manner. Oxygen was
bubbled at the rate of 200 ml/min to ice water (1 °C ~ 1 °C).
The container with
distilled water was kept in an ice bath during the ozonation. Ozone, bubbled
into
water or saline, was determined by measuring the absorbance at 260nm. Using a
SOmL sample, it takes a minimum of 30 minutes to reach an absorbance reading
of
2.0, although the time is dependent upon the oxygen input.
Since water that is saturated with oxygen will not become saturated with
ozone, the flow rate of input oxygen was ideally kept at equal to or less than
200mL/min. Once the ozonated water reaches an absorbance of 1.0 to 2.0, serial
dilutions of the ice cold ozonated water were made and measurements of the
absorbance at 260nm were made. The ozonated water was also used to measure
kinetics, and in particular, decay rate over the time. The serially diluted
water was
used to measure the ozone concentration by Accuvac method.
Water ozonated in this manner can be used to oxidize a formed polypeptide, in
- order to cleave the disulfide groups and render the polypeptide inactive.
Alternatively, and preferably, the ozonate water can be used to stabilize a
polypeptide
that is prepared in an inactive form by the genetic engineering method
described
above. In either case, the oxidized peptide can be compared to the original,
active
toxin using a variety of methodologies, including animal models and bioassays.
Tn a typical approach, the material to be stabilized (e.g., lyophilized salt
free
toxin) is weighed into 150 ml plastic bottles, each containing 600 mg of
toxin.
Approximately 800 ml of pure deionized water is allowed to chill in the
freezer until
ice crystals begin to form. The beaker of pure water is placed in an ice bath
and
ozonated by bubbling 03 from an ozone generator connected to an 02 source.
Measurements of OD are taken at 260 nm using a 1 cm light path until an ODz6o
of
2.0 is achieved.
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Sixty ml of .ice cold ozonated pure water is added to each bottle containing
600 mg of toxin, resulting in a 1 percent solution (a concentration of
lOmg/ml).
While waiting for the powder to dissolve, the bottles are stored in the
freezer and ice
crystals are again allowed to form. Once in solution, the bottles are placed
in an ice
bath where each bottle is ozonated for 30 seconds by bubbling ozone into the
solution.
Ten bottles are done at one time, such that each bottle is ozonated for 30
seconds
every five minutes. This is done to maintain an effective level of 03 and is
continued
for seven hours.
Periodic testing is done by injecting mice with the toxin suspension and
monitoring the time to death. When the mice no longer die (after seven hours
ozonation) alI disulfide bonds have been broken, and the material has been
effectively
converted from an active toxin to an atoxic toxoid.
It has been noted that if the original ozonated protein solution is maintained
at
4°C for 24 hours and, no fixrther ozonation is carried out, the
disulfide bonds are
likely not going to be broken, and the solution will remain toxic and able to
kill mice.
Also, when bacterial or viruses suspensions are added to ozonated water as
prepared
above, there is inunediate 6-8 log kill. Since bacterial and viral kill
appears to occur
well before oxidation of proteins, ozonated water prepared in this manner can
be used
to treat protein-containing formulations (e.g., monoclonal antibody
preparations) in
order to inactive any remaining animal viruses without damaging the antibody
itself
by breaking critical disulfide bonds.
The oxidized (or stabilized) toxin polypeptide can be compared to the native
alpha neurotoxin in a number of respects. It is found that the former is
atoxic is mice,
while the latter retains full toxicity. The molecular weights as measured on
SDS gels
are 7380 daltons for both the primary neurotoxin and the resultant oxidized
peptide.
The isoelectric point as measured by iso-electric focusing gels varies
substantially
because of the breaking (or stabilized failure to form) of the f ve disulfide
bonds
creating a net charge change of ten. The isoelectric point is the pH at which
a protein
migrates to in an ampholyte solution (continuous pH gradient) to which a
current is
applied. The primary alpha neurotoxin and resultant oxidized peptide also show
separate peaks when measured by HPLC and FPLC.
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Example 4
Immunokine Production
A preferred process for the production of an immunokine of this invention is
outlined below. Alpha-immunokine-NNS (immunokine) is a protein derived from
5 alpha-cobratoxin. Cobratoxin (CTX) has a molecular weight of 7821 and is
composed of 71 amino acids. The native protein is purified from the venom of
the
Thailand cobra, Naj a naj a siamensis. Alpha-cobratoxin from the Thailand
cobra
(Naja naja siamensis) was purchased from Biotoxins, Kississimi, Florida. The
published amino-acid sequence for cobratoxin employing single letter code is:
10 ICRFITPDITSKDCPNGHVCYTKTWCDAFCSIRGKRVDLGCAATCPTVKTGVD
IQCCSTDNCNPFPTRKRP
Employing the reactive molecule, ozone, the precursor protein is modified
through the addition of oxygen molecules. Ozone has the major advantage in
that
when the reaction is complete there is no residual material which requires
removal.
15 Unreacted ozone decays back to oxygen in a relatively short period of time.
The procedure below describes the dissolution of ozone into saline (0.9%) and
its addition to cobratoxin to form immunolane. The reaction is rapid being
completed
in minutes. In order to create a more homogeneous product consistently the
procedure
described below was developed whereby an ozone-saturated fluid is added
directly to
20 a solution of cobratoxin. It is expected that greater reproducibility can
be achieved
with this method. The critical point of the reaction centers on adding
sufficient ozone
to ensure that no native cobratoxin remains. When the reaction is deemed
complete
several parameters can be measured to be suggestive of successful preparation.
The
reaction can be conducted at ambient temperatures but the concentration of the
final
25 product is limited to below 350mg/ml. This arises because of the
limitations placed on
dissolving ozone in saline at these temperatures.
MATERIALS
Eguipment
Approved ozone generator - Haemozone or equivalent
30 Spectrophotometer - Beckman or equivalent
Peristaltic pump, digital input
Thermometer, degrees centigrade, range minimum of -5°C to
25°C
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41
Pipette, lml Gilsen or equivalent or disposable (Sml)
Quartz cuvette or similar, non absorbing at 260nm
Glassware, depyrogenated and autoclaved, flask or graduated cylinder
appropriate for reaction volume, minimum of 2 required.
S Insulated container capable of holding chosen glassware (optional)
Consumables
Gloves, disposables
Oxygen, medical (USP)
Saline, 0.9% for injection from approved source
Cobratoxin, from approved source
Disposable filters, 0.2~m for bacterial culture
Icepacks chilled to -20°C (optional)
Ice (optional)
Confirm that ozone generator has recently been validated for function and
1 S output. Turn on oxygen supply at outlet valve. Switch on ozone generator.
Adjust
oxygen flow at regulator to give a flow reading on generator of up to 0.25L
per
minute. Switch on or ensure sparking coil is operational (listen for auditory
beep).
Switch on peristaltic,pump and set flow to 10 -l Sml per minute. W spect
tubing for
defects. Attach bubbling frit to peristaltic output and place in container of
clean water.
Ensure frit output is functioning and satisfactory. Confirm that ozone
production has
commenced and is rising. Allow machine to operate for 30 minutes in order to
wann-
up.
Switch on spectrophotometer and/or UV lamp and allow to warm-up for 20
minutes. Set absorbance measurements at 260nm. The machine should be blanked
2S against an aliquot of saline (see below). With gloved hands clean frit
surface with
alcohol and place in saline. Increase peristaltic pump flow to 1 Sml per min.
The
obj ect is to supply as much ozone to the solution without inlvbiting ozone
production.
At 10 minute intervals remove aliquots from the solution with a gilsen
pipettor or
disposable pipette and record the absorbance at 260nm.
1. Chill saline to below 3.S°C prior to commencing. This may be
achieved by
storing saline solution at a suitable temperature. If saline temperature is
not
sufficiently low the solution can be stored in a -20°C refrigerator
until the saline has
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42
reached a temperature of -5°C or below. Do not freeze the saline
solution solid though
the presence of slush is quite acceptable.
2. While wearing gloves weigh-out cobratoxin either as 600mg lots or prepare a
60mg/ml solution in saline for injection.
3. Add lOml solution to depyrogenated 1L (or greater) flask. For larger
volumes
add l Oml of 60mg/ml cobratoxin per liter. Appropriately sized containers
should be
employed.
4. Conunence addition of ozone to saline which is below or being held at a
temperature of 3.5°C or below. Monitor ozone content in saline until
the absorbance
at 260nm is recorded at above 1.95 and below 2.05. Place alcohol-cleaned
thermometer in saline, measure temperature and record. If the saline
temperature
exceeds 4.0°C abandon process and return saline to refrigerator for
further chilling.
Should the 260nm reading reach above 2.1 then allow the saline solution stand
at
room temperature until it has.decreased to within the correct limits.
5. Immediately add ozone treated saline up to the correct mark on the flask
containing the cobratoxin solution. Alternatively remove sufficient saline
from the
ozone solution to leave 990m1. Mix by agitation and store overnight on the
bench
(>18 hours). If volumes greater than 1L are being prepared, ozone-treat the
quantity
desired and add to greater volume flasks. Do not make sequential 1 L lots from
the
same ozone treated solution unless it is confirmed by spectrophotometric means
that
the 260nm limits before each addition are satisfied.
6. Following overnight storage record pH of solution, perform spectral scans
from 215nm to 305nm and calculate the 260/280 ratio. Toxicity can be
determined by
injecting lml (600ug) into at least 2 mice via the intra-peritoneal route. For
this
purpose a 27 gauge, 0.5 inch insulin syringe is preferred. The mice should be
monitored for 24 hours. Alternatively or concurrently the absence of
cobratoxin can
be demonstrated by chromatographic analysis.
7. Remove l Oml aliquot for retention and place in sterile glass vial, seal,
crimp
and label.
8. Benzalkonium chloride can be added to a final concentration of 0.01%.
9. (Remove aliquot (1m1) with pipette or syringe and place in sterile
container for
analysis by mass spectrometry.)
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43
Spectrophotometric scans of the ozone treated cobratoxin from 200nm to
400nm were identical to those described by Chang et al. (1990) confirming the
modification of the tryptophan residue. Because ozone attacks tryptophan there
is a
significant reduction in the UV absorption at 280nm - approximately half that
measured fox the original cobratoxin solution and an increase in the
absorption at
260nm. This provides a simple method to determine if the chemical modification
is
sufficiently complete to produce a satisfactory product. If the A260 value is
divided
by A280 a ratio is developed. From our experience and validation, if the ratio
is
greater than 2.7 and the pH is 4.5 or less then the product is non-toxic. This
approach
towards an indication of potency is appropriate only for those proteins which
have
tryptophan residues. However it should be noted that there exists a
fluctuating curve
for the ratios which peak at 3.4 before dropping to levels below 2.6 and
rising again.
At this point the product is being deteriorated and fragmented by excess
ozone. It is
therefore best to combine these measurements with other assays for potency
and/or
toxicity. Potency of the modified neurotoxin was evaluated through a
modification of
the procedure described by Stiles et al. (1991).
The reaction can be conducted at room temperature if refrigeration is
unavailable but the concentration of final product will be substantially less
(approximately 300mcg/ml). This results because the solubility of ozone in
saline is
dependent on the temperature of the liquid. The lower the temperature the
higher the
ozone concentration and subsequently the greater the quantity of material that
can be
reacted at one time. To all intents and purposes the product produced at
300mcg/ml
and 600mcg/ml with the appropriate levels of ozone were identical and it is
known
that material produced at ambient and chilled temperatures by the previous
bubbling
method do not differ by mass spectrometry and sequence. The reaction is a
single step
one, easily reproducible and provided the correct conditions were employed it
can be
reasonably assumed that the drug produced is at the desired potency.
An immunokine solution prepared in this manner had an acidic pH and a pI of
approximately 4.5. Cobratoxin solutions are basic having pH of 8.5. In
solution, the
drug migrates through molecular sieving gels as monomers, dimers and
tetramers.
Cobratoxin migrates under these conditions as a monomer. Upon analysis on
NuPAGE (Novex) SDS polyacrylamide gel electrophoresis (PAGE) the cobratoxin
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44
migrates as a l4Kd and 8Kd protein with a reference to comparable proteins
under
unreduced and reduced conditions respectively. Immunokine migrates under
reduced
and unreduced conditions without change. A single protein band is not obtained
showing a diffuse smear from the loading geI down to a molecular weight
equivalent
to BKd. Additionally, the protein is resistant to staining with standard
coomassie
dyes. By ion exchange, cobratoxin and immunokine have generally opposite
properties consistent with the proteins' charges; Specialized ion-exchange
chromatographic resins and conditions can be employed to confirm the retention
of
positive charges which are considered critical for neuroactive properties.
As defined by mass spectrometry the average molecular weight of
immunokine is 7,933.3 ~ 30 daltons (determined from 7 lots, 5 consecutive
assays
each) with a molecular weight range of 7,600 to 8,400 daltons. This molecular
weight
variance is expected by the nature of the reaction and ozone. As indicated
above
excessive ozone application can fragment the protein and insufficient levels
do not
modify enough amino-acid residues to render the neurotoxin atoxic. The
calculated
average molecular suggests the addition of 6 oxygen residues with higher
molecular
weights having correspondingly more. Smaller than expected molecular weights
suggest protein fragmentation. Current analytical techniques allow for limited
structural identification of the number and location of oxygen residues being
added to
the protein and rely heavily on previously published information and current
chemical
theory. Amino acid analyzers do not recognize unnatural amino acids and have
limited capabilities for this application.
Example 5
FeLV Study
A group of 87 that had tested positive for either FeLV or FIV yielded 87 was
studied. Of these 87, 20 were found to be negative for both FeLV and FIV when
blood samples were submitted to the University of Miami Medical School
Laboratory.
These 20 were excluded. Fourteen cats presented in critical condition were
also
excluded. All of these cats died within ten days. The study therefore became a
study
of cats with chronic FeLV and/or chronic FIV. Confirmation of presence of
either or
both viral infections in each cat was determined by tests conducted by the
University
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of Miami Medical School Pathology Reference Laboratory by either IFA or ELISA
tests.
Thirty-seven cats were confirmed positive for either FeLV, FIV, or both as
follows: twenty-eight only FeLV, seventeen only FIV, seven both FeLV and FIV.
5 Hematocrits ranged from twenty eight to forty with a median range of thirty
to thirty-
five and there were no consistent abnormalities in the white cell counts or
differentials. The occasional cat would have a slightly elevated segmented
neutrophil
count and/or aslightly decreased lymphocyte count. Physiological abnormalities
include poor appetite resulting in weight loss, poor hair coat, diminished
activity,
IO frequent and sometimes continuing bouts of rhinitis and/or sinusitis,
gingivitis,
frequent abscesses. Interestingly, two cats were in excellent health with
glossy hair
coats, normal to slightly above normal weight, normal strength and activity,
etc. Both
of these cats had been vaccinated for FeLV as young adults and were only
mildly
positive to tests for FeLV. Cat~owners were instructed as to how to give the
mCTX
15 injections and a quantity sufficient for thirty days was dispensed.
Each cat was given a physical examination and the results recorded. History
included length of known infection and/or when cat was first discovered to be
FeLV
or FIV positive, previous or current therapy. Blood was drawn for CBC and test
for
FeLV and FIV. Criteria for entering the study was either IFA or ELISA positive
as
20 determined by Pathology reference Laboratory. Ploymerase Chain Reaction
(PCR)
testing was carried out by Dr. James Thompson, D. V. M. (University of Florida
Veterinary Teaching Hospital). At the end of each thirty day period blood
samples
were submitted for CBC's including differentials, and FeLV and/or FIV tests.
Placebo
controls were not utilized since these animals were privately owned. The
animals
25 were monitored for FeLV and FIV by ELISA and these acted as their own
control in
the objective sense due to absence of the "placebo" effect., subjectively,
improvement
was noted in found
The following concentrations were used to determine the IC50 FIV values for
MCTX; no MCTX (control), O.I, 0.4, 1, 4, 10, 20, 50, 100 and 200ug/ml. Results
are
30 given in Figure 1. Using the linear portion of the graph in Fig. 1 and ICSO
value of
804ug/ml was determined. It should be noted that the concentrations used in
the
determination were well below the calculated ICSO concentration, however, due
to the
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46
scarcity of material another ICSO determination was not possible. These data
suggest
that modified cobratoxin may not block effectively over 4 days.
The data presented in Fig. 2 shows the infectious virus yield over a four week
period. These data show that the total virus formation from cultures treated
with
MCTX were reduced compared to cultures with no drug. Fig. 3 is a re-plot of
data
from figure 2, showing tha percent inhibition of virus from cultures treated
with
MCTX compared to no drug control. From these data both concentrations of MCTX
appear somewhat effective over 4 weeks.
The approach to treating infected cats was empirical. To avoid any possible
adverse reactions to the MCTX, it was decided to administer small doses
initially
though the in-vitro testing indicated that higher doses would be required.
Also,
positive responses were seen in various animals with low concentrations of
MCTX
(Harnson, 1989 and Smith, 1991). As the MCTX appears to have broad anti-viral
properties, cats presenting with FeLV were included to evaluate if the MCTX
could
be utilized against other lentivirus infections. The treatment regime began
with 5
micrograms of MCTX every 12 hours by subcutaneous injection for a period of
thirty
days. At the end of the thirty days, tests for FeLV/FIV were to be conducted
and
compared with pre-treatment tests. Following thirty days of twice daily
treatment the
first group of cats returned for clinical appraisal and blood samples. Tn
every case
there were clinical improvements such as increased appetite, weight gain,
improved
hair coat, more playful, etc. There were no significant changes in IFA and
ELISA
titers after thirty days of treatment. Repeat blood tests were scheduled
thirty days
later. At the end of the second thirty day period (30 days from last
treatment) all in-
house tests were still positive and were confirmed positive at the University
of Miami
Medical School Laboratory. Clinical improvements, however, were being
maintained
without further treatment.
At this phase it was decided to resume treatments and to increase the dosage
to
10 micrograms every 12 hours and continue as long as necessary to obtain
negatives
or until the cat owner elected to drop out. The laboratory reported the IFA
titers for
FIV as 1:50 (borderline negative), 1:250, 1:500. IFA or ELISA for FeLV was
subjectively reported as l, 2, or 3 plus depending on depth and rapidity of
color
change in the tests. At this dosage level we began to see some reduction in
titers after
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47
each thirty days of treatment. Meanwhile all cats in the "chronic" study
continued to
do well and each month one or more owners elected to drop out either because
of
satisfaction with clinical results or the inability to continue twice daily
inj ections to
the cat.
The next dosage increase was to 25 micrograms every 12 hours. After one
month at this level the first negative for FeLV was attained, both for IFA and
ELISA,
in a cat that had been positive for both FIV and FeLV. The cat remained
positive for
FIV. Unfortunately, this owner dropped out after the FeLV negative tests. A
second
cat positive for both FIV and FeLV tested negative for both viruses at the end
of the
second month of the 25 microgram dosage. At this point a decision was made to
double the dose each month until more negatives were attained. To date all
cats that
have remained in the study, both FIV and FeLV, have become completely negative
with the exception of five cats that are IFA negative and ELISA positive for
FeLV.
All five of these cats have gone through at least 30 days of 200 micrograms
every 12
hours. Three of them finished 60 days at this level. Only one owner reported a
troublesome side effect. This cat was FIV positive. After three or four days
of
treatment the owner reported the cat had developed diarrhea. Treatment was
discontinued for a few days and the diarrhea subsided. Treatment was resumed
and
the diarrhea started again. Lactobacillus was prescribed twice daily. The
diarrhea
stopped and treatment was continued uneventfully.
From Table 1 the results can be summarized. From 28 chronic FeLV cats,
fourteen stopped treatment by owners due to satisfactory clinical improvement
in their
condition. Fourteen went to IFA negative. Nine of these also went to ELISA
negative while five remained ELISA positive. The laboratory reported these as
weak
positives. Of interest here also, the two cats that had been vaccinated
against FeLV as
young adults remained ELISA positive. Five cats tested PCR negative for FeLV.
From 24 cats with FIV, seventeen with FIV alone plus seven with both FIV and
FeLV, fourteen dropped out after satisfactory clinical improvement. Ten of the
remaining ten went to IFA negative. ELISA testing was not done in the last
months
of the study on the FIV cats.
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Table 1. Summary of Dosage Regime and Blood Analysis from 52 Cats
with FeLeuk and/or FIV
Time Dosage Total drug Losses Cumulative FeL/FIVFeL
(months) (ug/ml Administeredfrom (negative) (-ve)
B.LD) (ug/cat) study ELISA PCR
IFA
1 5 300 1 la nd
2 5 600 S 1 nd
3 10 1200 nd 2 nd
4 25 2700 3 7 3 nd
25 4200 28 nd nd nd
6 50 7000 28 7 6 1
7 100 13000 28 9 8 2
8 100 19000 28 19 24 nd
9 100 25000 28 19 24 5
12 100 31000 49 19 24e rid
Totals 31000 49 79% 100% 20%
5 nd = not determined, a: Cat vaccinated with FeLeuk, b: Previously negative
cat tests positive, c: Random data, not performed on aII cats, d: 3 cats
remained in
long term study (18 months) to observe ELISA responses, e: Includes 3 cats
positive
for FeLeuk and F1V.
Dosage values (given IM) are per animal irrespective of size. Percent values
calculated from animals remaining at end of trial(24). Three of the above cats
had
concurrent chronic conditions. Two of these cats had FeLeuk titers to both
ELISA and
IFA testing although they had been previously vaccinated for it.
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Table. 2 Summary of Response in S2 Cats with Feline Leukemia and FIV
Duration ImprovedWeight IncreasedConsumer Consumer
of TherapyappetiteGain Activity Satisfactioncessation
(months)
1 52 43 52 52 -
2 52 45 52 52 -
3 52 47 52 52 -
4 52 49 52 52 3
52 49 52 52 28
6 52 49 52 52 28
7 52 49 52 52 28
8 52 49 52 52 28
9 52 49 52 52 28
Total 100% 94% 100% 100% 54%
5 Example 6
CXCR4 Study
Replication endpoint concentration assay.
A TCIDso of: 1000 for HIV-lBal (CCRS-using) and 10,000 for HIV-1~;
CXCR4-using)was used to infect 107 PHA-stimulated peripheral blood mononuclear
cells in 24 well microtiter plates. The concentrations of recombinant,
ultrapure
irnmunokina used were 1-1000 ~.g/mL. All strains were tested in quadruplicate
wells
in three separate experiments. To correlate the replication endpoint
concentration with
a formal percent inhibitory concentration, we obtained that absolute p24
antigen
content for each drug concentration. The concentration of drug that reduced
the p24
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antigen value of the control well by 50% (ICSO) was calculated using non-
parametric
regression analysis. Immunokine inhibited infection by HIV-lBat by 87%
compared to
untreated controls and inhibited infection by HIV-1~; by 96% compared to
untreated
controls with an ICSO for CCRS-using isolates of 90 r~g/mL and an ICSO of 10
~,g/mL
5 for CXCR4-using isolates of HIV-1 (see figure). Immunokine did not affect
proliferation as measured by [3H]thymidine incorporation and was not cytotoxic
as
determined by the soluble fonnazan assay.
25
Example 7
Human Thymus Explant Culture
30 Human thymus removed for cardiac procedures from children ages 4.5 months
to 11 years was grown in culture up to 7 days without loss of cells. A minimum
of
three replicate tissue pieces were harvested for each time point or condition
normally
yielding 3-6 million cells per/fragments. The tissue fragments were pretreated
with
100 r~g/mL of immunokine for 1 hour at 37° C. The tissue fragments were
washed in
35 PBS, pH 7.4 and placed into sterile tubes contaiung 3000 TCIDSO of either
HIV-lBai
or HIV-lLai. The tissues were incubated at room temperature for 4 hours with
gentle
rocking. The tissue fragments were washed twice with PBS, pH 7.4 and
transferred to
0.45~.m nucleopore filters (Millipore) atop gelfoam boats (Upjohn) saturated
in media
[(YSSL's, 1% human serum, 50 ~,g/ml streptomycin, 50 U/ml penicillin G, 1X MEM
40 vitamin solution (GIBCO,BRL), 1X insulin/transfernn/sodium selenite media
supplement (Sigma)], in six well plates with a maximum of 16 pieces per raft.
The
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51
fragments were incubated at 37°C with 5% C02 for up to 3 days. At day
3, 3-4
fragments were removed and processed for flow cytometry. Quantitative
evaluation of
T-cell precursor subsets was performed to determine if immunokine protected
thymocytes from HIV-1 induced destruction in this in vivo model.'As shown in
the
figure, 100 r~g/mL of immunokine protected CD4 and CD8 single positive T-cell
precursors and CD8/CD4 dual positive T-cell precursors from the HIV-1 induced
destruction seen in untreated controls.
Untreated 100 ~,g/mL PP03
.SRI . R!.,
R2' . ,.,. /(f;
qr ,ta
HIV-lLai
_.- ; _r~..~..
. , . R3 ...R3.
10~~. " IOV ~ 10~.: ~.;10~:: -100,., .A 17.~., 'U':. :,..a
Rl RI
R2'
~',d
r.
'~ °' HIV-lBa1
N .' . ! ~'~' fry P ',
a'!r 4s
'! , f~~ ,
R3.., 'fir . .,R3.
~di.
poi ' .":j~ , 1~ . ..,t ~r ~~ ~ ,... ;fit '~16F' ~ ~1)# ' , ~,',io'~
CD4 --t
